lf-less acts can be found. The label was also politically convenient.Conclusions. A middle ground for reasonable expectations from HCW when treating diseases that have serious risk of infection should be expected. While all should act according to the ethic of beneficence not all persons should be expected to be martyrs for society. Abstract Computer analysis of the intrinsic membrane protein, myelin proteolipid, shows strong sequence similarities between the putative extramembranc segments of the proteolipid protein and a number of viral proteins, several of which infect humans. These similarities are even more striking than those reported previously between viral proteins and the encephalitogenic myelin basic protein (MBP). These findings, along with other reports of molecular mimicry by viruses, suggest that immunological cross-reactions between virusinduced antibodies or T-cells and analogous antigenic determinants (epitopes) in myelin protcolipid could be involved in the pathophysiology of multiple sclerosis or post-infectious dcmyclinating syndromes. Proteolipid prolein Multiple sclerosis Viral protein Myelin basic protein Amino acid sequenee Sequence homology Abstract The interferon (IFN)-inducing capacities of intact NDV virions, /3-propiolactone-inactivated particles and several structural components were compared, using human PBML as the IFN producing cells. Intact and inactivated virions as well as the nucleocapsid fraction did not differ significantly in their IFN-inducing capacity. In contrast, genomic RNA as well as M protein fraction and envelopes induced IFN titres to a level of about 10% of those achieved with virions. NDV-induced IFN production could be blocked specifically by incubation with polyclonal anti-NDV-monoclonal antibodies (mAbs) and with two of three anti-HN-mAbs, but not with anti-NDV-mAbs directed against the F, M or NP protein. In addition, IFN induction by fixed MDBK cells, expressing NDV surface proteins after infection with NDV Ulster, was inhibited by one of two anti-F-mAbs. The results suggest that the induction of IFN synthesis in human PBML is a complex process involving not only the HN protein but also the uncleaved F protein precursor, a component of the M protein fraction and -once having entered the cell -the genomic RNA. Abstract An average of 70 samples were collected from 80 dairy farms in England and Wales, from cattle, co-grazed sheep, wildlife and farm wastes, to investigate prevalence, potential sources and transmission routes of Cryptosporidium. At least one positive sample was detected on 74 of the farms (92.5%) by IFAT microscopy. The prevalence in cattle was 10.2% (95% CI 9.4-11.1%), with greater prevalences detected in calf samples, especially from those under 1 month (45.1%). Young calves were also more likely to be shedding Cryptosporidium parvum and larger concentrations of oocysts, whereas older calves and adult cattle were more likely to be shedding Cryptosporidium bovis and Cryptosporidium andersoni, respectively. The C. parvum subtypes detected were predominantly from types commonly identified in UK cattle (67% were either IIaA15G2R1 or IIaA17G1R1). A novel subtype, IIaA17G1R2, was identified from one cattle sample.The prevalence in co-grazed sheep was low (4%). Birds and rodents may represent significant reservoirs of Cryptosporidium due to high prevalence, large oocyst concentrations, and the detection of a C. parvum subtype known to be present in human populations, identified in samples from these wildlife. Cryptosporidium were detected in dirty water and manure, and also from pasture samples where slurry had been spread.On 64% of the farms, identical Cryptosporidium species were detected (mainly C. parvum or C. bovis) from different cattle groups on the farms, although no direct or indirect contact between the groups were recorded, apart from sharing staff. The same Cryptosporidium species were found in cattle, farm wastes and bird samples on the same farms, but rarely, or not at all, present in sheep or rodent samples. The matching of species/subtypes was also related to the proximity of the different sample sources which may indicate a potential transmission route.Crown Abstract Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease 1 . The host factors required for alphavirus entry remain poorly characterized 2 . Using a genome-wide CRISPR/Cas9-based screen, we identified the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses including chikungunya (CHIKV), Ross River, Mayaro, and O'nyong nyong (ONNV) viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced viral infection of cells, and reciprocally, ectopic expression resulted in increased infection. Mxra8 bound directly to CHIKV particles and enhanced virus attachment and Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature. Abstract The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin Ldeficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form. Abstract Supplementary Data SUPPLEMENTARY FIG. S1. High Performance Liquid Chromatography trace of branched PepC7 cyclized product. (Top Panel) Branched PepC7 with first disulfide bond formed by dissolving the linear peptide in 0.1 M NH 4 HCO 3 (1 mg/ 3 mL, pH 8) followed by the addition of 2,2¢-dipyridyldisulfide (1 mM) in methanol (0.2 mL/1 mg). (Middle Panel) Iodine oxidation of the Acm-protected Cys residues using a 10-fold excess of I2 per Acm as 20 mM I2 solution in acetic acid, 60 min at room temperature. (Bottom Panel) Iodine oxidation using a 20-fold excess of I2 per Acm, 30 min at room temperature. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ~0 1000 strains of C. perfringens from cases of enteritis in domestic animals were type E, and all (n ¼ 45) were from neonatal calves with hemorrhagic enteritis. Furthermore, type E isolates represented nearly 50% of all isolates submitted from similar clinical cases in calves. Commercial toxoids available in North America have no label claims for efficacy against type E infections. Consideration should be given to type E-associated enteritis when planning for the health care of calves. r Abstract More than 1 million sexually transmitted infections (STIs) are acquired each day globally. Etiotropic drugs cannot effectively control infectious diseases therefore, there is a dire need to explore alternative strategies especially those based on the regulation of immune system. The review discusses all rational approaches to develop better understanding towards immunotherapeutic strategies based on modulation of immune system in an attempt to curb the elevating risk of infectious diseases such as HIV, HPV and HSV because of their high prevalence. Development of monoclonal antibodies, vaccines and several other immune based treatments are promising alternative strategies that are offering new opportunities to eradicate pathogens. Abstract This study was conducted to evaluate the quantities of medical waste generated and the factors associated with the generation rate at medical establishments in Taiwan. Data on medical waste generation at 150 health care establishments were collected for analysis in 2003. General medical waste and infectious waste production at these establishments were examined statistically with the potential associated factors. These factors included the types of hospital and clinic, reimbursement payment by National Health Insurance, total number of beds, bed occupancy, number of infectious disease beds and outpatients per day. The average waste generation rates ranged from 2.41 to 3.26 kg/bed/day for general medical wastes, and 0.19-0.88 kg/bed/day for infectious wastes. The total average quantity of infectious wastes generated was the highest from medical centers, or 3.8 times higher than that from regional hospitals (267.8 vs. 70.3 Tons/ yr). The multivariate regression analysis was able to explain 92% of infectious wastes and 64% of general medical wastes, with the amount of insurance reimbursement and number of beds as significant prediction factors. This study suggests that large hospitals are the major source of medical waste in Taiwan. The fractions of medical waste treated as infectious at all levels of healthcare establishments are much greater than that recommended by the USCDC guidelines. Abstract Although the brain has been considered as an immunologically privileged site, the evidence to date suggests that this is no longer the case. Cytokines such as interferon (IFN)-g, tumor necrosis factor (TNF)-a, and interleukin (IL)-3 induce class I major histocompatibility complex (MHC) antigen expression on neural cells. IFN-g, the most potent inducer of MHC antigen, also induces class II MHC antigen expression on microglia and astrocytes, which enable them to function as antigen-presenting cells. Thus, in some pathological conditions, invading T cells can interact with neural cells to induce central nervous system (CNS) damage. Glial cells have also been shown to produce various cytokines and chemokines. Almost all cytokines and chemokines known to occur in the immune system are also produced in the CNS. In this chapter, the glial responses contributing to neuroimmune interactions are reviewed, with a focus on production and functions of cytokines in the CNS.Cytokines and the Brain 289 Edited by C. Phelps and E. Korneva Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.INTESTINAL BACTERIABacterial culture Cultivation methods are most useful when targeting a specific pathogen in clinical specimens (eg, Salmonella). Culture assesses the viability of organisms and allows antimicrobial susceptibility testing. Isolates can be genotyped for epidemiologic studies. Culture is also valuable for characterizing the metabolic properties of isolates and their virulence factors.The author has nothing to disclose. Abstract Due to a combination of ecological, political, and demographic factors, the emergence of novel pathogens has been increasingly observed in animals and humans in recent decades. Enhancing global capacity to study and interpret infectious disease surveillance data, and to develop data-driven computational models to guide policy, represents one of the most cost-effective, and yet overlooked, ways to prepare for the next pandemic. Epidemiological and behavioral data from recent pandemics and historic scourges have provided rich opportunities for validation of computational models, while new sequencing technologies and the 'big data' revolution present new tools for studying the epidemiology of outbreaks in real time. For the past two decades, the Division of International Epidemiology and Population Studies (DIEPS) of the NIH Fogarty International Center has spearheaded two synergistic programs to better understand and devise control strategies for global infectious disease threats. The Multinational Influenza Seasonal Mortality Study (MISMS) has strengthened global capacity to study the epidemiology and evolutionary dynamics of influenza viruses in 80 countries by organizing international research activities and training workshops. The Research and Policy in Infectious Disease Dynamics (RAPIDD) program and its precursor activities has established a network of global experts in infectious disease modeling operating at the research-policy interface, with collaborators in 78 countries. These activities have provided evidence-based recommendations for disease control, including during large-scale outbreaks of pandemic influenza, Ebola and Zika virus. Together, these programs have coordinated international collaborative networks to advance the study of emerging disease threats and the field of computational epidemic modeling. A global community of researchers and policy-makers have used the tools and trainings developed by these programs to interpret infectious disease patterns in their countries, understand modeling concepts, and inform control policies. Here we reflect on the scientific achievements and lessons learnt from these programs (hindex = 106 for RAPIDD and 79 for MISMS), including the identification of outstanding researchers and fellows; funding flexibility for timely research workshops and working groups (particularly relative to more traditional investigator-based grant programs); emphasis on group activities such as large-scale modeling reviews, model comparisons, forecasting challenges and special journal issues; strong quality control with a light touch on outputs; and prominence of training, data-sharing, and joint publications. Abstract During the first year of enhanced MERS coronavirus surveillance in England, 77 persons traveling from the Middle East had acute respiratory illness and were tested for the virus. Infection was confirmed in 2 travelers with acute respiratory distress syndrome and 2 of their contacts. Patients with less severe manifestations tested negative. Abstract Positive-strand RNA genomes function as mRNA for viral protein synthesis which is fully reliant on host cell translation machinery. Competing with cellular protein translation apparatus needs to ensure the production of viral proteins, but this also stifles host innate defense. In the present study, we showed that porcine reproductive and respiratory syndrome virus (PRRSV), whose replication takes place in the cytoplasm, imprisoned host cell mRNA in the nucleus, which suggests a novel mechanism to enhance translation of PRRSV genome. PRRSV nonstructural protein (nsp) 1β was identified as the nuclear protein playing the role for host mRNA nuclear retention and subversion of host protein synthesis. A SAP (SAF-A/B, Acinus, and PIAS) motif was identified in nsp1β with the consensus sequence of 126 -LQxxLxxxGL-135 . In situ hybridization unveiled that SAP mutants were unable to cause nuclear retention of host cell mRNAs and did not suppress host protein synthesis. In addition, these SAP mutants reverted PRRSV-nsp1β-mediated suppression of interferon (IFN) production, IFN signaling, and TNF-α production pathway. Using reverse genetics, a series of SAP mutant PRRS viruses, vK124A, vL126A, vG134A, and vL135A were generated. No mRNA nuclear retention was observed during vL126A and vL135A infections. Importantly, vL126A and vL135A did not suppress IFN production. For other arteriviruses, mRNA nuclear accumulation was also observed for LDV-nsp1β and SHFV-nsp1β. EAV-nsp1 was exceptional and did not block the host mRNA nuclear export. Abstract RNA interference (RNAi) plays a pivotal role in the regulation of gene expression to control cell development and differentiation. In plants, insects and nematodes RNAi also functions as an innate defence response against viruses. Similarly, there is accumulating evidence that RNAi functions as an antiviral defence mechanism in mammalian cells. Viruses have evolved highly sophisticated mechanisms for interacting with the host cell machinery, and recent evidence indicates that this also involves RNAi pathways. The cellular RNAi machinery can inhibit virus replication, but viruses may also exploit the RNAi machinery for their own replication. In addition, viruses can encode proteins or RNA molecules that suppress existing RNAi pathways or trigger the silencing of specific host genes. Besides the natural interplay between RNAi and viruses, induced RNAi provides an attractive therapy approach for the fight against human pathogenic viruses. Here, we summarize the latest news on virus-RNAi interactions and RNAi based antiviral therapy. Abstract The widespread use of compound pelleted feeds and chemical fertilizers in modern food production contribute to a vast amount of residual nutrients into the production system and adjacent ecosystem are major factors causing eutrophication. Furthermore, the extensive development and application of chemical compounds (such as chemical pesticides, disinfectants and hormones used in enhancing productivity) in food production process are hazardous to the ecosystems, as well as human health. These unsustainable food production patterns cannot sustain human living in the long run. Wetlands are perceived as self-decontamination ecosystems with high productivities. This review gives an overview about wetlands which are being integrated with food production processes, focusing on aquaculture. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. The re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. Ongoing efforts to develop MERS-CoV vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. Extending on our current knowledge of MERS, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish MERS-CoV transmission to humans. In addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next CoV outbreak. Abstract Ribonucleases are counterweights in the balance of gene expression and are also involved in the maturation of functional RNA. Recent structural data reveal how ribonucleases recognize and cleave targets, in most cases with the catalytic assistance of metal cofactors. Many of these enzymes are 'processive', in that they make multiple scissions following the binding of substrates; crystallographic data can account for this solution behaviour. These data not only explain how ribonucleases turn over transcripts, but also provide hints about how they often play dual roles in quality control checks on structured RNA. Abstract Epidemiologic investigations showed that 2 of 4 patients with severe acute respiratory syndrome (SARS) identified in the winter of 2003-2004 were a waitress at a restaurant in Guangzhou, China, that served palm civets as food and a customer who ate in the restaurant a short distance from animal cages. All 6 palm civets at the restaurant were positive for SARS-associated coronavirus (SARS-CoV). Partial spike (S) gene sequences of SARS-CoV from the 2 patients were identical to 4 of 5 S gene viral sequences from palm civets. Phylogenetic analysis showed that SARS-CoV from palm civets in the restaurant was most closely related to animal isolates. SARS cases at the restaurant were the result of recent interspecies transfer from the putative palm civet reservoir, and not the result of continued circulation of SARS-CoV in the human population. Abstract Moraillon, A., Barrr-Sinoussi, F., Parodi, A., Moraillon, R. and Dauguet, C., 1991. In vitro properties and experimental pathogenic effect of three strains of feline immunodeficiency viruses (FIV) isolated from cats with terminal disease. Vet. Microbiol., 31:41-54.Three strains of virus isolated from peripheral blood mononuclear cells (PBMC) of sick cats were identified as feline immunodeficiency virus (FIV) on the basis of in vitro cytopathic effect, T-lymphotropism, ultrastructural morphology and magnesium-dependent reverse-transcriptase activity. The pathogenic properties of two isolates were studied in 13 experimentally infected cats. The primary phase of infection was characterised by a range of haematological (neutropenia, lymphopenia, presence of atypical lymphocytes) and clinical alterations (fever, various signs lasting several weeks, generalised lymphadenopathy persisting for several months) and specific seroconversion. A correlation between the inoculated dose of virus and the intensity and duration of clinical signs was observed. The primary phase was followed in the 10 surviving cats by a stage of asymptomatic seropositivity of undetermined duration but which has persisted for over 35 months for the earliest infections. Viruses reisolated several weeks or months after experimental infection retained the same in vitro properties as the initial isolates.0378-1135/92/$05.00 Abstract The pharmacological inhibitor SP600125 [anthra(1,pyrazol-6(2H)-one 1,9-pyrazoloanthrone] has been largely employed as a c-JUN N-terminal kinase (JNK1/2) inhibitor. In this study, we evaluated whether pretreatment with SP600125 was able to prevent Orthopoxviruses Vaccinia virus (VACV), Cowpox virus (CPXV) and modified Vaccinia virus Ankara (MVA) replication. We found that incubation with SP600125 not only blocked virus-stimulated JNK phosphorylation, but also, significantly reduced virus production. We observed 1-3 log decline in viral yield depending on the cell line infected (A31, BSC-40 or BHK-21). The reduction in viral yield correlated with a dramatic impact on virus morphogenesis progress, intracellular mature viruses (IMV) were barely detected. Despite the fact that SP600125 can act as an efficient anti-orthopoxviral compound, we also provide evidence that this antiviral effect is not specifically exerted through JNK1/2 inhibition. This conclusion is supported by the fact that viral titers measured after infections of JNK1/2 knockout cells were not altered as compared to those of wild-type cells. In contrast, a decline in viral titers was verified when the infection of KO cells was carried out in the presence of the pharmacological inhibitor. SP600125 has been the focus of recent studies that have evaluated its action on diverse viral infections including DNA viruses. Our data support the notion that SP600125 can be regarded as a potential antipoxviral compound. Abstract The complete genome sequence of the first equine coronavirus (ECoV) isolate, NC99 strain was accomplished by directly sequencing 11 overlapping fragments which were RT-PCR amplified from viral RNA. The ECoV genome is 30,992 nucleotides in length, excluding the polyA tail. Analysis of the sequence identified 11 open reading frames which encode two replicase polyproteins, five structural proteins (hemagglutinin esterase, spike, envelope, membrane, and nucleocapsid) and four accessory proteins (NS2, p4.7, p12.7, and I). The two replicase polyproteins are predicted to be proteolytically processed by three virus-encoded proteases into 16 non-structural proteins (nsp1-16). The ECoV nsp3 protein had considerable amino acid deletions and insertions compared to the nsp3 proteins of bovine coronavirus, human coronavirus OC43, and porcine hemagglutinating encephalomyelitis virus, three group 2 coronaviruses phylogenetically most closely related to ECoV. The structure of subgenomic mRNAs was analyzed by Northern blot analysis and sequencing of the leader-body junction in each sg mRNA. Abstract Background: The role of respiratory viruses in cystic fibrosis (CF) exacerbations is incompletely understood. Methods: Cross-sectional study of CF children with a pulmonary exacerbation. Mid-turbinate swabs were tested by a direct immunofluorescent antibody assay and a multiplex PCR panel (ResPlex II v2.0, Qiagen). Resplex II was also applied to sputum or throat swab samples. Pulmonary function tests and quality of life and severity scores were recorded. Sputum cell counts, bacterial density and cytokines were measured. Results: 26/43 (60.5%) subjects tested positive for at least one respiratory virus by any diagnostic method applied to any sample type. Viruspositive patients were younger (p = 0.047), more likely to be male (p = 0.029), and had higher CF clinical severity (p = 0.041) and lower quality of life (physical) scores (p = 0.023) but similar IL-8, neutrophil percentage and elastase levels.Conclusions: Compared to non-viral exacerbations, viral-related exacerbations were associated with worse severity and quality of life scores but similar pulmonary inflammation. Abstract Enveloped viruses enter cells by inducing fusion of viral and cellular membranes, a process catalyzed by a specialized membrane-fusion protein expressed on their surface. This review focuses on recent structural studies of viral fusion proteins with an emphasis on their metastable prefusion form and on interactions with neutralizing antibodies. The fusion glycoproteins have been difficult to study because they are present in a labile, metastable form at the surface of infectious virions. Such metastability is a functional requirement, allowing these proteins to refold into a lower energy conformation while transferring the difference in energy to catalyze the membrane fusion reaction. Structural studies have shown that stable immunogens presenting the same antigenic sites as the labile wild-type proteins efficiently elicit potently neutralizing antibodies, providing a framework with which to engineer the antigens for stability, as well as identifying key vulnerability sites that can be used in next-generation subunit vaccine design. Abstract A 2-year-old male neutered cat presented for further investigation of biliothorax. The cat was initially treated for pyothorax, including bilateral chest drains for lavage of the pleural space. Five days later, the pleural effusion turned clear-yellow and had a bilirubin concentration of 427 mmol/l compared to the serum bilirubin concentration of 15 mmol/l. Exploratory surgery revealed a 2 mm tear in the diaphragm, with a corresponding 2 mm defect in the diaphragmatic surface of the gall bladder, creating a fistula between the gall bladder and the pleural cavity. The defects were repaired routinely and the cat made a full recovery. It was suspected that the tears had been created at the time of the thoracostomy tube placement. Biliothorax has not been described before in a cat, and appears to be a rare complication following thoracostomy tube placement. Abstract Introduction: Traditional Chinese medicine (TCM) has been used to treat upper respiratory tract infections/common colds (URTIs) in Asian countries for over 2000 years. However, Chinese medicine doctors (CMDs) follow the traditional treatment rules to select or administer these diverse Chinese medicine formulae. The main purpose of our study was to explore data on the frequency of medication and medication habits by CMDs for the treatment of URTIs with Chinese herbs and Chinese medicine formulae. Methods: The TCM treatments for patients consulting with an URTIs were analyzed from the National Health Insurance Research Database using the appropriate codes from the International Classification of Diseases, Ninth Revision, Clinical Modification diagnoses for Taiwan in 2009. A data mining and association rules, were used to analyze co-prescriptions of TCM for patients with URTIs. Results: For 472,005 patients who sought the treatment of URTIs, a total of 46,805 patients with URTIs received TCM treatments, of these 29,052 patients sought both TCM and Western medication treatments. Of the URTIs patients who had received a TCM treatment, 79% presented with an acute common cold, 9% had influenza, and 9% had acute upper respiratory infections. Furthermore, 53.89% of the sample were aged between 20 and 49 years, and 62.84% were women, 3.56% of the patients used Yin-Qiao-San and 2.76% used Jie-Geng. Yin-Qiao-San and Ma-Xing-Gan-Shi-Tang were the most commonly combinations of prescriptions for patients with URTIs. Conclusions: The patients experiencing URTIs were more likely to request TCM treatment if their symptoms were mild and they were women. The Chinese medicine doctors treating URTIs generally followed TCM theory. A coding system for TCM diagnostic classifications could improve evaluations of TCM treatments. Abstract Predicting the movement of contaminants in the indoor environment has applications in tracking airborne infectious disease, ventilation of gaseous contaminants, and the isolation of spaces during biological attacks. Markov matrices provide a convenient way to perform contaminant transport analysis. However, no standardized method exists for calculating these matrices. A methodology based on set theory is developed for calculating contaminant transport in real-time utilizing Markov matrices from CFD flow data (or discrete flow field data). The methodology provides a rigorous yet simple strategy for determining the number and size of the Markov states, the time step associated with the Markov matrix, and calculation of individual entries of the Markov matrix. The procedure is benchmarked against scalar transport of validated airflow fields in enclosed and ventilated spaces. The approach can be applied to any general airflow field, and is shown to calculate contaminant transport over 3000 times faster than solving the corresponding scalar transport partial differential equation. This near real-time methodology allows for the development of more robust sensing and control procedures of critical care environments (clean rooms and hospital wards), small enclosed spaces (like airplane cabins) and high traffic public areas (train stations and airports). Abstract The Chinese herbal prescription JZ-1 induces autophagy to protect against herpes simplex Virus-2 in human vaginal epithelial cells by inhibiting the PI3K/Akt/mTOR pathway, Journal of Ethnopharmacology (2020), doi: https://doi. Abstract Recombinant fragments of S proteins from the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARA-CoV) were generated and used in a Western blot (WB) assay that was compared to a commercial SARS ELISA method. In 85% of confirmed SARS cases (n = 20), the S2 recombinant fragment based WB was positive and this was comparable to the commercial ELISA using heat killed SARS-CoV. WB using the other four recombinant fragments in confirmed SARS cases generated lower rates of detection (S1-75%, S1-N-25%, S1-C-55%). Evaluation of sera from healthy controls (n = 60) resulted in two weakly positive ELISA results with the remainder being negative while the S2 protein WB demonstrated three positive results from the 20 controls with a history of SARS contact and no positive results in 40 noncontact controls. A discrepancy between the ELISA and S2 WB arose when evaluating per-2003 sera from individuals (n = 10) with SARS-like symptoms (ELISA-100% positive, S2 WB-30% positive). These data suggest that the S2 WB assay may be particularly useful in ELISA-negative SARS cases and in some ELISA-positive non-SARS cases. D Abstract Porcine epidemic diarrhea virus (PEDV), a coronavirus discovered more than 40 years ago, regained notoriety recently by its devastating outbreaks in East Asia and the Americas, causing substantial economic losses to the swine husbandry. The virus replicates extensively and almost exclusively in the epithelial cells of the small intestine resulting in villus atrophy, malabsorption and severe diarrhea. Cellular entry of this enveloped virus is mediated by the large spike (S) glycoprotein, trimers of which mediate virus attachment to the target cell and subsequent membrane fusion. The S protein has a multidomain architecture and has been reported to bind to carbohydrate (sialic acid) and proteinaceous (aminopeptidase N) cell surface molecules. PEDV propagation in vitro requires the presence of trypsin(-like) proteases in the culture medium, which capacitates the fusion function of the S protein.Here we review the current data on PEDV entry into its host cell, including therein our new observations regarding the functional role of the sialic acid binding activity of the S protein in virus infection. Moreover, we summarize the recent progress on the proteolytic activation of PEDV S proteins, and discuss factors that may determine tissue tropism of PEDV in vivo. Abstract Respiratory viral diseases can be spread when a virus-containing particle (droplet) from one individual is aerosolized and subsequently comes into either direct or indirect contact with another individual. Increasing numbers of studies are examining the occupational risk to healthcare workers due to proximity to patients. Selecting the appropriate air sampling method is a critical factor in assuring the analytical performance characteristics of a clinical study. The objective of this study was to compare the physical collection efficiency and virus collection efficiency of a 5 mL compact SKC BioSampler ® , a gelatin filter, and a glass fiber filter, in a laboratory setting. The gelatin filter and the glass fiber filter were housed in a home-made filter holder. Submersion (with vortexing and subsequent centrifugation) was used for the gelatin and glass fiber filters. Swabbing method was also tested to retrieve the viruses from the glass fiber filter. Experiments were conducted using the H1N1 influenza A virus A/Puerto Rico/8/1934 (IAV-PR8), and viral recovery was determined using culture and commercial real-time-PCR (BioFire and Xpert). An atomizer was used to aerosolize a solution of influenza virus in PBS for measurement, and two Scanning Mobility Particle Sizers were used to determine particle size distributions. The SKC BioSampler demonstrated a U-shaped physical collection efficiency, lowest for particles around 30-50 nm, and highest at 10 nm and 300-350 nm within the size range examined. The physical collection efficiency of the gelatin filter was strongly influenced by air flow and time: a stable collection across all particle sizes was only observed at 2 L/min for the 9 min sampling time, otherwise, degradation of the filter was observed. The glass fiber filter demonstrated the highest physical collection efficiency (100% for all sizes) of all tested samplers, however, its overall virus recovery efficiency fared the worst (too low to quantify). The highest viral collection efficiencies for the SKC BioSampler and gelatin filter were 5% and 1.5%, respectively. Overall, the SKC BioSampler outperformed the filters. It is important to consider the total concentration of viruses entering the sampler when interpreting the results. Abstract Disease is still regarded as a major constraint to aquaculture production globally. Rapid disease diagnosis and vaccination play a huge part in the control of bacterial diseases, and there has been signifi cant progress in both of these areas. This chapter considers the limitations of existing methods and reviews recent advances made in pathogen detection technologies and vaccine development methodologies. Future directions are discussed, including nanotechnology and reversed vaccinology. Abstract A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases. PLOS Pathogens | https://doi.Increasing evidence has been gathered to support the bat origin of SARS coronavirus (SARS-CoV) in the past decade. However, none of the currently known bat SARSr-CoVs is thought to be the direct ancestor of SARS-CoV. Herein, we report the identification of a diverse group of bat SARSr-CoVs in a single cave in Yunnan, China. Importantly, all of the building blocks of SARS-CoV genome, including the highly variable S gene, ORF8 and ORF3, could be found in the genomes of different SARSr-CoV strains from this single location. Based on the analysis of full-length genome sequences of the newly identified bat SARSr-CoVs, we speculate that the direct ancestor of SARS-CoV may have arisen from sequential recombination events between the precursors of these bat SARSr-CoVs prior to spillover to an intermediate host. In addition, we found bat SARSr-CoV strains with different S proteins that can all use the receptor of SARS-CoV in humans (ACE2) for cell entry, suggesting diverse SARSr-CoVs capable of direct transmission to humans are circulating in bats in this cave. Our current study therefore offers a clearer picture on the evolutionary origin of SARS-CoV and highlights the risk of future emergence of SARS-like diseases.A gene pool of bat SARS-related coronaviruses PLOS Pathogens | https://doi. Abstract A virulent infectious bronchitis virus (IBV), designated as CK/CH/GD/QY16 (referred as QY16), was isolated from a diseased chicken farm in Guangdong province, China, in 2016. The complete genome of the strain was sequenced and analyzed. The results show that the genome of QY16 consists of 27,670 nucleotides, excluding poly (A) tail, and that its genome organization is 5' UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3' UTR-poly (A) tail. Sequence comparison among QY16 and other IBV strains was conducted and its results demonstrate that the S1 gene of QY16 has the highest nucleotide sequence identity with that of 4/91, and the other part of its genome is highly similar to that of YX10. The results of the phylogenic analysis show that the entire genome of QY16 and most of the QY16 genes are located in the same cluster as those of YX10, except for the S1 gene which is located in the same cluster with that of 4/91. It has been further confirmed by the RDP and SimPlot analysis that QY16 is a recombinant strain deriving from YX10 (as the major parental sequence) and 4/91 (as the minor parental sequence), and that the recombination occurs in a region which includes the 3'-terminal 1b sequence (85 nt) and the 5'-terminal S1 protein gene sequence (1,466 nt). The results of the vaccination-challenge test suggest that QY16 is a nephropathogenic strain of IBV and that the vaccine strains-H120 and 4/91-cannot provide effective protection against it. These results indicate that the continuing evolution of IBV strains by genetic drift and genetic recombination may lead to IBV outbreaks even among the vaccinated chickens in China. Abstract Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line. Abstract Porcine circovirus type 2 (PCV2) is the pathogen that causes postweaning multisystemic wasting syndrome, which leads to significant economic losses for swine farms worldwide. However, the infection mechanism of PCV2 is not completely understood yet. Vimentin is a part of the cytoskeleton network and plays an important role in several virus infections. It is not clear whether vimentin has a role in PCV2 infection nor how it affects PCV2 infection. In this study, the function of vimentin in PK-15 cells infected with PCV2 has been elucidated. We found that vimentin had a restrictive effect on the replication of PCV2 in PK-15 cells. Overexpression of vimentin by transferred pCAGGS-vimentin and down-regulation by the respective scrambled small interfering RNA showed that vimentin restricted the replication and virion production of PCV2. A special interaction between vimentin and PCV2 Cap protein was observed using laser confocal microscopy and immunoprecipitation assay. Moreover, overexpression of vimentin could decrease NF-κB activity and increase PCV2-induced caspase-3 activity in PK-15 cells. These data suggest that vimentin is involved in the replication of PCV2 and has a restrictive effect on it, which is helpful in the study of the replication mechanism of PCV2. Abstract Paralichthys olivaceus interferon-inducible transmembrane 1 (PoIFITM1) Antiviral effect Rana grylio virus (RGV) Scophthalmus maximus rhabdovirus (SMRV) Golgi localization a b s t r a c t Interferon-inducible transmembrane (IFITM) protein family is novel viral restriction factors with representative transmembrane structure. These proteins also exist in fish, however, their roles in the innate immune response remain unknown. Here, we report a characterization of teleost IFITM1 from flounder Paralichthys olivaceus (PoIFITM1), which exhibits conserved structure characteristic of the IFITM family but comprises a relatively longer N-terminal region. The expression and promoter activity of PoIFITM1 are markedly induced by aquatic animal viruses: Rana grylio virus (RGV) and Scophthalmus maximus rhabdovirus (SMRV). Overexpression and siRNA-mediated knockdown demonstrate that PoI-FITM1 exhibits strong antiviral effects against both DNA virus (RGV) and RNA virus (SMRV), expanding the spectrum of viruses inhibited by IFITM proteins. Further analysis shows that PoIFITM1 suppresses viral entry into host cells, confirming that the IFITM-mediated restriction is conserved from lower vertebrates to mammals. Deletion mutagenesis reveals that PoIFITM1 exerts antiviral activity by targeting to Golgi complex and the N-terminal region is required for its subcellular localization, which is not observed in other known IFITM family members. Our current data provide the first evidence that IFITM1 functions as a key effector of the innate immune to restrict virus replication in lower vertebrates, through the action of impeding viral entry. Abstract The effect of tumor necrosis factor (TNF) on expression of major histocompatibility complex (MHC) antigens was examined in mouse glial cells in vitro. TNF induced MHC class I, but not class II, antigen expression on the surface of astrocytes but not on oligodendrocytes. Gliai cells do not normally express detectable amounts of MHC antigens. Thus TNF may play a role in the immunopathogenesis of neurologic diseases that involve MHC class l-restricted reactions. Abstract Immunological equivalence between mouse brain-derived and Vero cell-derived Japanese encephalitis vaccines (121) 152 Abergel, C., see Claverie, J.-M. (117) 133 Abraham, S., R. Manjunath, Induction of classical and nonclassical MHC-I on mouse brain astrocytes by Japanese encephalitis virus (119) 216 cellular localisation analysis of the severe acute respiratory syndrome coronavirus nucleocapsid protein using monoclonal antibodies (122) 119 Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications (120) 36 Genetic recombination at different points in the N pro -coding region of bovine viral diarrhea viruses and the potentials to change their antigenicities and patho- Abstract Mice aged 1, 4 or 8 weeks were inoculated with haemagglutinating encephalomyelitis virus (HEV), strain 67N, by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) or oral route, with different doses. In 1-week-old mice, mortality and mean time to death were mostly the same regardless of the inoculation route, except for the oral route, which appeared to be the least effective. The virus killed 4-week-old mice readily by all routes of inoculation except the oral, and 8-weekold mice by i.c., i.n. or s.c. inoculation. In descending order of efficacy, the routes of HEV infection were: i.c., i.n., s.c., i.p., i.v. and oral. To follow the spread of HEV from peripheral nerves to the central nervous system (CNS), the virus was inoculated subcutaneously into the right hind leg of 4-week-old mice. The virus was first detected in the spinal cord on day 2, and in the brain on day 3. The brain titres became higher than those of the spinal cord, reaching a maximum of 10 7 PFU/0.2 g when the animals were showing CNS signs. Viral antigen was first detected immunohistochemically in the lumbar spinal cord and the dorsal root ganglion ipsilateral to the inoculated leg; it was detected later in the pyramidal cells of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum but not in the ependymal cells, choroid plexus cells or other glial cells. The infected neurons showed no cytopathological changes. q Abstract Extraction Health benefits A B S T R A C T Cordyceps sinensis (CS) is a well-known entamophagus fungus, naturally distributed in the Tibetan Plateau of Asia and Himalayas. Recently this synonym is transferred to Ophiocordyceps by both scientific and non-scientific communities. It is widely used as a tonic and medicinal food in traditional Chinese medicine (TCM), as it possess wonderful health benefits. To support its functional attributes, various investigations have been carried out to find out its adaptogenic, aphrodisiac, anti-oxidant, anti-aging, neuroprotective, nootropic, immunomodulatory, anti-cancer and hepatoprotective role. Its fruiting portion as well as the larvae possesses potent bio-active fractions and their composition almost found to be similar in both. The bioactive principles are nucleosides, exo-polysaccharides, sterols and, proteins, among others. Among nucleosides, adenosine and cordycepin are the major biochemical markers. Further, different types of solvent extracts and their mixtures exhibit wide range of pharmacological activities, while the water and methanol extracts with the richest sources of nucleosides and polysaccharides also show wide range of pharmacological activities. This review gives a panoramic view of potential health benefits of various classes of bio-active fractions along with the need for sustainable management of CS for human wellness. Abstract A B S T R A C T Viral metagenomics, modeling of protein structure, and manipulation of viral genetics are key approaches that have laid the foundations of our understanding of coronavirus biology. In this review, we discuss the major advances each method has provided and discuss how future studies should leverage these strategies synergistically to answer novel questions. Abstract Mucosal surfaces are common sites of pathogen colonization/entry. Effective mucosal immunity by vaccination should provide protection at this primary infection site. Our aim was to develop a new vaccination strategy that elicits a mucosal immune response. A new strain of Enterococcus faecium, a non pathogenic lactic acid bacteria (LAB) with strong cell adhesion ability, was identified and used as a vaccine vector to deliver two model antigens. Specifically, sigma () C protein of avian reovirus (ARV), a functional homolog of mammalian reovirus 1 protein and responsible for M-cell targeting, was administered together with a subfragment of the spike protein of infectious bronchitis virus (IBV). Next, the effect of immunization route on the immune response was assessed by delivering the antigens via the LAB strain. Intranasal (IN) immunization induced stronger humoral responses than intragastic (IG) immunization. IN immunization produced antigen specific IgA both systemically and in the lungs. A higher IgA titer was induced by the LAB with ARV C protein attached. Moreover, the serum of mice immunized with LAB displaying divalent antigens had much stronger immune reactivity against ARV C protein compared to IBV-S1. Our results indicate that ARV C protein delivered by LAB via the IN route elicits strong mucosal immunity. A needle-free delivery approach is a convenient and cost effective method of vaccine administration, especially for respiratory infections in economic animals. Furthermore, ARV C, a strong immunogen of ARV, may be able to serve as an immunoenhancer for other vaccines, especially avian vaccines. Abstract The propagation of a mouse-adapted strain (67N) of haemagglutinating encephalomyelitis virus in infected mice and murine cells was examined by viral re-isolation and immunostaining. Viral propagation was strictly limited to the neurons and to an established line ofneuroblastoma cells in in-vivo and in-vitro experiments. These results provide adequate evidence that this virus is neurotroplc. Abstract An increasing number of insect-specific viruses are found around the world. Very recently, a new group of insectspecific viruses, the Mesoniviridae family, was discovered in Africa, Asia, North America and Australia. Here we report the first detection and isolation of a new virus belonging to Mesonivirus genus in Senegal, West Africa. The so-called Dianke virus was detected in 21 species of arthropods trapped in the eastern part of the country. Male individuals were also infected, supporting vertical transmission assertion of insect specific viruses. As described for other mesoniviruses, no viral replication was observed after inoculation of mammalian cells. Viral replication in mosquito cells was blocked at a temperature of 37°C, highlighting the importance of thermal conditions in Mesonivirus host restriction. Similar to our study, where a diverse range of arthropod vectors were found infected by the new virus, several studies have detected mesonivirus infection in mosquitoes with concerns for human health. It has been shown that dual infections in mosquito can alter viral infectivity. Due to their extensive geographic distribution and host range, as well as their use as potential disease control agents in vector populations, more studies should be done for a better knowledge of arthropod-restricted viruses prevalence and diversity. Abstract The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112-and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae. Abstract Although the feasibility of oral tacrolimus administration in the presence of jejunostomy has already been reported, few studies monitoring tacrolimus trough blood levels have been analyzed in detail, either during or after a jejunostomy closure. We report on our experience with a 34-year-old patient who underwent liver transplantations, with a proximal jejunostomy constructed a few days prior to the second transplantation. He was administered tacrolimus by a predominantly oral route, and less frequently received it by jejunostomy. The aim of this paper is to discuss this administration strategy and whether a different method could have been more suitable. This case report highlights that during the jejunostomy period, the tacrolimus doses that were required to maintain trough concentrations within the therapeutic range were four times higher than those administered after the closure of the jejunostomy. We observed an increase in the Dose-Normalized Trough Concentration (DNTC) values when tacrolimus was administered for 4 consecutive days by jejunostomy as compared to oral administration, indicating that the relative bioavailability of tacrolimus increased. Moreover, when returning to oral administration, the subsequent DNTC value was halved, highlighting a reduction in the tacrolimus bioavailability. Thus, in such a case, administration by jejunostomy could be more appropriate. Abstract This review article outlines the essentials of production medicine (factors affecting production colony output) in rabbits and rodents, emphasizing the importance of routine management methods and record-keeping as well as disease control. Common management regimes and production diseases of the important rodent species are covered.Copyright 9 2001 by W.B. Saunders Company. Abstract Protein-protein interactions dictate the assembly of the macromolecular complexes essential for functional networks and cellular behavior. Elucidating principles of molecular recognition governing important interfaces such as coiled coils is a challenging goal for structural and systems biology. We report here that two valine-containing mutants of the GCN4 leucine zipper that fold individually as fourstranded coiled coils associate preferentially in mixtures to form an antiparallel, heterotetrameric structure. X-ray crystallographic analysis reveals that the coinciding hydrophobic interfaces of the hetero-and homotetramers differ in detail, explaining their partnering and structural specificity. Equilibrium disulfide exchange and thermal denaturation experiments show that the 50-fold preference for heterospecificity results from a combination of preferential packing and hydrophobicity. The extent of preference is sensitive to the side chains comprising the interface. Thus, heterotypic versus homotypic interaction specificity in coiled coils reflects a delicate balance in complementarity of shape and chemistry of the participating side chains. Abstract Gross examination of the gastrointestinal tract (GIT) of cattle is one of the most challenging parts of a necropsy. The GIT is one of the largest and heaviest organ systems of the animal. It is initially divided into the upper GIT (oral cavity, esophagus, forestomachs, abomasum) and the lower GIT (intestines, rectum, and anus). In healthy cattle, the forestomachs occupy more than half the abdominal cavity. The rumen ( Fig. 1) alone has a capacity range of 102 to 148 L (which approximates to a similar weight in kilograms), whereas the abomasal volume ranges from 10 to 20 L. 1 The intestines occupy a much smaller space in the peritoneal cavity but have a length that is considerable, ranging from 33 to 63 m. 1 Thus, a thorough examination is physically demanding because of its size and requires a significant proportion of time to adequately examine its entirety.In addition, the GIT is one of the few organ systems that normally contain a mixture of commensal and potentially pathogenic organisms. In health, there is continuous interaction between the host and microorgansims resulting in a perpetual steady state Abstract Partitiviridae Evolutio a b s t r a c tThe population genetics of the family Partitiviridae was studied within the European race of the conifer pathogen Gremmeniella abietina. One hundred sixty-two isolates were collected from different countries, including Canada, the Czech Republic, Finland, Italy, Montenegro, Serbia, Spain, Switzerland, Turkey and the United States. A unique species of G. abietina RNA viruseMS1 (GaRV-MS1) appears to occur indistinctly in G. abietina biotypes A and B, without a particular geographical distribution pattern. Forty-six isolates were shown to host GaRV-MS1 according to direct specific RT-PCR screening, and the virus was more common in biotype A than B. Phylogenetic analysis based on 46 partial coat protein (CP) cDNA sequences divided the GaRV-MS1 population into two closely related clades, while RNA-dependent RNA polymerase (RdRp) sequences revealed only one clade. The evolution of the virus appears to mainly occur through purifying selection but also through recombination. Recombination events were detected within alignments of the three complete CP and RdRp sequences of GaRV-MS1. This is the first time that recombination events have been directly identified in fungal partitiviruses and in G. abietina in particular. The results suggest that the population dynamics of GaRV-MS1 do not have a direct impact on the genetic structure of its host, G. abietina, though they might have had an innocuous ancestral relationship. Abstract The porcine small intestinal epithelial cell line, IPEC-J2, is useful to characterize the interactions of enterocytes with enteric viruses in vitro. We investigated whether IPEC-J2 cells are susceptible to porcine deltacoronavirus (PDCoV) infection. We conducted quantification of infectious virus or viral RNA, immunofluorescent (IF) staining for the detection of PDCoV antigens, and TUNEL assay in IPEC-J2 cells inoculated with the strain OH-FD22-P8 grown in LLC-PK cells, and supplemented with 10 μg/ml of trypsin in the cell culture medium. Cytopathic effects (CPE) that consisted of enlarged and rounded cells followed by cell shrinkage and detachment, were identified by the 3rd viral passage in the IPEC-J2 cells. PDCoV antigen was detected in the cells showing CPE. By double IF and TUNEL staining, most PDCoV antigen-positive IPEC-J2 cells failed to show TUNELpositive signals, indicating that PDCoV-infected IPEC-J2 cells may not undergo apoptosis, but rather necrosis, similar to necrotic cell death of infected enterocytes in vivo. There was increased interleukin-6 in PDCoV-infected IPEC-J2 cell culture supernatants at post-inoculation hour (PIH) 48-96, as evaluated by ELISA, concurrent with increased titers of PDCoV at PIH 24-72. The susceptibility of IPEC-J2 cells to PDCoV infection supports their usefulness to characterize the interactions of enterocytes with PDCoV. We also demonstrated that IPEC-J2 cell culture-passaged PDCoV (OH-FD22-P8-I-P4) was enteropathogenic in 10-day-old gnotobiotic pigs, and induced systemic innate and pro-inflammatory cytokine responses during the acute PDCoV infection. Abstract High-flow nasal therapy Pathogen transmission Droplet infectionHigh-flow nasal therapy is increasingly used in hospitals because of its effectiveness and patient comfort. However, pathogens in the patient's nasal and oral cavities may be dispersed by forced air. This study aimed to investigate the risk of pathogen dispersal during high-flow nasal therapy. Liquid and bacterial dispersal were assessed via in-vitro experimental set-ups using a manikin. Thickened water or fresh yeast solution mimicked saliva and nasal mucus secretions. Dispersal was limited to the proximal area of the face and nasal cannula, suggesting that high-flow nasal therapy does not increase the risk of droplet and contact infection. Abstract The relation between the complexity of organisms and proteins and their evolution rates has been discussed in the context of multiple generic models. The main robust claim from most such models is the negative relation between complexity and the accumulation rate of mutations.Viruses accumulate escape mutations in their epitopes to avoid detection and destruction of their host cell by CD8+ T cells. The extreme regime of immune escape, namely, strong selection and high mutation rate, provide an opportunity to extend and validate the existing models of relation between complexity and evolution rate as proposed by Fisher and Kimura.Using epitope prediction algorithms to compute the epitopes presented on the most frequent human HLA alleles in over 100 fully sequenced human viruses, and over 900 non-human viruses, we here study the correlation between viruses/proteins complexity (as measured by the number of proteins in the virus and the length of each protein, respectively) and the rate of accumulation of escape mutation. The latter is evaluated by measuring the normalized epitope density of viral proteins.If the virus/protein complexity prevents the accumulation of escape mutations, the epitope density is expected to be positively correlated with both the number of proteins in the virus and the length of proteins. We show that such correlations are indeed observed for most human viruses. For non-human viruses the correlations were much less significant, indicating that the correlation is indeed induced by human HLA molecules. Abstract Enterovirus 71 (EV71) has emerged as a clinically important neurotropic virus that can cause acute flaccid paralysis and encephalitis, leading to cardiopulmonary failure and death. Recurring outbreaks of EV71 have been reported in several countries. The current lack of approved anti-EV71 therapy has prompted intense research into antiviral development. Several strategies -ranging from target-based chemical design to compound library screenings -have been employed, while others revisited compound series generated from antiviral developments against poliovirus and human rhinoviruses. These efforts have given rise to a diversity of antiviral candidates that include small molecules and nonconventional nucleic-acid-based strategies. This review aims to highlight candidates with potential for further clinical development based on their putative modes of action.Enterovirus 71 (EV71) is described as one of the human enterovirus A species under the genus Enterovirus in the Picornaviridae family of viruses, which includes poliovirus [1] . While efforts to eradicate poliovirus through vaccination programs have limited the number of polio-endemic countries to just four (Afghanistan, India, Nigeria and Pakistan) [2], EV71 has emerged as an important non-polio neurotropic enterovirus. EV71 was first isolated from patients with central nervous system diseases in California between 1969 and 1974 [3]. The same authors also described the EV71 prototype strain, BrCr, isolated from a twomonth-old patient who presented with aseptic meningitis. Since then, EV71 outbreaks have been reported in several countries beyond North America, including Taiwan, Australia, Malaysia and Singapore [4]. These outbreaks have mainly involved young children, with most cases displaying mild, self-limiting hand, foot and mouth disease; however, EV71 outbreaks have also been associated with a variety of severe neurological complications that can deteriorate rapidly to involve cardiopulmonary failure with high mortality rates. Transmission of EV71 can occur rapidly via the fecal-oral and droplet and/or aerosol routes. During the largest EV71 outbreak to date, in Taiwan in 1998, more than 100,000 children were affected, with 405 severe cases involving neurological or cardiopulmonary complications, of which 78 were fatal [5] . Abstract Background: On-site vaccination arrangements were introduced in 2005 to improve influenza vaccination rate among employees of a 1500-bed tertiary hospital in Singapore. Methods: On-site arrangements include mobile teams to 3 distant departments and same-service area vaccination for employees at 4 service areas.Results: Influenza vaccination rate in 2005 was 66.4% (versus 56.8% in 2004, odds ratio 1.50, 95% confidence interval 1.39-1.62). Employees who attended on-site arrangements had higher influenza vaccination rate (97.0%). Conclusion: On-site vaccination arrangements improved influenza vaccination rate among hospital employees. (Am J Infect Control 2007;35:481-3.) Abstract Objectives: Our objective was to evaluate whether vaccination with the 13-valent pneumococcal conjugate vaccine (PCV13) prevents the incidence of community-acquired pneumonia (CAP) caused by influenza (influenza-associated CAP, IA-CAP) or other respiratory viruses in the elderly. Methods: This analysis was part of the Community-Acquired Pneumonia immunization Trial in Adults (CAPiTA); a double blind, randomized, placebo-controlled trial in 84 496 immunocompetent individuals aged !65 years. CAP was defined by clinical and radiological criteria, and oropharyngeal swabs were collected from all individuals referred to a sentinel centre with a clinical suspicion of pneumonia. Presence of influenza A and B, parainfluenza 1, 2, 3 and 4, human adeno-, boca-, corona-, metapneumo-, rhino-and respiratory syncytial viruses was determined by real-time PCR. Results: Of 3209 episodes of suspected pneumonia, viral aetiology was tested in 2917 and proportions with influenza virus, human metapneumovirus and respiratory syncytial virus were 4.6%, 2.5% and 3.1%, respectively. There were 1653 oropharyngeal swabs for PCR testing available from 1814 episodes that fulfilled criteria for CAP, yielding 23 first episodes of IA-CAP in the PCV13 and 35 in the in placebo groupdvaccine efficacy for IA-CAP of 34.4% (95% CI e11.1% to 61.2%; p 0.117). Annual influenza vaccination was received by 672 (87.2%) in the PCV13 group and 719 (87.7%) in the placebo group of the confirmed CAP cases. Conclusion: In a randomized study of 84 496 elderly individuals with a high uptake of influenza vaccination, PCV13 was not associated with a statistically significant reduction of influenza or virus-associated CAP. Overall incidence of non-influenza viral pneumonia was low. S.M. Huijts, Clin Microbiol Infect 2018;24:764 Abstract Coronaviridae. To evaluate the lung pathology in this life-threatening respiratory illness, we studied postmortem lung sections from 8 patients who died from SARS during the spring 2003 Singapore outbreak. The predominant pattern of lung injury in all 8 cases was diffuse alveolar damage. The histology varied according to the duration of illness. Cases of 10 or fewer days' duration demonstrated acute-phase diffuse alveolar damage (DAD), airspace edema, and bronchiolar fibrin. Cases of more than 10 days' duration exhibited organizing-phase DAD, type II pneumocyte hyperplasia, squamous metaplasia, multinucleated giant cells, and acute bronchopneumonia. In acute-phase DAD, pancytokeratin staining was positive in hyaline membranes along alveolar walls and highlighted the absence of pneumocytes. Multinucleated cells were shown to be both type II pneumocytes and macrophages by pancytokeratin, thyroid transcription factor-1, and CD68 staining. SARS-CoV RNA was identified by reverse transcriptase-polymerase chain reaction in 7 of 8 cases in fresh autopsy tissue and in 8 of 8 cases in formalin-fixed, paraffin-embedded lung tissue, including the 1 negative case in fresh tissue. Understanding the pathology of DAD in SARS patients may provide the basis for therapeutic strategies. Further studies of the pathogenesis of SARS may reveal new insight into the mechanisms of DAD. HUM PATHOL 34:743-748. Abstract In vitro interferon production by peripheral blood mononuclear cells from 50 children suffering from recurrent upper respiratory tract infections was examined, and compared with that of 50 healthy children. Five respiratory pathogenic viruses and Mycoplasma pneumoniae were used as inducers. Cells from every child responded to at least three out of the six inducers by interferon production. As a group, cultures prepared from patient cells showed decreased production of IFN when stimulated with adeno, rhino, corona or RS viruses or with the mycoplasma. Similar trend between the two groups of children was seen as regards influenza A virus-induced IFN production in leukocyte cultures. These results corroborate our previous findings that relative deficiency in interferon production appears to be inducer-specific, and suggest that this phenomenon may have a role in the pathogenesis of recurrent respiratory infections. Abstract Background: It is increasingly recognized that human rhinoviruses (HRV) can be associated with severe infections. However, conflicting results have been reported on the relative prevalence and severity of the three HRV species. Objectives: The relative prevalence and clinical characteristics of HRV-A, B and C, in children attending a South London teaching hospital were investigated retrospectively. Study design: Children aged <16 years with episodes of respiratory tract infections and detectable entero/rhinovirus RNA in respiratory samples between November 2009 and December 2010 were investigated. Retrospective case review was performed and patients' characteristics recorded. Results: Entero/rhinoviruses were the commonest viral pathogens (498/2316; 21.5%). Amongst 204 infection episodes associated with entero/rhinovirus, 167 were typed HRV, HRV-C was the most prevalent (99/167, 59.3%) followed by HRV-A (60/167; 35.9%) and HRV-B (8/167, 4.8%). The severity spectrum of HRV-A and HRV-C infections were similar and affected all parts of the respiratory tract. Co-pathogens were observed in 54 (26.5%) episodes. Severity was increased in patients with non-viral co-pathogens and those with an underlying respiratory condition. Univariate and multiple regression analyses of potential prognostic variables including age, co-pathogens and underlying respiratory illnesses showed that monoinfection with HRV-C, as compared with other HRV species, was associated with more severe disease in young children <3 years. Conclusions: HRV-C was the most prevalent species and on its own was associated with severe disease in children <3 years. The association between infection with HRV species and clinical presentation is complex and affected by many confounding factors. Abstract The coronavirus avian Infectious bronchitis virus (IBV) poses economic threats to poultry farms worldwide, affecting the performance of both meat-type and egg-laying birds. To define the evolution of recent IBVs in Iran, a genetic analysis based on hypervariable nucleotide sequences of S1 gene was carried out. Tracheal swab samples were collected from 170 Broiler flocks during 2017. Ten tracheal swabs from each flock pooled. From a total number of 170 flocks tested, 84.71% found to be positive. Phylogenetic tree analysis revealed the presence of D274 as a first time in Iran. IS/1494/06 was showed to be dominant IBV type circulating in broiler farms with a significantly higher prevalence than other four genotypes. Considering fluctuations in QX-type prevalence in recent years, continuous monitoring is necessary to reduce economic consequences in layer and broiler farms. The findings highlight the importance of using modified vaccination strategies that are adapted to the changing disease scenario. Abstract . The treatment of fresh water and additives, control of the residence time in the storage tanks, and control of the stagnant flow areas should be done to control the contamination sources and the microbiological populations. Systematic maintenance and cleaning of the system by using different chemicals or treatments to reduce the formation of deposits and to eliminate the already formed deposits is required. A microbiological control program requires a good knowledge of the system and the main sources of contamination. Once the sources of contamination have been reduced, the paper manufacturer can control the microbiological populations of the system. Good housekeeping and regular inspection of all areas, effective boilouts, and regularly scheduled washup reduces slime development.To reduce the number of maintenance shutdowns in the paper machines, chemical products are introduced into the system to get rid of the microorganisms by preventing their growth or by reducing the harmful effects they produce. The chemical products most commonly used are dispersants and biocides. Other control methods are the enzymatic degradation of microbiological deposits and controlling the population by limiting nutrients Abstract High levels of interleukin-6 (IL-6) in the acute stage associated with lung lesions were found in SARS patients. To evaluate the mechanisms behind this event, we investigated the roles of SARS-CoV proteins in the regulation of IL-6. Results showed that the viral nucleocapsid (N) protein activated IL-6 expression in a concentration-dependent manner. Promoter analyses suggested that NF-κB binding element was required for IL-6 expression regulated by N protein. Further studies demonstrated that N protein bound directly to NF-κB element on the promoter. We also showed that N protein activated IL-6 expression through NF-κB by facilitating the translocation of NF-κB from cytosol to nucleus. Mutational analyses revealed that two regions of N protein were essential for its function in the activation of IL-6. These results provided new insights into understanding the mechanism involved in the function of SARS-CoV N protein and pathogenesis of the virus. Abstract Peripheral T cells can be polarized towards type 1 or type 2 cytokine immune responses during TCR engagement. Because T cell selection by peptide plus self-MHC in the thymus requires TCR engagement, we hypothesized that type 1 cytokines may polarize developing T cells. We cultured thymi from BBDR rats in adult thymus organ cultures (ATOC) under type 1 cytokine conditions in the absence of exogenous antigen. Type 1 cytokine-conditioned ATOC generated cells that spontaneously secreted high levels of IFN , but not IL-4. A second exposure to type 1 cytokines further increased IFN secretion by these cells, most of which were blasts that expressed the activation markers CD25, CD71, CD86, and CD134. Studies using blocking antibodies and pharmacological inhibitors suggested that both IL-18 and cognate TCR-MHC/ligand interactions were important for activation. Blocking anti-MHC class I plus anti-MHC class II antibodies, neutralizing anti-IL-18 antibody, and the p38 MAP-kinase inhibitor SB203580 each reduced IFN production by w75-80%. Cyclosporin A, which prevents TCR signaling, inhibited IFN production by w50%. These data demonstrate that exposure to type 1 cytokines during intrathymic development can polarize differentiating T cells, and suggest a mechanism by which intrathymic exposure to type 1 cytokines may modulate T cell development. Abstract Background Respiratory virus infection is a common cause of hospitalisation in adults. Rapid point-of-care testing (POCT) for respiratory viruses might improve clinical care by reducing unnecessary antibiotic use, shortening length of hospital stay, improving influenza detection and treatment, and rationalising isolation facility use; however, insufficient evidence exists to support its use over standard clinical care. We aimed to assess the effect of routine POCT on a broad range of clinical outcomes including antibiotic use.In this pragmatic, parallel-group, open-label, randomised controlled trial, we enrolled adults (aged ≥18 years) within 24 h of presenting to the emergency department or acute medical unit of a large UK hospital with acute respiratory illness or fever higher than 37·5°C (≤7 days duration), or both, over two winter seasons. Patients were randomly assigned (1:1), via an internet-based allocation sequence with random permuted blocks, to have a molecular POC test for respiratory viruses or routine clinical care. The primary outcome was the proportion of patients who received antibiotics while hospitalised (up to 30 days). Secondary outcomes included duration of antibiotics, proportion of patients receiving single doses or brief courses of antibiotics, length of stay, antiviral use, isolation facility use, and safety. Analysis was by modified intention to treat, excluding patients who declined intervention or were withdrawn for protocol violations. This study is registered with ISRCTN, number 90211642, and has been completed. , we enrolled 720 patients (362 assigned to POCT and 358 to routine care). Six patients withdrew or had protocol violations. 301 (84%) of 360 patients in the POCT group received antibiotics compared with 294 (83%) of 354 controls (difference 0·6%, 95% CI −4·9 to 6·0; p=0·84). Mean duration of antibiotics did not differ between groups (7·2 days [SD 5·1] in the POCT group vs 7·7 days [4·9] in the control group; difference −0·4, 95% CI −1·2 to 0·4; p=0·32). 50 (17%) of 301 patients treated with antibiotics in the POCT group received single doses or brief courses of antibiotics (<48 h) compared with 26 (9%) of 294 patients in the control group (difference 7·8%, 95% CI 2·5 to 13·1; p=0·0047; number needed to test=13). Mean length of stay was shorter in the POCT group (5·7 days [SD 6·3]) than in the control group (6·8 days [7·7]; difference −1·1, 95% CI −2·2 to −0·3; p=0·0443). Appropriate antiviral treatment of influenza-positive patients was more common in the POCT group (52 [91%] of 57 patients) than in the control group (24 [65%] of 37 patients; difference 26·4%, 95% CI 9·6 to 43·2; p=0·0026; number needed to test=4). We found no differences in adverse outcomes between the groups (77 [21%] of 360 patients in the POCT group vs 88 [25%] of 354 patients in the control group; −3·5%, −9·7 to 2·7; p=0·29).Interpretation Routine use of molecular POCT for respiratory viruses did not reduce the proportion of patients treated with antibiotics. However, the primary outcome measure failed to capture differences in antibiotic use because many patients were started on antibiotics before the results of POCT could be made available. Although POCT was not associated with a reduction in the duration of antibiotics overall, more patients in the POCT group received single doses or brief courses of antibiotics than did patients in the control group. POCT was also associated with a reduced length of stay and improved influenza detection and antiviral use, and appeared to be safe. Abstract All members of the herpesviridae contain within their large tegument protein a cysteine protease module that displays deubiquitinating activity. We report the crystal structure of the cysteine protease domain of murine cytomegalovirus M48 (M48 USP ) in a complex with a ubiquitin (Ub)based suicide substrate. M48 USP adopts a papain-like fold, with the active-site cysteine forming a thioether linkage to the suicide substrate. The Ub core participates in an extensive hydrophobic interaction with an exposed b hairpin loop of M48 USP . This Ub binding mode contributes to Ub specificity and is distinct from that observed in other deubiquitinating enzymes. Both the arrangement of active-site residues and the architecture of the interface with Ub lead us to classify this domain as the founding member of a previously unknown class of deubiquitinating enzymes. Abstract We report the synthesis of a series of novel 2 0 -deoxy-2 0 ,2 0 -difluoro-5-halouridines and their corresponding phosphoramidate ProTides. All compounds were evaluated for antiviral activity and for cellular toxicity. Interestingly, 2 0 -deoxy-2 0 ,2 0 -difluoro-5-iodo-and -5-bromo-uridines showed selective activity against feline herpes virus replication in cell culture due to a specific recognition (activation) by the virus-encoded thymidine kinase.Crown Abstract In this contribution, the classification of protein binding sites using the physicochemical properties exposed to their pockets is presented. We recently introduced Cavbase, a method for describing and comparing protein binding pockets on the basis of the geometrical and physicochemical properties of their active sites. Here, we present algorithmic and methodological enhancements in the Cavbase property description and in the cavity comparison step. We give examples of the Cavbase similarity analysis detecting pronounced similarities in the binding sites of proteins unrelated in sequence. A similarity search using SARS M pro protease subpockets as queries retrieved ligands and ligand fragments accommodated in a physicochemical environment similar to that of the query. This allowed the characterization of the protease recognition pockets and the identification of molecular building blocks that can be incorporated into novel antiviral compounds. A cluster analysis procedure for the functional classification of binding pockets was implemented and calibrated using a diverse set of enzyme binding sites. Two relevant protein families, the a-carbonic anhydrases and the protein kinases, are used to demonstrate the scope of our cluster approach. We propose a relevant classification of both protein families, on the basis of the binding motifs in their active sites. The classification provides a new perspective on functional properties across a protein family and is able to highlight features important for potency and selectivity. Furthermore, this information can be used to identify possible cross-reactivities among proteins due to similarities in their binding sites. Abstract A simple, cell-based, membrane fusion assay system that uses split green fluorescent proteins (spGFPs) as an indicator was developed. The attachment of the pleckstrin homology (PH) domain to the N-termini of each spGFP not only localized the reporter signal to the plasma membrane but also helped the stable expression of the smaller spGFP of seventeen amino acid residues. It was shown that this system allowed real-time monitoring of membrane fusion by HIV-1 envelope protein (Env) without the addition of external substrates. This method can be adapted to the analyses of other viral membrane fusion. Abstract Only a month after the outbreak of pneumonia caused by 2019-nCoV, more than forty-thousand people were infected. This put enormous pressure on the Chinese government, medical healthcare provider, and the general public, but also made the international community deeply nervous. On the 25th day after the outbreak, the Chinese government implemented strict traffic restrictions on the area where the 2019-nCoV had originateddHubei province, whose capital city is Wuhan. Ten days later, the rate of increase of cases in Hubei showed a significant difference (p ¼ 0.0001) compared with the total rate of increase in other provinces of China. These preliminary data suggest the effectiveness of a traffic restriction policy for this pandemic thus far. At the same time, solid financial support and improved research ability, along with network communication technology, also greatly facilitated the application of epidemic prevention measures. These measures were motivated by the need to provide effective treatment of patients, and involved consultation with three major groups in policy formulationdpublic health experts, the government, and the general public. It was also aided by media and information technology, as well as international cooperation. This experience will provide China and other countries with valuable lessons for quickly coordinating and coping with future public health emergencies. Abstract IEF, isoelectric focusing; NC, nitrocellulose; TBS, Tris-buffered saline pH 8.3; TBS-milk, TBS containing 3% nonfat dry milk and phenyl-methyl sulfonyl fluoride; PBS, phosphate-buffered saline, pH 7.2. Abstract Background: Respiratory syncytial virus and other respiratory tract viruses lead to common colds in most infants, whereas a minority develop acute severe bronchiolitis often requiring hospitalization. We hypothesized that such an excessive response to respiratory tract viral infection is caused by host factors reflected in pre-existing increased bronchial responsiveness. Objective: We sought to compare bronchial responsiveness and lung function in 1-month-old neonates who later develop acute severe bronchiolitis with those who do not. Methods: We measured infant lung function (n 5 402) and bronchial responsiveness to methacholine (n 5 363) using the raised-volume rapid thoracoabdominal compression technique before any respiratory symptoms in 1-month-old neonates from the Copenhagen Prospective Study of Asthma in Childhood birth cohort born to mothers with asthma. The children were prospectively monitored for respiratory symptoms and given a diagnosis of acute severe bronchiolitis according to a fixed algorithm. Results: Thirty-four (8.5%) infants had acute severe bronchiolitis before 2 years of age, 21 (62%) were hospitalized, Abstract Macrolide have enjoyed continued use for over 40 years, being increasingly usedfor the treatment ofre :piratory tract infection. Newer macrolide have been introduced that show improved ab orption after oral administration, better gastrointestinal tolerance, and delivery of increased amount ofdrug to the infection ite. Macrolides are commonly used in community-acquired pneumonia, as ,'.'ell as in atypical pneumonia and legionello is. The newer macrolides, in comparative studies, have been shown to be as effective as the conventional therapies for treating acute otitis media, acute sinusitis and acute pharyngitis, with a low incidence of side-effect. HOlvever, dosing can be simplified because of their unique pharmacokinetic properties. Limitations in the use of macrolides for respiratory infections include rather marginal activity in the most severe cases ofHaemophilu influenzae infections, lack of activity against Kleb iella and other coliforms, which precludes their lise as single agents in the therapy of pneumonia in patient with Significant underlying disease or in the elderly, and development of resistance in treptococci and taphylococci. Abstract Coronavirus envelope (E) protein is a small integral membrane protein with multi-functions in virion assembly, morphogenesis and virus-host interaction. Different coronavirus E proteins share striking similarities in biochemical properties and biological functions, but seem to adopt distinct membrane topology. In this report, we study the membrane topology of the SARS-CoV E protein by immunofluorescent staining of cells differentially permeabilized with detergents and proteinase K protection assay. It was revealed that both the N-and C-termini of the SARS-CoV E protein are exposed to the cytoplasmic side of the membranes (N cyto C cyto ). In contrast, parallel experiments showed that the E protein from infectious bronchitis virus (IBV) spanned the membranes once, with the N-terminus exposed luminally and the C-terminus exposed cytoplasmically (N exo(lum) -C cyto ). Intriguingly, a minor proportion of the SARS-CoV E protein was found to be modified by N-linked glycosylation on Asn 66 and inserted into the membranes once with the C-terminus exposed to the luminal side. The presence of two distinct membrane topologies of the SARS-CoV E protein may provide a useful clue to the pathogenesis of SARS-CoV. Abstract Upon virus infection, host cells mount a concerted innate immune response involving type I interferon and pro-inflammatory cytokines to enable elimination of the pathogen. Recently, cGAS and STING have been identified as intracellular sensors that activate the interferon pathway in response to virus infection and thus mediate host defense against a range of DNA and RNA viruses. Here we review how viruses are sensed by the cGAS-STING signaling pathway as well as how viruses modulate this pathway. Mechanisms utilized by viral proteins to inhibit cGAS and/or STING are also discussed. On the flip side, host cells have also evolved strategies to thwart viral immune escape. The balance between host immune control and viral immune evasion is pivotal to viral pathogenesis, and we discuss this virus-host stand-off in the context of the cGAS-STING innate immune pathway. Abstract A swine-origin H1N1 triple-reassortant influenza A virus found to be a distant relative of the 1918 "Spanish flu" virus emerged in April 2009 to give rise to the first influenza pandemic of the 21st century. Although disease was generally mild and similar to seasonal influenza, severe manifestations including respiratory failure were noted in some, particularly those with underlying conditions such as asthma, pregnancy and immunosuppression. Children and younger adults accounted for most cases, hospitalizations and deaths. A reverse transcriptase-polymerase chain reaction assay was superior to antigenbased rapid tests for diagnosis. All 2009 H1N1 pandemic influenza strains were susceptible to 1 or more neuraminidase inhibitors. Monovalent, unadjuvanted 2009 H1N1 vaccines were licensed in the United States in September 2009 and initially targeted to younger individuals, pregnant women, caretakers of infants and healthcare providers. The 2009 H1N1 pandemic highlights the need for modernization of influenza vaccines, improved diagnostics and more rigorous evaluation of mitigation strategies. Abstract Background: Proper hand hygiene helps prevent infectious diseases, while health literacy plays a critical role in preventive health behaviors. It remains unclear as to why proper hand hygiene practices cannot be sustained and what role health literacy plays in older adults fight against infectious diseases. Methods: A convenience sample of 433 old adults aged 65 and above was recruited. Their hygiene practices and health literacy were evaluated using a structured questionnaire adopted from the Centre for Health Protection and the Chinese version of the HLS-Asia-Q questionnaire. Results: The percentage distribution of the hand hygiene performance, from always to never, was 18%-10%. A majority 63.28% (274) of them were classified as having inadequate health literacy (0-25), while a meager 1.62% (7) of them as having excellent health literacy (42.01-50). The Spearman correlation showed a significant positive relationship (P < 0.05) between the participants' health literacy and their hand hygiene practices. Conclusions: Health literacy and hand hygiene are positively related in helping the older adults fight against infectious diseases. To sustain proper hand hygiene practices is to provide frequent hand hygiene training to the older adults. Abstract Objectives: To confirm this, using conventional and molecular detection methods, and expanding the study to younger children.Study design: One hundred and thirty-two nasal aspirates from 75 children hospitalized for a severe attack of asthma were studied (32 infants, mean age 9.1 months; and 43 children, mean age 5.6 years). According to the virus, a viral isolation technique, immunofluorescence assays (IFA) or both were used for the detection of rhinovirus, enterovirus, respiratory syncytial (RS) virus, adenovirus, coronavirus 229E, influenza and parainfluenza virus. Polymerase chain reaction (PCR) assays were used for the detection of rhinovirus, enterovirus, RS virus, adenovirus, coronavirus 229E and OC43, Chlamydia pneumoniae and Mycoplasma pneumoniae.Results: Using IFA and viral isolation techniques, viruses were detected in 33.3% of cases, and by PCR techniques, nucleic acid sequences of virus, Chlamydia pneumoniae and Mycoplasma pneumoniae were obtained in 71.9% of cases. The combination of conventional and molecular techniques detects 81.8% of positive samples. Two organisms were identified in the same nasal sample in 20.4% of the cases. The percentage of detections was higher (85.9%) in the younger group than in the other (77%). The most frequently detected agents were rhinovirus (46.9%) and RS virus (21.2%). Using PCR rather than conventional techniques, the detection rates were increased 5.8-and 1.6-fold in rhinovirus and RS virus infections, respectively. The detection levels of the other organisms are as follows: 9. 8, 5.1, 4.5, 4.5, 4.5, 3.7, and 2.2% for enterovirus, influenza virus, Chlamydia pneumoniae, adenovirus, coronavirus, parainfluenza virus, and Mycoplasma pneumoniae, respectively.Conclusion: These results confirm the previously reported high frequency of rhinovirus detection in asthmatic exacerbations in children. They also point out the frequency of RS virus detection, and emphasize the fact that PCR assays may be necessary to diagnose respiratory infections in asthma. Abstract Following the anthrax attacks of 2001 and the recent SARS outbreak, concerns about emerging and re-emerging infectious diseases have catalyzed a renewed interest in developing new vaccination strategies that provide rapid and flexible response options to future threats. Because the probability of encountering one of these exotic agents is unknown, it is essential that new vaccine formulations employ methods that provide effective protection and extremely good safety profiles if they are to be used by either military or civilian populations. One approach, which potentially satisfies these criteria, is the use of live recombinant Gram-positive commensal bacteria as expression vectors. This review provides an overview of the system, its advantages and limitations, and details an example of how Gram-positive commensal bacteria are being developed as a fifth generation vaccine against a Class A biowarfare pathogen, namely smallpox. D Abstract Chemokines are an extensive family of small proteins which, in conjunction with their receptors, guide the chemotactic activity of various immune cells throughout the body. CCL28, βor CC chemokine, is involved in the host immunity at various epithelial and mucosal linings. The unique roles of CCL28 in several facets of immune responses have attracted considerable attention and may represent a promising approach to combat various infections. CCL28 displays a broad spectrum of antimicrobial activity against gram-negative and gram-positive bacteria, as well as fungi. Here, we will summarize various research findings regarding the antimicrobial activity of CCL28 and the relevant mechanisms behind it. We will explore how the structure of CCL28 is involved with this activity and how this function may have evolved. CCL28 displays strong homing capabilities for B and T cells at several mucosal and epithelial sites, and orchestrates the trafficking and functioning of lymphocytes. The chemotactic and immunomodulatory features of CCL28 through the interactions with its chemokine receptors, CCR10 and CCR3, will also be discussed in detail. Thus, in this review, we emphasize the dual properties of CCL28 and suggest its role as an anchoring point bridging the innate and adaptive immunity. Abstract Whether influenza vaccination influences the severity of illness in cases of clinical failure in solid organ transplant (SOT) recipients receiving influenza vaccine has not been extensively studied. Our goal was to evaluate the frequency of influenza vaccination among SOT recipients with influenza disease and its impact on the illness severity during the 2010-2011 season. Adult SOT recipients with confirmed influenza infection were included from December 2010 to April 2011. Follow-up data were recorded and antibody titres were determined using a microneutralization assay. Sixty-four SOT recipients were included in the study, ten (15.6%) with severe disease, requiring admission to intensive care units, of whom four (6.3%) died. In all, 34 (53.1%) received the 2010-2011 seasonal influenza vaccine and 32 (50.0%) received the 2009-H1N1 pandemic vaccine, and none had detectable antibodies against influenza at the time of diagnosis of influenza infection. Twenty-three (67.6%) of the patients that received the vaccine required hospital admission and presented less dyspnoea (10, 29.4% versus 14 (50.0%), p 0.09) and pneumonia (8, 23.8% versus 15, 50.0%, p 0.03, relative risk 0.3, 95% CI 0.1-0.9) than unvaccinated patients, with relative risk reductions of 60% and 70%, respectively. Although influenza vaccination confers protection on SOT recipients against developing influenza pneumonia, the rate of clinical failure is still high. New strategies to improve influenza immunization are needed for this group of patients. Abstract A diagnostic test for canine coronavirus (CCV) infection based on a nested polymerase chain reaction (n-PCR) assay was developed and tested using the following coronavirus strains: CCV (USDA strain), CCV (45/93, field strain), feline infectious peritonitis virus (FIPV, field strain), trasmissible gastroenteritis virus (TGEV, Purdue strain), bovine coronavirus (BCV, 9WBL-77 strain), infectious bronchitis virus (IBV, M-41 strain) and fecal samples of dogs with CCV enteritis. A 230-bp segment of the gene encoding for transmembrane protein M of CCV is the target sequence of the primer. The test described in the present study was able to amplify both CCV and TGEV strains and also gave positive results on fecal samples from CCV infected dogs. n-PCR has a sensitivity as high as isolation on cell cultures, and can therefore be used for the diagnosis of CCV infection in dogs. (M. Tempesta) 0166-0934/99/$ -see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 -0 9 3 4 ( 9 9 ) 0 0 0 1 7 -8 Abstract A novel coronavirus has been associated with a worldwide outbreak of atypical pneumonia referred to as Severe Acute Respiratory Syndrome (SARS-CoV). SARS-CoV nucleocapsid (N) protein has been cloned sequenced and expressed in Escherichia coli strain. Purified N protein was used to measure the SARS-CoV specific IgG antibodies from 16 SARS-CoV infected patients' sera and from 131 control subjects using ELISA assay. Specific antibody responses to the purified recombinant N protein after 10, 20, and 30 days of disease onset were observed in 13 of 16 (81.3%), 16 of 16 (100%) and 16 of 16 (100%) SARS patients sera, respectively. Comparison of detection results with a commercially available diagnostic kit coated with a mixture of SARS-CoV viral proteins showed 9 of 16 (56.3%), 13 of 16 (81.3%), and 15 of 16 (93.7%) positive responses, respectively. None of 131 control sera gave positive reaction in either assay. This data suggests that the N protein of SARS-CoV is immunodominant and this ELISA based test assay for detecting the SARS-CoV N antigen may hold a significant value for SARS diagnosis. Abstract The objective of the experiment was to evaluate effects of increased milk replacer feeding on growth, intake, feed efficiency, and health parameters in stressed calves. Holstein bull calves (n = 120; approximately 3 to 8 d of age) were purchased from sale barns and dairy farms and housed in fiberglass hutches. In addition, wood shavings contaminated with coronavirus were mixed with clean shavings and added to each hutch before the start of the experiment. Calves were fed either a fixed amount (454 g/d) of a 20% crude protein (CP), 20% fat milk replacer to weaning at 28 d or a variable amount (454, 681, 908, and 454 g/d on d 0 to 7, 8 to 14, 15 to 31, and 32 to 41, respectively) of a milk replacer containing 28% CP and 17% fat without or with added dietary supplement containing bovine serum. Calves were also fed commercial calf starter and water ad libitum. Plasma IgG concentration in most calves on arrival at the facility was <10 g/L. Intake, change in body weight, feed efficiency, morbidity and mortality, and selected plasma metabolites were determined. Body weight at 28 d, 56 d, daily body weight gain, intake of milk replacer, fecal scores, days with diarrhea, and days treated with antibiotics were increased with feeding variable amount of milk replacer over the 56-d study. Starter intake from d 1 to 56 was reduced from 919 to 717 g/d in calves fed fixed and variable amounts of milk replacer, respectively. Morbidity, measured as the number of days that calves had diarrhea, was increased by 53% when a variable amount of milk replacer was fed. Calves fed variable milk replacer were treated with antibiotics for 3.1 d compared with 1.9 d for calves fed 454 g of milk replacer/ d. Concentrations of plasma glucose, urea N, and insulin-like growth factor-I were increased when calves 207 were fed variable amount of milk replacer. Dietary supplement containing bovine serum had no effect on any parameter measured. There was no effect of milk replacer feeding on concentrations of nonesterified fatty acids, total protein, or growth hormone concentrations. Plasma tumor necrosis factor-α was highest in calves with the highest plasma IgG concentrations on the day of arrival and might be related to the calf's ability to identify pathogens in the environment. Under conditions of this study, calves fed variable amount of milk replacer and exposed to immunological challenge before weaning had greater BW gain, but also increased incidence of diarrhea that required added veterinary treatments. Abstract Xenotropic murine leukemia virus-related virus (XMRV) has been proposed to be associated with prostate cancer and chronic fatigue syndrome (CFS). This proposition has been controversial because many investigators have failed to replicate the reported associations. Here, we explore whether XMRV is an authentic human pathogen in the light of recent findings that indicate otherwise. Abstract Travellers' compliance with influenza vaccines and antiviral drugs remains very poor despite influenza being a frequent vaccine-preventable disease with levels of compliance varying from 58% to 72% depending on the beliefs and perceptions of travellers' regarding the prevention of influenza. Abstract None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this article. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Abstract Booman, P., Tieman, M., Van Zaane, D., Bosma, A.A. and De Boer, G.F., 1990. Construction of a bovine-murine heteromyeloma cell line; production of bovine monoclonal antibodies against rotavirus and pregnant mare serum gonadotrophin. Vet. Immunol. Immunopathol., 24: 211-Bovine-murine heteromyeloma cell lines were prepared by fusing lymphoid cells from a bovine leukemia virus (BLV)-infected cow with mouse myeloma cells. Selection of hybrid cell colonies was based on the ratio of bovine and murine chromosomes, the presence of cell-surface immunoglobulins and growth characteristics. First-generation fusion partners were compared for fusion efficiency and the number of antigen-specific antibody-producing clones generated. Hybrid cell colonies that initially secreted antibodies were selected from first-generation heteromyelomas to function as second-generation fusion partners. Although fusion efficiencies for both generations did not differ, the second-generation heteromyelomas yielded a higher number of specific antibody-producing clones. Fusion of heteromyelomas with either lymph node cells or splenocytes indicated that fusion with lymph node cells results in a higher number of specific antibody-producing clones, whereas fusion efficiency was found to be higher with splenocytes. The optimal time intervals between the final booster injection and fusion were found to be 4 days for splenocytes and 7 days for lymph node cells. Finally, the characterization of bovine monoclonal antibodies against bovine rotavirus and pregnant mare serum gonadotrophin and their neutralizing capacities in vitro are described. Abstract . Problems of calf rearing in connection with their mortality and optimal growth: a review. Livest. Prod. Sci., 10: 339--349.Protection of calves against enteric and respiratory disorders is dependent on the passive immunity that the calf has received, its innate resistance to infection, the burden of infection in the environment and the nutrition of the calf. Superimposed on these, are the effects of management and physical environment.The plane of nutrition required for dairy heifers during rearing depends on the age at first calving, and for meat animals depends on the carcass weight and fat deposition required at a particular slaughter date in relation to time of birth.0301-6226/83/$03.00 Abstract Objective: We examined the relationship between hospital structural characteristics and system-level activities for patient safety and infection control, for use in designing an incentive structure to promote patient safety. Methods: This study utilized a questionnaire to collect institutional data about hospital infrastructure and volume of patient safety activities from all 1039 teaching hospitals in Japan. The patient safety activities were focused on meetings and conferences, internal audits, staff education and training, incident reporting and infection surveillance. Generalized linear modeling was used. Results: Of the 1039 hospitals surveyed, 418 (40.2%) hospitals participated. The amount of activities significantly increased by over 30% in hospitals with dedicated patient safety and infection control full-time staff (P < 0.001 and P < 0.01, respectively). High profit margins also predicted the increase of patient safety programs (P < 0.01). Perceived lack of administrative leadership was associated with reduced volume of activities (P < 0.05), and the economic burden of safety programs was found to be disproportionately large for small hospitals (P < 0.05). Conclusions: Hospitals with increased resources had greater spread of patient safety and infection control activities. To promote patient safety programs in hospitals, it is imperative that policy makers require the assignment of dedicated full-time staff to patient safety. Economic support for hospitals will also be required to assure that safety programs are sustainable. Abstract Aminopeptidase N, or CD13, is a receptor for serologically related coronaviruses of humans, pigs, and cats. A mouse line transgenic for the receptor of human coronavirus-229E (HCoV-229E) was created using human APN (hAPN) cDNA driven by a hAPN promoter. hAPNtransgenic mice expressed hAPN mRNA in the kidney, small intestine, liver, and lung. hAPN protein was specifically expressed on epithelial cells of the proximal convoluted renal tubules, bronchi, alveolar sacs, and intestinal villi. The hAPN expression pattern within transgenic mouse tissues matched that of mouse APN and was similar in mice heterozygous or homozygous for the transgene. Primary embryonic cells and bone marrow dendritic cells derived from hAPN-transgenic mice also expressed hAPN protein. Although hAPN-transgenic mice were resistant to HCoV-229E in vivo, primary embryonic cells and bone marrow dendritic cells were infected in vitro. hAPN-transgenic mice are valuable as a source of primary mouse cells expressing hAPN. This hAPN-transgenic line will also be used for crossbreeding experiments with other knockout, immune deficient, or transgenic mice to identify factors, in addition to hAPN, that are required for HCoV-229E infection. D Abstract A B S T R A C T SARS coronavirus (CoV) papain-like protease (PLpro) reportedly induced the production of TGF-β1 through p38 MAPK/STAT3-meidated Egr-1-dependent activation (Sci. Rep. 6, 25754). This study investigated the correlation of PLpro-induced TGF-β1 with the expression of Type I collagen in human lung epithelial cells and mouse pulmonary tissues. Specific inhibitors for TGF-βRI, p38 MAPK, MEK, and STAT3 proved that SARS-CoV PLpro induced TGF-β1-dependent up-regulation of Type I collagen in vitro and in vivo. Subcellular localization analysis of SMAD3 and SMAD7 indicated that non-SMAD pathways in TGF-β1 signaling involved in the production of Type I collagen in transfected cells with pSARS-PLpro. Comprehensive analysis of ubiquitin-conjugated proteins using immunoprecipitation and nanoLC-MS/MS indicated that SARS-CoV PLpro caused the change in the ubiquitination profile of Rho GTPase family proteins, in which linked with the increase of Rho-like GTPase family proteins. Moreover, selective inhibitors TGF-βRI and STAT6 (AS1517499) ascertained that STAT6 activation was required for PLpro-induced TGF-β1-dependent up-regulation of Type I collagen in human lung epithelial cells. The results showed that SARS-CoV PLpro stimulated TGF-β1-dependent expression of Type I collagen via activating STAT6 pathway. Abstract In order to understand the spatio-temporal structure of epidemics beyond that permitted with classical SIR (susceptible-infectiverecovered)-type models, a new mathematical model for the spread of a viral disease in a population of spatially distributed hosts is described. The positions of the hosts are randomly generated in a rectangular habitat. Encounters between any pair of individuals are according to a Poisson process with a mean rate that declines exponentially as the distance between them increases. The contact rate allows the mean rates to be set at a certain number of encounters per day on average. The relevant state variables for each individual at any time are given by the solution of a pair of coupled differential equations for the viral load and the quantity of general immune system effectors which reduce the viral load. The parameters describing within-host viral-immune system dynamics are generated randomly to reflect variability across a population. Transmission is assumed to depend on the viral loads in donors and occurs with a probability p trans . The initial conditions are such that one randomly chosen individual carries a randomly chosen amount of the virus, whereas the rest of the population is uninfected. Simulations reveal local or whole-population responses. Whole-population disease spread may be in the form of isolated or multiple occurrences, the latter often being approximately periodic. The mechanisms of this oscillatory behaviour are analyzed in terms of several parameters and the distribution of critical points in the host dynamical systems. Increased contact rate, increased probability of transmission and decreased threshold for viral transmission, decreased immune strength and increased viral growth rate all increase the probability of multiple outbreaks and the distribution of the critical points also plays a role. Abstract Background: Pneumonia, a leading cause of childhood mortality, is associated with household air pollution (HAP) exposure. Mechanisms between HAP and pneumonia are poorly understood, but studies suggest that HAP may increase the likelihood of bacterial, instead of viral, pneumonia. We assessed the relationship between HAP and infant microbial nasal carriage among 260 infants participating in the Ghana Randomized Air Pollution and Health Study (GRAPHS).Methods: Data are from GRAPHS, a cluster-randomized controlled trial of cookstove interventions (improved biomass or LPG) versus the 3-stone (baseline) cookstove. Infants were surveyed for pneumonia during the first year of life and had routine personal exposure assessments. Nasopharyngeal swabs collected from pneumonia cases (n = 130) and healthy controls (n = 130) were analyzed for presence of 22 common respiratory microbes by MassTag polymerase chain reaction. Data analyses included intention-to-treat (ITT) comparisons of microbial species presence by study arm, and exposure-response relationships.Results: In ITT analyses, 3-stone arm participants had a higher mean number of microbial species than the LPG (LPG: 2.71, 3-stone: 3.34, p < 0.0001, n = 260). This difference was driven by increased bacterial (p < 0.0001) rather than viral species presence (non-significant). Results were pronounced in pneumonia cases and attenuated in healthy controls. Higher prevalence bacterial species were Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Exposure-response relationships did not yield significant associations between measured CO and nasal microbial carriage.Our intention-to-treat findings are consistent with a link between HAP and bacterial nasal carriage. No relationships were found for viral carriage. Given the null results in exposure-response analysis, it is likely that a pollutant besides CO is driving these differences. Abstract This study measures the travelers' perceived change in utility by accepting one of the modes of transport air, rail, or bus as one component of a packaged city trip. The part-worth values for the trip product elements are expected to depend on a number of traveler characteristics. The predictors hypothesized are city travel experience, general modal preference, socio-economic status, and car ownership. In the survey, the combinations of trip attributes differed between the two subgroups of leisure and business travelers. The leisure travelers rated three levels of mode, length of stay, and price, but only one level of the hotel category. The business travelers were shown four mode alternatives and only two levels for each of the other trip product elements. The conjoint measurements were elaborated by fitting an Extended Bradley-Terry Model. Demonstrating the application of the EBTM is the main purpose of the paper. The EBTM offers several advantages over the more popular versions of conjoint analysis. It correctly treats ties and allows for simultaneous estimation of the trip package ('object') parameters, object covariates (trip attributes), subject covariates (traveler characteristics) and their interactions. For both the business and the leisure travelers, the mode of transport dominated the assessment of a city trip package. For leisure tourists, e.g., switching from train 2nd class to an economy flight boosted the trip package more than twice as much as replacing train for bus. A variation of the package price was much more important for the leisure than for the business travelers. The socio-economic status proved to be an important factor and was particularly influential among the business travelers. In the leisure tourists' sub-sample age was not only important for valuing the mode of transport, but had a preferential impact for all trip components. Finally, the limitations of this demonstration study that discourage extrapolation to city travelers in general are emphasized. Abstract Studying the emergence of novel infectious agents involves many processes spanning host species, spatial scales, and scientific disciplines. Mathematical models play an essential role in combining insights from these investigations and drawing robust inferences from field and experimental data. We describe nine challenges in modelling the emergence of novel pathogens, emphasizing the interface between models and data. Abstract Hotel business in Hong Kong has been badly affected by the outbreak of the Severe Acute Respiratory Syndrome (SARS) since March 2003. Medical reports suggest that a direct contact with the secretion or respiratory droplets from a patient through a short distance is very likely to be the possible way of transmitting the disease. World Health Organization (WHO), hence, has advised international travellers to avoid visiting several regions that have the most number of SARS cases, including Hong Kong. Since then, the number of hotel guests has largely dropped to a level that Hong Kong has never experienced before. As a totally new experience to the hotel industry in Hong Kong and elsewhere, hoteliers basically do not have enough knowledge to respond to epidemic crisis. In view of the lack of published articles in the hospitality literature that examine the issue, this paper investigates the impact of the SARS outbreak on the hotel industry in Hong Kong. In addition, this paper addresses the issue of SARS crisis management in risk identification, assessment and alleviation. The paper is expected to benefit the entire hotel industry from better understanding and planning to face the mysterious illness. r Abstract Sensitive molecular techniques are needed for rapid detection of the R292K oseltamivir-resistant mutant of influenza A(H7/N9) virus strain to monitor its transmission and guide antiviral treatment. We developed a real-time reverse transcription PCR and single nucleotide polymorphism probes to differentiate this mutant strain in mixed virus populations in human specimens. Abstract Pneumonia virus of mice (PVM) infection of BALB/c mice induces bronchiolitis leading to a fatal pneumonia in a dose-dependent manner, closely paralleling the development of severe disease during human respiratory syncytial virus infection in man, and is thus a recognised model in which to study the pathogenesis of pneumoviruses. This model system was used to investigate delivery of the internal structural proteins of PVM as a potential vaccination strategy to protect against pneumovirus disease. Replicationdeficient recombinant human adenovirus serotype 5 (rAd5) vectors were constructed that expressed the M or N gene of PVM pathogenic strain J3666. Intranasal delivery of these rAd5 vectors gave protection against a lethal challenge dose of PVM in three different mouse strains, and protection lasted for at least 20 weeks post-immunisation. Whilst the PVM-specific antibody response in such animals was weak and inconsistent, rAd5N primed a strong PVM-specific CD8 + T cell response and, to a lesser extent, a CD4 + T cell response. These findings suggest that T-cell responses may be more important than serum IgG in the observed protection induced by rAd5N. Abstract Coronavirus Autophagy ER stress Unfolded protein response Mitogen-activated protein kinase A B S T R A C T Coronavirus infection induces the generation of autophagosomes, and certain host proteins regulating cellular autophagy are hijacked by some coronaviruses to facilitate the formation of double membrane vesicles. However, mechanisms underlying coronavirus-induced autophagy remain largely unknown. In this study, we demonstrate that autophagosome formation and apparent autophagic flux are induced in cells infected with infectious bronchitis virus (IBV)a gammacoronavirus. Notably, IBV-induced autophagy was dependent on autophagy related 5 (ATG5) but not beclin1 (BECN1), although both are essential proteins in the canonical autophagy pathway. Moreover, the ER stress sensor inositol requiring enzyme 1 (IRE1), but not its substrate Xbox protein 1 (XBP1), was also essential for the induction of autophagy during IBV infection. Finally, the antiapoptotic extracellular signal-regulated kinase 1/2 (ERK1/2) also contributed to IBV-induced autophagy. Our findings add new knowledge to the regulatory mechanisms governing coronavirus-induced autophagy, highlighting an extensive cross-talk among cellular signaling pathways during coronavirus infection. Abstract The successive emergence of highly pathogenic coronaviruses (CoVs) such as the Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 has stimulated a number of studies on the molecular biology. This research has provided significant new insight into functions and activities of the replication/transcription multi-protein complex. The latter directs both continuous and discontinuous RNA synthesis to replicate and transcribe the large coronavirus genome made of a single-stranded, positive-sense RNA of ∼30 kb.In this review, we summarize our current understanding of SARS-CoV enzymes involved in RNA biochemistry, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3 -5 exoribonuclease activities involved in RNA capping, and RNA proofreading, respectively. The recent discoveries reveal fascinating RNA-synthesizing machinery, highlighting the unique position of coronaviruses in the RNA virus world. Abstract To investigate the potential of RNA interference (RNAi) as antiviral agent against rabies, two small interfering RNAs (siRNAs) targeting rabies virus (RABV) nucleoprotein (N) and polymerase (L) genes were designed and evaluated. Both siRNAs knockdown or silenced the target RABV genes as evaluated in a plasmid based transient expression model. For efficient delivery, adenoviruses expressing the siRNAs were constructed and antiviral potential of the delivered siRNAs was investigated in BHK-21 cells. When cells treated with adenoviruses expressing siRNAs were challenged with RABV, there was 88.35 ± 2.4% and 41.52 ± 9.3% reduction in RABV multiplication in infected cells with siRNAs targeting RABV-N and L genes, respectively. Relative quantification of RABV transcripts using real-time PCR revealed knockdown of both RABV-N and L gene transcripts, however, significant reduction was observed only with adenovirus expressing siRNA against RABV-N. When mice treated intracerebrally with adenoviruses expressing siRNAs were challenged peripherally with lethal RABV by the intramuscular route in masseter muscle, there was 66.6% and 33.3% protection with adenoviruses expressing siRNAs against RABV-N and L genes, respectively. These results demonstrated that adenovirus expressing siRNA against RABV-N efficiently inhibited the RABV multiplication both, in vitro and in vivo and conferred significant protection against lethal RABV challenge. This supported the hypothesis that RNAi, based on siRNA targeting RABV-N gene can prevent RABV infection and holds the potential of RNAi as an approach to prevent rabies infection. Abstract We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)envelope and -liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse model compared to the BCG. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality. The BCG prime and HSP65+IL-12/HVJ vaccine (boost) by the prime-boost method showed a synergistic prophylactic effect in the monkey. Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys.HVJ-Envelope/HSP65 DNA+IL-12 DNA vaccine increased the body weight of TB-infected monkeys, improved the ESR, and augmented the immuneresponses (proliferation of PBL and IL-2 production). The enhancement of IL-2 production from monkeys treated with this vaccine was correlated with the therapeutic efficacy of the vaccine. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials. Abstract A total of 123 community paediatricians and 23 microbiology laboratories studied the clinical and bacteriological efficacy of treatment of group A streptococcal pharyngitis in Italy. Of 1065 patients, from whom Streptococcus pyogenes was isolated, 723 returned to follow up and of these 138 (19%) still had a positive throat culture. The erythromycin resistance (ER) rate was 23.7% with resistance phenotype distribution of: 31.7% constitutive (CR), 26.6% inducible (IR) and 41.7% efflux pump (M) resistance phenotype. All strains were susceptible to the b-lactam agents tested. CR strains were highly resistant to all 14, 15 and 16 membered macrolides with the exception of rokitamycin which showed activity against 37.8% of isolates. All phenotype M and some IR isolates were susceptible to clindamycin, rokitamycin, josamycin and spiramycin; clarithromycin was active against a small percentage of strains belonging to the IR and M phenotype. Bacterial eradication was found in 85.5, 78.7 and 75.8% of the penicillin, macrolide and cephalosporin treated groups. Genotyping of strains showed that 8.7% of the 19% of cases classified as 'failed bacterial eradication' were due to recolonization with a different isolate, observed exclusively among b-lactams treated patients. Clinical cure was achieved in a high percentage of cases, irrespective of the antibiotic prescribed, with the best clinical efficacy being found following therapy with amoxycillin and clarithromycin (90.9%). Abstract The great advance in the field of diagnosis of avian viruses is reflecting the highly sophisticated molecular assays of the human and general virology in providing highly sensitive and fast methods of diagnosis. The present review will discuss the biotic factors and the complexities that became evident with the evolution of the novel molecular diagnostic assays with emphasis on 4 avian viruses, chicken anemia, infectious laryngotracheitis, turkey meningoencephalitis, but mainly on Marek's disease virus.To create a biologically meaningful diagnosis, attention should be dedicated to various biotic factors and not only of the diagnostic assay. Included among the important factors are, (a) the sample examined and the sampling strategy, (b) the outcomes of the pathogen amplification ex vivo, (c) the sampling time and its reflection on the disease diagnosis, (d) the impact of simultaneous multiple virus-infections regarding the ability to demonstrate all pathogens and inter-and intra-interactions between the pathogens. A concerted consideration of the relevant factors and the use of advanced molecular diagnostic assay would yield biologically significant diagnosis in real-time that would beneficiate the poultry industry. Recently Shojaei et al. (2015) and Astill et al. (2018) described novel methodologies developed to detect rapidly avian viruses, such as Abstract Background: Immunocompromised adults are more vulnerable to a complicated course of viral respiratory tract infections (RTI). Objectives: Provide evidence on the effect of implementation of rapid molecular diagnostics for viruses on use of in-hospital isolation facilities, oseltamivir and antibiotic usage, and other clinical outcomes in immunocompromised patients. Study design: A before-after study during two consecutive respiratory viral seasons, including immunocompromised adult patients presenting at a tertiary care emergency department with clinical suspicion of RTI. During the first season (2016/2017), respiratory viruses were detected using inhouse real-time PCR. The second season (2017/2018), we implemented a diagnostic flowchart including a rapid molecular test for 15 respiratory viruses (FilmArray®). We assessed the effect of this implementation on need for isolation, antivirals and empirical antibiotics. Results: We included 192 immunocompromised adult patients during the first and 378 during the second season. Respiratory viral testing was performed in 135 patients (70%) during the first and 284 (75%) during the second season (p = 0.218) of which 213 (75%) using the rapid test. After implementation, use of in-hospital isolation facilities was reduced (adjusted odds ratio 0.35, 95%CI 0.19-0.64). Furthermore, adequate use of oseltamivir improved, with fewer prescriptions in influenza negative patients (0.15, 95%CI 0.08-0.28) and more in influenza positive patients (11.13,. No effect was observed on empirical antibiotic use, hospital admissions, length of hospital stay or safety outcomes. Conclusions: Implementation of rapid molecular testing for respiratory viruses in adult immunocompromised patients results in more adequate use of oseltamivir and in-hospital isolation facilities without compromising safety. Abstract Orthopoxviruses code for numerous immunomodulatory proteins, the structure and function of which are clarified inadequately. Antibodies constitute a potent tool to study such proteins, enabling conclusions on protein location and time course of expression. However, common antibody production in mice or rabbits requires tedious protein expression and injection, as well as blood collection at regular intervals. To simplify this procedure, IgY antibodies specific for poxviral proteins (F1L and p28) were generated by immunisation of chickens, because antibody retrieval from eggs allows the noninvasive generation of huge amounts of antibodies. The main intentions were (i) to decrease invasiveness, (ii) to immunise with native forms of proteins and (iii) to circumvent previous protein expression and purification. Therefore, chicken were immunised with DNA expression vectors coding for conserved domains of the selected proteins delivered for the first time by a gene gun. Four weeks after initial immunisation specific antibodies were found in the egg yolk as proven by immunofluorescence staining of poxvirus-infected cells. The specific IgY titre rose to 1:80,000 and was stable for more than 120 days. With this investigation we present an universal procedure for IgY design and production that can be applied for various issues in the future. Abstract In his homage to Lucretius ('Georgica'), Vergil is credited with stating: 'Felix qui potuit rerum cognoscere causas' ('Fortunate is he who knows the causes of things'). Based on numerous commentaries and publications it is obvious that clinicians, diagnosticians and biomedical research scientists continue to struggle with disease causation, particularly in the assessment of the pathogenic role of 'stealth pathogens' that produce persistent infections in the host. Bartonella species, because of their evolutionary ability to induce persistent intravascular infections, present substantial challenges for researchers attempting to clarify the ability of these stealth bacteria to cause disease. By studying the comparative biological and pathological behaviour of microbes across mammalian genera, researchers might be able more rapidly to advance medical science and, subsequently, patient care by undertaking focused research efforts involving a single mammalian species or by attempting to recapitulate a complex disease in an rodent model. Therefore, in an effort to further assist in the establishment of disease causation by stealth pathogens, we use recent research observations involving the genus Bartonella to propose an additional postulate of comparative infectious disease causation to Koch's postulates. Abstract Therapeutic medicine today includes a vast number of procedures involving the use of biomaterials, transplantation of therapeutic cells or cell clusters, as well as of solid organs. These treatment modalities are obviously of great benefit to the patient, but also present a great challenge to the innate immune system, since they involve direct exposure of non-biological materials, cells of non-hematological origin as well as endothelial cells, damaged by ischemia-perfusion in solid organs to proteins and cells in the blood. The result of such an exposure may be an inappropriate activation of the complement and contact/kallikrein systems, which produce mediators capable of triggering the platelets and PMNs and monocytes, which can ultimately result in thrombotic and inflammatory (i.e., a thrombo-inflammatory) response to the treatment modality. In this concept review, we give an overview of the mechanisms of recognition within the innate immunity system, with the aim to identify suitable points for intervention. Finally, we discuss emerging and promising techniques for surface modification of biomaterials and cells with specific inhibitors in order to diminish thromboinflammation and improve clinical outcome. Abstract FIGURE 25.2 Normal patagium, Pteropus vampyrus. The patagium consists of two thin layers of epidermis separated by collagen, vessels, and bundles of elastic fibers. FIGURE 25.1 FIGURE 25.4 Normal brain, Pteropus hypomelanus. The brain is largely lissencephalic, with rare shallow sulci. (Photo Courtesy of E. Kieran, University of Florida) FIGURE 25.5 Iron overload in the liver, Rousettus aegyptiacus. (A) Bridging fibrosis with iron overload. Abundant brown pigment is present within and at the margins of the connective tissue. (B) Abundant hemosiderin is present in macrophages at the margins of and within the fibrous connective tissue, with lesser amounts in hepatocytes, bile ducts, and Kupffer cells. Prussian blue. Abstract The aim of this study was to explore the role of the Diffusion of Innovations framework in adopting an infection prevention and control program (IPCP) in a low and middle income (LMI) country, the Republic of Kiribati.Methods: Case-study methodology was used to examine and contextualise the analysis of the Republic of Kiribati's adoption of the IPCP from 2003 to 2010. Data were collected from multiple sources including semi-structured interviews, IPCP documentation, program evaluation and a healthcare worker survey. Data were subjected to thematic analysis and descriptive statistics where relevant to the study design.Results: It was found that the self-initiated progression of activities and stimuli has resulted in the successful adoption of a comprehensive IPCP. The process followed the staged model of the classic Diffusion of Innovations process in organisations described by Everett Rogers.Conclusion: This case study provides an illustration of how a comprehensive IPCP can be adopted in a LMI country setting with little involvement from external agencies. It identifies key stimuli, opportunities and activities which could be similarly adopted and implemented by other LMI countries. Abstract This study employs an extended gravity model to analyse the complementarity or competitiveness relationship of the number of inbound tourists and corresponding tourism revenue between China and 19 other nations under the implementation of China's Open-door Tourism Policy to Taiwan in 2008. A simulation for 2018-2021 demonstrates the sustained impact of this policy. The results show that the number of tourists to Taiwan from China reached its peak in 2015 at 41% and will decrease to 9% by 2021. The corresponding tourism revenue will decrease from 49% to 11% over the same period. The results also show that if the number of tourists from China remains above 836,772, the number of tourists from Japan, Hong Kong, Australasia, North America, and Europe will still increase. However, the number of tourists from South Korea and South and Southeast Asia will increase continuously regardless of tourists from China, even far below 836,772. Abstract We determined the prevalence of antibodies to infectious bovine rhinotracheitis virus (IBRV) and bovine viral diarrhea virus (BVDV) in sera of dairy cows on 4 different farms in the Republic of Croatia. A high percentage (60.8%) of cows had various reproductive disorders. The results showed that seroprevalence of infectious bovine rhinotracheitis (IBR) was 85.8% and that of bovine viral diarrhea (BVD) was 79.2% in tested cows. Antibodies to both viruses werefound in 80.8% of cows with reproductive disorders but in only 46.8% of cows without reproductive disorders. This difference was statistically siLmificant (P<0.01), and indicated a connection between reproductive disorders and simultaneous infections with IBR and BVD viruses in dairy COWS. O 1999 by Elsevier Science Inc. Abstract This study numerically investigated the thermal effect of human body on the time-dependent dispersion of cough droplets with evaporation process. The thermal flow of human body was imitated using a 3D thermal manikin with real body features, while a recent developed multi-component Eulerian-Lagrangian approach was used to address the effects of inhomogeneous temperature and humidity fields on droplet evaporation. By comparing the results yielded without and with the human body heat, the outcomes demonstrated strong impact of human body heat on the droplets mass fraction and local air velocity distributions. Inspirable droplets could potentially drop into respirable droplets by evaporation, although the evaporation rate was not significantly affected by body heat. The thermal effect of human body revealed its vital impacts on the time-dependent droplets dispersion. Due to the buoyancy driven thermal flow, both the vertical velocity and displacement of small droplets (≤20 μm) were completely reversed from descending to ascending, while the deposition time of large droplets (≥50 μm) were significantly delayed. With the reduced droplet size by evaporation and droplets lifted into breathing zone by human thermal effect, the inhalability and infection risks of cough droplets would be much higher in real occupied indoor spaces. Abstract In Poland between several thousand and several million cases of influenza and suspected influenza cases are registered, depending on the epidemic season. A variety of methods are available for the detection of the influenza viruses responsible for respiratory infection starting with the isolation of the virus in chick embryos or in cell lines such as MDCK, VERO, etc., and finishing with a variety of modifications of the classical PCR molecular biology such as PCR multiplex and Real-Time. The most effective way to combat influenza is through vaccination. Regular vaccination is one of the few steps that may be taken to protect individuals, especially in high-risk groups, from the potential and serious complications of influenza. In many countries, including Poland, despite the recommendations, the rate of vaccination against influenza is still low in all age groups. In the epidemic season 2011/2012, the level of distribution of the seasonal influenza vaccines was 4.5% of the population. Abstract In outbreak detection, one of the key issues is the need to deal with the weakness of early outbreak signals because this causes the detection model to have has less capability in terms of robustness when unseen outbreak patterns vary from those in the trained model. As a result, an imbalance between high detection rate and low false alarm rate occurs. To solve this problem, this study proposes a novel outbreak detection model based on danger theory; a bio-inspired method that replicates how the human body fights pathogens. We propose a signal formalization approach based on cumulative sum and a cumulative mature antigen contact value to suit the outbreak characteristic and danger theory. Two outbreak diseases, dengue and SARS, are subjected to a danger theory algorithm; namely the dendritic cell algorithm. To evaluate the model, four measurement metrics are applied: detection rate, specificity, false alarm rate, and accuracy. From the experiment, the proposed model outperforms the other detection approaches and shows a significant improvement for both diseases outbreak detection. The findings reveal that the robustness of the proposed immune model increases when dealing with inconsistent outbreak signals. The model is able to detect new unknown outbreak patterns and can discriminate between outbreak and non-outbreak cases with a consistent high detection rate, high sensitivity, and lower false alarm rate even without a training phase. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.Enteric protozoal infection is the commonest cause of diarrhoea in HIV-seropositive persons (table) and is associated with apoptosis, occasional crypt abscesses, and much more severe villus atrophy than that seen with HIV infection alone. 18 Malabsorption and maldigestion increase bowel frequency. However, symptoms persist despite Abstract This is a phenomenological study that examined nursing students' perception of nursing professional identity during severe acute respiratory syndrome (SARS) outbreak in Hong Kong in 2003. The aim of the study was to find out how the impact of the SARS event might have affected nursing students in identification with the nursing profession. A total of 10 nursing students were interviewed. This study showed that the SARS crisis enhanced a reconstruction of worldview and affirmed the professional identity of nursing students. Central themes derived from the interview were (1) appreciation and sharing of nursing identity; (2) a sense of moral duty; (3) a change of worldview and feeling of self-growth. This study provided insights to nursing education that acquisition of professional identity could be enhanced through reflective appreciation of critical events such as SARS. Abstract An infection of pigs with the porcine respirator)' coronavirus ( PRCV) induces antibodies which neutralize the enteropathogenic transmissible gastroenteritis virus ( TGEV) and PRCV to the same titre. In the present study, lO-week-old seronegative pigs (n = 8), pigs immune ./o/lowing TGEV inoculation (n = 4) or pigs immune following aerosol (n = 8) or intragastrie inoculation (n = 4) with PRCV were challenged with TGEV. Whereas TGEV-immune pigs were completely protected against challenge, all PRCV-immune pigs showed serological evidence of TGEV replication. Nevertheless, the aerosol or intragastric inoculation with PRCV primed the humoral immune system against TGEV and the TGEV challenge induced a secondary antibody response in most PRCV-immune pigs. Furthermore, all PRCV-immune pigs showed a decrease in the duration of the excretion of infectious TGEV (0-4 days) in comparison with the duration of the virus excretion by seronegative pigs (5-6 days). Abstract We consider the mathematical model for the viral dynamics of HIV-1 introduced in Rong et al. (2007) [37]. One main feature of this model is that an eclipse stage for the infected cells is included and cells in this stage may revert to the uninfected class. The viral dynamics is described by four nonlinear ordinary differential equations. In [37], the stability of the infected equilibrium has been analyzed locally. Here, we perform the global stability analysis using two techniques, the Lyapunov direct method and the geometric approach to stability, based on the higher-order generalization of Bendixson's criterion. We obtain sufficient conditions written in terms of the system parameters. Numerical simulations are also provided to give a more complete representation of the system dynamics. Abstract Feline infectious peritonitis (FIP) is a feline coronavirus (FCoV)-induced fatal disease in wild and domestic cats. There are two FCoV serotypes. Both type I and II FCoV can replicate in Felis catus whole fetus (fcwf)-4 cells, but the replicability of type I FCoV in feline cell lines is lower than that of type II FCoV, the reason for which is unclear. Inhibition of IFNβ production by non-structural and structural proteins, excluding spike protein has been reported in many coronavirus infections. In this study, we investigated whether IFNβ is involved in the difference in replicability in feline cell lines between types I and II FCoV. When fcwf-4 cells were infected with FCoV, the virus titer of type II FCoV in the culture supernatant was higher than that of type I FIPV. When the IFNβ expression level in FCoV-infected fcwf-4 cells was semi-quantitatively analyzed, infection with type I FIPV, excluding type I FIPV UCD-1, highly induced IFNβ expression. In contrast, induction of IFNβ by type II FCoV infection was significantly lower than that by type I FIPV. In addition, when fcwf-4 cells were adsorbed by FIPV and then stimulated with Poly(I:C), type II FCoV infection inhibited Poly(I:C)-induced IFNβ gene expression. Also, the proliferation of type I FIPV was enhanced by a IFN inhibitor. These findings clarified that, unlike type I FIPV, type II FCoV strongly inhibits IFNβ expression in infected cells. It was also suggested that the IFNβ-inducing ability is different among type I FIPV strains. Abstract of the long tracts of the spinal cord by macrophages. The clinical and morphological findings of 8 AIDS-patients with vacuolar myelopathy are reported here. The syndrome developed during the final stages of AIDS and was associated with HI V-encephalopathy in 5 cases. The vacuoles were mainly due to intramyelinic swelling and vacuolation. Vacuolated macrophages and axons contributed only to a minor degree. In one case only, HIV-antigens were detected immunohistochemical/yo The results are discussed in the light of modern pathogenetical concepts of HIV-related diseases. Abstract Radiology in low-and middle-income (developing) countries continues to make progress. Research and international outreach projects presented at the 2015 annual RAD-AID conference emphasize important global themes, including (1) recent slowing of emerging market growth that threatens to constrain the advance of radiology, (2) increasing global noncommunicable diseases (such as cancer and cardiovascular disease) needing radiology for detection and management, (3) strategic prioritization for pediatric radiology in global public health initiatives, (4) continuous expansion of global health curricula at radiology residencies and the RAD-AID Chapter Network's participating institutions, and (5) technologic innovation for recently accelerated implementation of PACS in low-resource countries. Abstract Applying a combination of semi-nested PCR and immunohistology (IHC), the presence of exogenous feline leukemia virus infection was studied in 302 necropsied cats with various disorders. 9% showed the classical outcome of persistent productive FeLV infection which was represented by FeLV antigen expression in different organs. 152 cats (50%) harboured exogenous FeLV-specific proviral sequences in the bone marrow but did not express viral antigen. These cats were considered as horizontally but non-productively infected.Statistical evaluation showed a significant association of non-productive horizontal FeLV infection with a variety of parameters. Non-productively infected cats were statistically significantly older and more often originated from animal shelters than cats without exogenous FeLV infection. Furthermore, some pathological disorders like anemia, panleukopenia, and purulent inflammation showed significant association with non-productive FeLV infection. No significant association was found with lymphosarcoma, known for a long time to be induced by productive FeLV infection. Abstract Glucocorticoid (GC) usage is the most common non-traumatic cause of osteonecrosis of the femoral head (ON). Despite the strong association of GC with ON, the underlying mechanisms have been unclear. Investigators have proposed both direct and indirect effects of GC on cells. Indirect and direct mechanisms remain intimately related and often result in positive feedback loops to potentiate the disease processes. However, the direct effects, in particular apoptosis, have recently been shown to be increasingly important. Suppression of osteoblast and osteoclast precursor production, increased apoptosis of osteoblasts and osteocytes, prolongation of the lifespan of osteoclasts and apoptosis of endothelial cells (EC) are all direct effects of GC usage. Elevated blood pressure through several pathways may raise the risk of clot formation. High-dose GC also decreases tissue plasminogen activator activity (t-PA) and increases plasma plasminogen activator inhibitor-1 (PAI-1) antigen levels increasing the procoagulant potential of GC. Inhibited angiogenesis, altered bone repair and nitric oxide metabolism can also result. Also, GC treatment modulates other vasoactive mediators such as endothelin-1, noradrenalin and bradykinin. Thus, GCs act as a regulator of local blood flow by modulating vascular responsiveness to vasoactive substances. Vasoconstriction induced in intraosseous femoral head arteries causes femoral head ischemia. GCs also cause ischemia through increased intraosseous pressure, which subsequently decreases the blood flow to the femoral head by apoptosis of ECs as well as elevating the level of adipogenesis and fat hypertrophy in the bone marrow.It is difficult to predict which patients receiving a specific dose of GC will develop ON, indicating individual differences in steroid sensitivity and the potential of additional mechanisms. The textbook model of ON is a multiple hit theory in which, with a greater number of risk factors, the risk of ON increases. While more effort is needed to better comprehend the role of GC in ON, newer data on GC action upon the endothelial cell and the regional endothelial bed dysfunction theory sheds new light on particular GC mechanisms. Better understanding of GC pathomechanisms can lead to better treatment options. Abstract When unexpected diseases such as the severe acute respiratory syndrome (SARS) and avian influenza become a serious threat to public health, an immediate response is imperative. This should take into consideration existing licensed antiviral drugs against other viral diseases already known to be safe for use in humans. In this report, evidence is presented that HIV-1 protease inhibitors (PIs) currently used in anti-HIV-1 therapies might exert some effects on SARS and perhaps, on avian influenza. Evidence for the potential benefits of PIs against the SARS coronavirus (SARS-CoV) is provided by empirical clinical studies, in vivo viral inhibition assays and computational simulations of the docking of these compounds to the active site of the main SARS-CoV protease. As suggested by in silico docking of these molecules to a theoretical model of a subunit of type A influenza virus RNA-dependent RNA polymerase, there also exists a remote possibility that these PIs may have an effect on avian influenza viruses. Although this evidence is still far from being definitive, the results so far obtained suggest that PIs should be seriously taken into consideration for further testing as potential therapeutic agents for SARS and avian influenza. Abstract Urban Emergency Response System (UERS) is a modernization symbol of a city. With acceleration of urbanization process and constant expansion of city size in China, China cities must respond to various emergencies timely and effectively to satisfy urban residents' needs for public security. In recent years, many China cities made trials and efforts in setting up and improving the UERS. At the same time, the China government began to build Emergency Response Systems (ERS) in some cities to deal with various possible emergencies. In this paper, using Petri net (PN), we study the performance of China typical UERS and establish its PN model for performance analysis. Based on the Markov chain (MC) of the model, the performance of China typical UERS is analyzed. Results from our simulation are in conformity with practical operation of China current UERS. Abstract Please cite this article as: D.A. Patel, A.M. Akinsete, J. de la Fuente, A.A. Kassim, Haploidentical bone marrow transplant with posttransplant cyclophosphamide for sickle cell disease: An update, Hematology/Oncology and Stem Cell Therapy (2020), doi: https://doi.Hematopoietic cell transplant (HCT) can cure both children and adults with sickle cell disease. Outcomes have historically been poor for the vast majority of patients who lack a matched sibling donor. However, the development of haploidentical HCT (haplo-HCT) with high doses of posttransplant cyclophosphamide (PTCy) has allowed for curative long-term potential with favorable transplantrelated outcomes, though this has not obviated the potential for graft rejection from human leukocyte antigen mismatch and repeated 2 red blood cell transfusions. Accordingly, multiple strategies have been developed to improve outcomes, the majority of which are based on the Johns Hopkins platform from 2012. Presently, we aim to discuss results from pertinent studies and compare outcomes with the two most recent approaches involving either thiotepa plus 200-cGy total body irradiation or 400-cGy total body irradiation.Direct comparisons are required to determine the optimized curative potential. Transplant-eligible patients must be referred to tertiary medical centers for consideration of haplo-HCT. Abstract Background: Viral upper respiratory tract infection (vURI) may or may not present with a cold/flu-like illness (CFLI). Objectives: For common upper respiratory viruses that cause vURIs, to determine the relative frequencies of virus detection by PCR in subjects with and without CFLIs. Study design: Prospective follow-up of 170 children aged 1-8.6 years through the CFLI season by daily parental diary for CFLI episodes and nasal secretion sampling using PCR assays for adenovirus, coronavirus (types 229E and OC43), influenza virus (types A and B), parainfluenza (types 1-3) virus, rhinovirus, and respiratory syncytial virus (RSV). Results: Virus was detected in 415 of 956 independent assays: 425 CFLI episodes and 531 non-CFLI periods were sampled; samples from 270 (64%) CFLI episodes and 145 (27%) non-CFLI periods contained virus detected by PCR. Rhinovirus was most frequently detected at 64%, followed by mixed viruses at 12%, RSV at 7%, and the other viruses at 3-5% of all detections. About 85% of RSV, influenza A and adenovirus detections were associated with a CFLI, whereas less than 62% of other virus detections were associated with CFLI. Conclusions: The frequency of PCR virus detection without CFLI was different among viruses. This introduces virus-specific biases to estimating the frequencies of specific complications attributable to a vURI when ascertained by CFLI identification. Abstract An immunocompetent toddler came to medication attention with gastroenteritis, complicated by encephalopathy and hepatitis. Multiplexed testing using a polymerase chain reaction meningitis panel was positive for human herpesvirus 6 (HHV-6). Clinical correlation, quantitative HHV-6 polymerase chain reaction, and metagenomic next-generation sequencing supported a likely diagnosis of primary HHV-6B infection. (J Pediatr 2019;212:228-31). Abstract Global health enables the harmonisation of international and domestic-health concerns-its outlook is much wider than a development or foreign-assistance perspective alone. Engaging globally in health requires the creation of relevant and eff ective partnerships to implement solutions for shared or common problems. To build on the UK's achievements and leadership in global health, the central government Department of Health is now leading the development of a UK Government-wide global strategy. This paper describes the rationale and process for developing the new UK Government-wide strategy for global health and highlights some of the issues that must be discussed. Abstract In the Netherlands a major part of preparedness planning for an epidemic or pandemic consists of maintaining essential public services, e.g., by the police, fi re departments, army personnel, and healthcare workers. We provide estimates for peak demand for healthcare workers, factoring in healthcare worker absenteeism and using estimates from published epidemiologic models on the expected evolution of pandemic infl uenza in relation to the impact on peak surge capacity of healthcare facilities and intensive care units (ICUs). Using various published scenarios, we estimate their effect in increasing the availability of healthcare workers for duty during a pandemic. We show that even during the peak of the pandemic, all patients requiring hospital and ICU admission can be served, including those who have non-infl uenza-related conditions. For this rigorous task differentiation, clear hierarchical management, unambiguous communication, and discipline are essential and we recommend informing and training non-ICU healthcare workers for duties in the ICU. I n the Netherlands a major part of preparedness planning for an epidemic or pandemic, e.g., avian infl uenza A, consists of maintaining essential services provided by the police, fi re departments, army personnel, and healthcare workers (HCWs). Even if an effective vaccine against avian infl uenza (H5N1) would be available (1), preparation for a pandemic is still vital to maintain optimal care for acutecare patients and those with infl uenza-like illness (ILI). The preparation for excess workloads among HCWs becomes even more important with the emergence of highly pathogenic avian infl uenza strains.We present a model to show the impact of the increased demand in HCWs with the increase in the number of hospi-talized patients. We factor in the notion that the number of HCWs will be reduced because of increased absenteeism, which in turn affects the utilization of intensive-care beds and mechanical ventilation capacity. We present scenarios aiding in temporarily increasing the work force of HCWs in the intensive-care unit (ICU) environment using different additive strategies. Because the surge capacity of intensive-care resources is typically limited (2), we explore what training and preparation HCWs and managers at different levels will need to face the challenges posed by pandemic infl uenza.MethodsThe University Medical Center Groningen (UMCG) is a tertiary-care university hospital covering ≈12% of the total Dutch population and ≈30% of the total surface area of the Netherlands. Under Dutch law, UMCG plays a dominant role in the region to organize and coordinate healthcare surge capacity during a catastrophe such as an avian infl uenza pandemic. With regional and municipal health authorities, general practitioners, and medical and managerial representatives of all 15 hospitals in the northern Netherlands region, training courses were organized for pandemic infl uenza. These courses emphasized the need for enhanced collaboration, sharing of information, and communication. Part of this training course was the development of an epidemiologic model to access the regional impact of a pandemic and the extent of possible preparations (3) at both managerial and medical domains.We used FluSurge 2.0 (4) and a computer model in an Excel (Microsoft, Redmond, WA, USA) fi le developed by one of the authors to calculate the impact of an infl uenza Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ¶ I -24-310-D-20Urgenze I -24-310-D-20 ¶ Bronchiolite del lattante Abstract surrounded by his devoted wife Margaret and his 5 beloved children. He talked about his children constantly, even in academic presentations in front of thousands of strangers. Jay always said, "Humor is an important tool in the practice of medicine. In teaching, I use it to engage the learner; in practice, it creates a relationship between me and the patient, which levels the playing field and puts them at ease." To clinical colleagues, he was the go-to clinician for tropical medicine cases; to trainees, he was the master professor to whom those from all over Canada came to work and learn; and to audiences worldwide, he was a marquee attraction for his legendary humor and wit (1). His clever political incorrectness never failed to augment the delivery of the key points on tropical and emerging exotic diseases. Abstract This short-term observational study of infection control practice was performed in the medical emergency outpatient department (EMOPD) of a tertiary-care hospital in India when threatened by an outbreak of severe acute respiratory syndrome (SARS). An investigator attended the lobby daily to screen patients with symptoms for SARS. Patient/attendant load, patient flow, medical staff working practices and position in the EMOPD were observed. Infection control measures such as fumigation and cleaning were noted, as was the EMOPD laboratory function, use of personnel protection and display of information on infectious diseases. A total of 162 (7.4%) of the 2165 patients surveyed had respiratory symptoms but no cases of SARS were found. The flow of patients and their attendants was not systematic. No laboratory tests for SARS were available, and no educational material on SARS was displayed. The EMOPDs in key hospitals need be able to screen for infectious diseases, especially in view of the threats from SARS and Avian influenza. Abstract The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE, Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice. Abstract Graphical Abstract Highlights d High-contiguity genome assembly for Rousettus aegyptiacus, a host of Marburg virus d Expansion of NK cell receptors, MHC class I genes, and type I interferons d Genetic signatures of unique signaling in NK cell receptors in multiple bats d Enhanced infection tolerance may be an antiviral defense strategy in bats Abstract In a recent report by risk assessment experts on the identification of food safety priorities using the Delphi technique, foodborne viruses were recognized among the top rated food safety priorities and have become a greater concern to the food industry over the past few years. Food safety experts agreed that control measures for viruses throughout the food chain are required. However, much still needs to be understood with regard to the effectiveness of these controls and how to properly validate their performance, whether it is personal hygiene of food handlers or the effects of processing of at risk foods or the interpretation and action required on positive virus test result. This manuscript provides a description of foodborne viruses and their characteristics, their responses to stress and technologies developed for viral detection and control. In addition, the gaps in knowledge and understanding, and future perspectives on the application of viral detection and control strategies for the food industry, along with suggestions on how the food industry could implement effective control strategies for viruses in foods. The current state of the science on epidemiology, public health burden, risk assessment and management options for viruses in food processing environments will be highlighted in this review. Abstract In the past decade, Asia has been actively engaged in human genomic studies and has made great contributions to the field. There is an increase in the number of genomics institutes, consortiums, and initiatives across the continent to study the association between genetic variation and disease. Despite these laudable efforts, Asia faces tremendous challenges in terms of funding, regulation, collaboration, and ethical, legal, and social issues related to genomics. These need to be addressed in the near future to promote the development of genomic medicine. Abstract Human Bocavirus (HBoV) is a novel virus which can cause respiratory tract disease in infants or children. In this study, the codon usage bias and the base composition variations in the available 11 complete HBoV genome sequences have been investigated. Although, there is a significant variation in codon usage bias among different HBoV genes, codon usage bias in HBoV is a little slight, which is mainly determined by the base compositions on the third codon position and the effective number of codons (ENC) value. The results of correspondence analysis (COA) and Spearman's rank correlation analysis reveals that the G + C compositional constraint is the main factor that determines the codon usage bias in HBoV and the gene's function also contributes to the codon usage in this virus. Moreover, it was found that the hydrophobicity of each protein and the gene length are also critical in affecting these viruses' codon usage, although they were less important than that of the mutational bias and the genes' function. At last, the relative synonymous codon usage (RSCU) of 44 genes from these 11 HBoV isolates is analyzed using a hierarchical cluster method. The result suggests that genes with same function yet from different isolates are classified into the same lineage and it does not depend on geographical location. These conclusions not only can offer an insight into the codon usage patterns and gene classification of HBoV, but also may help in increasing the efficiency of gene delivery/expression systems. Abstract The 3 0 end of a primer is a key component of PCR primer design. Many recommendations for the composition and sequence of the 3 0 end have been suggested based on theoretical considerations, but have not been verified experimentally. We analyzed 3 0 end triplets of PCR primer sequences obtained from refereed journal articles, to test those recommendations and to make empirical recommendations for primer design. The frequencies of the 64 possible 3 0 end triplets among 2137 PCR primers from the VirOligo database were not uniformly distributed. From the analysis, we found that unfavored and preferred 3 0 end triplets existed, and that the apparent preferences were not due to base compositions in viral genome sequences. Comparison of the sequences preferred by practitioners to those recommended, suggested that no single recommendation is entirely satisfactory. We suggest that recommendations be replaced with a scoring system incorporating empirical frequencies such as those reported here. q Abstract Zoonoses are infections in humans transmitted by animal pathogens or animal infections transmitted to humans. Viruses are the main etiological agents of emerging or re-emerging zoonoses. This chapter will discuss the most relevant foodborne and waterborne viral zoonotic infections along with their specifi c etiological agent, the issue of global and local infections, climate change, clinical manifestations and epidemiology and possible control and prevention measures. Abstract Rhinovirus (RV) is a frequent pathogen in young children, eliciting symptoms ranging from common colds to wheezing illnesses and lower respiratory tract infections. The recently identified RV-C seems to be associated with asthma exacerbations and more severe disease, but results vary. We studied the prevalence and severity of infection with RV in an unselected birth cohort. Children with respiratory symptoms entered the symptomatic arm of the cohort and were compared with asymptomatic children. Severity of wheezing and other respiratory symptoms was registered. Respiratory viruses were evaluated using throat and nasopharyngeal swabs on first presentation and after recovery (wheezing children). RV genotyping was performed on RV-PCR positive samples. RV was the most prevalent respiratory virus and was found in 58/140 symptomatic children (41%), 24/96 (25%) control children and 19/74 (26%) wheezing symptomatic children after recovery (p <0.05) and did not differ between wheezing and non-wheezing symptomatic childrendrespectively, 42% (38/90) and 40% (20/50). RV-A was the most commonly detected species (40/68, 59%), followed by RV-C (22/68, 32%) and RV-B (6/68, 9%). RV-B was more frequently detected in asymptomatic children (5/6, p <0.05). There was no significant difference in the frequency of RV species between wheezing and non-wheezing symptomatic children. Children with RV mono-infection had more severe symptoms, but no association between RV species and severity of disease was seen. In an unselected birth cohort from the Netherlands with mild respiratory disease RV was the most prevalent respiratory virus. RV(-C) infection was not associated with more severe disease or wheezing. J.G. Wildenbeest, CMI 2016;22:736.e9e736.e15 Abstract Objective: Lung transplantation is therapeutic for end-stage lung disease, but survival is limited due to bronchiolitis obliterans syndrome and restrictive chronic lung allograft dysfunction. We sought a common denominator in lung transplant recipients, analyzing risk factors that trigger immune responses that lead to bronchiolitis obliterans syndrome.We collected blood from patients who underwent lung transplant at our institution. Exosomes were isolated from the sera of recipients with risk factors for chronic rejection and from stable recipients. Exosomes were analyzed with western blot, using antibodies to lung self-antigens K alpha 1 tubulin and collagen-V, costimulatory molecules (costimulatory molecule 80, costimulatory molecule 86), transcription factors (nuclear factor kappa-light-chain-enhancer of activated B cells, hypoxia-inducible factor 1a, Class II Major Histocompatibility Complex Transactivator), and 20S proteasome. Abstract The 3C-like protease (3CL pro ) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays key roles in viral replication and is an attractive target for anti-SARS drug discovery. In this report, a fluorescence resonance energy transfer (FRET)-based method was developed to assess the proteolytic activity of SARS-CoV 3CL pro . Two internally quenched fluorogenic peptides, 1NC and 2NC, corresponding to the N-terminal and the C-terminal autocleavage sites of SARS-CoV 3CL pro , respectively, were used as substrates. SARS-CoV 3CL pro seemed to work more efficiently on 1NC than on 2NC in trans-cleavage assay. Mutational analysis demonstrated that the His41 residue, the N-terminal 7 amino acids, and the domain III of SARS-CoV 3CL pro were important for the enzymatic activity. Antibodies recognizing domain III could significantly inhibit the enzymatic activity of SARS-CoV 3CL pro . The effects of class-specific protease inhibitors on the trans-cleavage activity revealed that this enzyme worked more like a serine protease rather than the papain protease. Abstract Recently, amiloride was shown to potently suppress Coxsackievirus B3 (CVB3) replication. In the current study, we investigated whether amiloride could also exhibit antiviral activity against foot-and-mouth disease virus (FMDV), which belongs to the same family (Picornaviridae) as CVB3. We found that amiloride exerted antiviral activity in a dose-dependent manner against two strains of FMDV in IBRS-2 cells, with slight cytotoxicity at 1000 μM. Besides, amiloride did not inhibit the attachment and entry of FMDV in IBRS-2 cells, but prevented early viral replication. These data implied that amiloride could be a promising candidate for further research as a potential antiviral drug against FMDV infection. Abstract CONTRIBUTORS FUNDAMENTALS CH. 7 ENVIRONMENTAL AND SAFETY ISSUES WITH NANOPARTICLES FUNDAMENTALS CH. 7 ENVIRONMENTAL AND SAFETY ISSUES WITH NANOPARTICLES 396 Figure 7.2.10Size distribution of nanoparticles generated from a laser printer. Abstract To improve understanding of severe group A streptococcal (GAS) disease in Europe and achieve an integrated picture of these infections, the Strep-EURO project comprising 11 countries started in September 2002. A European case definition for severe invasive GAS disease was agreed upon amongst all participants. In order to obtain nation-wide coverage, information on the project was given to infectioncontrol units, relevant hospitals and microbiological laboratories. A clinical questionnaire containing background identification data, possible predisposing factors, portal of entry of infection, epidemiological risk factors, source of isolate, clinical condition, complications and outcome, was used for each case. Enhanced surveillance of GAS invasive disease commenced on 1 January 2003 for a two-year period. By July 2004, over 5000 invasive GAS isolates were collected of which more than 3000 were from UK. The disease incidence rate in the different countries seems to vary from 2 to 3.8 per 100,000 population. Molecular methods for typing and clonal identification of clinical isolates were used. An overall increase of new invasive types is noted. Higher emm types (77, 78, 81, 82, 83, 87, 89) were thus reported from almost all partners, in contrast to types 1, 3 and 28 being predominant for several years. In Sweden, for example, emm types 81 and 89 accounted for a majority of isolates. This unusual change of type distribution in Europe is of serious concern. A high level of MLS antibiotic resistance in some countries (France, Italy) and very frequent tetracycline resistance in almost all countries was noted. Furthermore, pathogenic aspects of severe GAS disease, such as proteolytic activation of the contact system, and selected virulence factors, such as surface proteins and superantigens, were investigated. A new Strep-EURO website (www.strep-euro.lu.se) was constructed and updated regularly. The website is linked with other, relevant websites and contains both an "intra-net" site accessible to those persons participating within the project, and an "extra-net" site for the general public. A central database for severe GAS cases, receiving clinical and laboratory information from the local databases was also established. Strep-EURO has now created a European network for epidemiological analysis and surveillance of severe streptococcal disease in ten EU and one Associated Countries.Objectives: Strep-EURO is an EU FP-5 funded programme designed to greatly enhance our understanding of severe group A streptococcal (GAS) infections. We present early epidemiological findings gathered by the UK team.Methods: Enhanced population-based surveillance was implemented in the UK in part fulfilment of the strep-EURO objectives. Two pre-existing data sources were reconciled to maximise case ascertainment: routine national surveillance based on local microbiological investigation; isolate referral to the national reference laboratory. Individual cases were followed-up to capture clinical and epidemiological information. Preliminary data relating to cases diagnosed in 2003 were analysed to determine the burden, clinical presentation and risk factors for disease and microbiological characteristics of strains.Results: A total of 2064 cases were reported by 223 laboratories across England and Wales, an overall incidence of 3.93 per 100,000 population. A clear seasonal pattern was apparent, with peak incidence from mid-March to -April. The observed incidence in males was higher than in females (RR = 1.29; 95% CI: 1.18-1.41). Twelve percent of cases occurred in children (<18 years). Ninety percent of cases were bacteraemic, 10% had septic arthritis and 9% pneumonia. Other clinical manifestations included toxic shock-like syndrome (6%), necrotising fasciitis (5%), puerperal sepsis (3%) and meningitis (1%). Skin lesions/wounds were the most common predisposing factor, with injecting drug use reported in 30% of cases; 28% had no predisposing/risk factors identified. Nineteen percent of cases were reported as having died within 7 days of diagnosis. Local microbiological analysis identified 4% of GAS isolates as erythromycin resistant. Type characterisation of strains using a combination of serological and sequencing techniques revealed over 35 M-types, with M1, M3 and M87most common.Conclusion: Enhanced surveillance of severe GAS infections is beginning to yield information of value in targeting public health initiatives. Our estimation of disease burden has been substantially improved, as has our understanding of the role of specific risk factors such as injecting drug use. Comparison with other European countries will further our understanding of these data.The objectives of our study were to create an animal model of aerosol infection with Coxiella burnetiiphase I (Nine Mile strain) and to determine the pathogenic potential of the organism in C57BL/6 mice exposed to an infectious aerosol with an inoculum of 2 × 10 9 bacteria. Mice (N = 15) were inoculated using an aerosolisation device for 45 min. Five mice were sacrificed at day 0, 7 and 14 of the study. C. burnetii detection was performed by Light Cycler Nested PCR assay in lungs and blood samples. Antibodies to phases I and II C. burnetii whole cell antigens were determined in the sera by immunofluorescence testing. Inflammatory reactions in lungs and spleen samples were investigated by histological and immunohistological analysis. C. burnetii was detected by genomic amplification in the blood of mice sacrificed at day 7 and in all lung samples. All infected mice sacrificed at day 14 had developed antibodies to C. burnetii phases I and II antigens (titer of 1:1600). Inflammatory infiltrates were seen in the lungs of mice sacrificed at day 7 and 14, bacteria were detected by immunohistochemistry in the macrophages cells. In our study, we demonstrated that the infection first resulted in an interstitial pneumonia. C. burnetii was then released and may spread hematogenously to other organs. As a conclusion, this murine model of aerosol-induced experimental Q fever is the first step for studies on acute Q fever, especially in terms of pathogenicity. Abstract Several tiny organisms of various size ranges present in air are called airborne particles or bioaerosol which mainly includes live or dead fungi and bacteria, their secondary metabolites, viruses, pollens, etc. which have been related to health issues of human beings and other life stocks. Bio-terror attacks in 2001 as well as pandemic outbreak of flue due to influenza A H1N1 virus in 2009 have alarmed us about the importance of bioaerosol research. Hence characterization i.e. identification and quantification of different airborne microorganisms in various indoor environments is necessary to identify the associated risks and to establish exposure threshold. Along with the bioaerosol sampling and their analytical techniques, various literatures revealing the concentration levels of bioaerosol have been mentioned in this review thereby contributing to the knowledge of identification and quantification of bioaerosols and their different constituents in various indoor environments (both occupational and non-occupational sections). Apart from recognition of bioaerosol, developments of their control mechanisms also play an important role. Hence several control methods have also been briefly reviewed. However, several individual levels of efforts such as periodic cleaning operations, maintenance activities and proper ventilation system also serve in their best way to improve indoor air quality. Abstract CMNEEJ 11(9)65-72,1989Elsevier 01%-4399/89/$0.00 + 02.20 Abstract Emerging infectious diseases represent a major challenge to human health worldwide. The risk of evolving new infectious pathogens has been intensifying due to urbanization, demographic changes, air travel, inappropriate use of antibiotics, and climate change. These pathogens can affect humans from urban centers to the remotest corners of the globe. Far from being a scourge of the past, infectious diseases are relevant for the world today. Abstract The development of information technology highly affects various business sectors including Small and Medium Enterprises (SMEs). Social media implementation, as one of the most widely used IT application, is expected to support SMEs' performances. Many types of research in social media have focused on the impact of its implementation on the performance of an organization at a macro level. Research that investigates the linkage between social media and how the company performs its business process is lacking in the literature to date. This study aims to fill this gap by conducting a literature review on social media implementation particularly in SMEs. The result of this study is a conceptual model that follows the proposition of Task-Technology Fit in exploring the impact of social media on SMEs business process performances. As a technology, social media functionality must fit the tasks that SMEs must perform. The model proposed social media alignment with business process domain based on the Process Classification Framework (PCF). The performances are measured at a business process level using four indicators i.e. cost, time, quality and flexibility of the business process.The development of information technology highly affects various business sectors including Small and Medium Enterprises (SMEs). Social media implementation, as one of the most widely used IT application, is expected to support SMEs' performances. Many types of research in social media have focused on the impact of its implementation on the performance of an organization at a macro level. Research that investigates the linkage between social media and how the company performs its business process is lacking in the literature to date. This study aims to fill this gap by conducting a literature review on social media implementation particularly in SMEs. The result of this study is a conceptual model that follows the proposition of Task-Technology Fit in exploring the impact of social media on SMEs business process performances. As a technology, social media functionality must fit the tasks that SMEs must perform. The model proposed social media alignment with business process domain based on the Process Classification Framework (PCF). The performances are measured at a business process level using four indicators i.e. cost, time, quality and flexibility of the business process. Abstract Member of the dynamin family of large GTPases, dynamin-related protein 1 (Drp1) dependent mitochondrial fission is an intricate process regulating both cellular and organ dynamics. Present study shows that NNV perturbs mitochondrial dynamics by promoting Drp-1 dependent mitochondrial fission, which attenuates MAVS mediated downstream signaling. NNV infected SISS cells revealed induction in Drp1 expression and subsequent translocation into mitochondria. The level of MAVS expression was up-regulated over a period of 24 hpi and declined with the progression of NNV infection at 48 and 72 hpi confirmed by western blot and mRNA transcript analysis. Drp-1 displayed its association with fragmented mitochondria and the transcript abundance was significant post infection along with Mff. Expression levels of IRF-3 IFN-1 and Mx followed a similar pattern with abundant expression at 48 hpi and diminished expression during the further period. Importantly, silencing of Drp1 caused significant elevation in the RLR downstream molecules and reduction in viral RNA expression. These results suggest that NNV-induced mitochondrial fission serve to attenuate host RLR signaling. This provides an illustration of host-pathogen interaction in which the virus evades innate immunity by enhancing mitochondrial fission and perturbs MAVS, and the downstream molecules. Abstract Despite a general decline in late life suicide rates over the last 30 years, older people have the highest rates of suicide in most countries. In contrast, non-fatal suicidal behaviour declines with age and more closely resembles suicide than in younger age groups. There are difficulties in the detection and determination of pathological suicidal ideation in older people. Multiple factors increase suicide risk ranging from distal early and mid-life issues such as child abuse, parental death, substance misuse and traumatic life experiences to proximal precipitants in late life such as social isolation and health-related concerns. Clinical depression is the most frequently identified proximal mental health concern and in many cases is a first episode of major depression. Recent studies have identified changes on neuroimaging and neurocognitive factors that might distinguish suicidal from non-suicidal depression in older people. Strategies for suicide prevention need to be 'whole of life' and, as no single prevention strategy is likely to be successful alone, a multi-faceted, multi-layered approach is required. This should include optimal detection and management of depression and of high risk individuals as available evidence indicates that this can reduce suicidal behaviour. How best to improve the quality of depression management in primary and secondary care requires further research. Abstract Myelin basic protein (MBP) appears frequently in the cerebrospinal fluid (CSF) of mice with chronic demyelination following intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV); antibody to MBP can frequently be found in the sera. The peaks of the immune responses to both MBP and TMEV coincide with the time course of the clinical signs of disease. Adsorption of mouse sera with TMEV or MBP indicate the non-identity of the antigens and the specificity of the antisera as measured by ELISA. Immunoblot analysis of sera confirmed the ELISA findings. The mechanism of induction of antibody directed against MBP and its role in TMEV-associated demyelination remain to be determined. Abstract The aim of this study was to compare the efficacy and patient discomfort between four techniques for obtaining nasal secretions. Nasal secretions from 58 patients with symptoms of a common cold, from three clinical centers (Amsterdam, Lodz, Oslo), were obtained by four different methods: swab, aspirate, brush, and wash. In each patient all four sampling procedures were performed and patient discomfort was evaluated by a visual discomfort scale (scale 1-5) after each procedure. Single pathogen RT-PCRs for Rhinovirus (RV), Influenza virus and Adenovirus, and multiplex real-time PCR for RV, Enterovirus, Influenza virus, Adenovirus, Respiratory Syncytial Virus (RSV), Parainfluenza virus, Coronavirus, Metapneumovirus, Bocavirus and Parechovirus were performed in all samples. A specific viral cause of respiratory tract infection was determined in 48 patients (83%). In these, the detection rate for any virus was 88% (wash), 79% (aspirate), 77% (swab) and 74% (brush). The degree of discomfort reported was 2.54 for swabs, 2.63 for washes, 2.68 for aspirates and 3.61 for brushings. Nasal washes yielded the highest rate of viral detection without excessive patient discomfort. In contrast, nasal brushes produced the lowest detection rates and demonstrated the highest level of discomfort. Abstract Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory disease among calves in the Danish cattle industry. An experimental BRSV infection model was used to study the pathogenesis of the disease in calves. Broncho alveolar lung lavage (BAL) was performed on 28 Jersey calves, of which 23 were experimentally infected with BRSV and ®ve were given a mock inoculum. The presence of the cytokine tumor necrosis factor a (TNF-a) in the BAL¯uids was detected and quanti®ed by a capture ELISA. TNF-a was detected in 21 of the infected animals. The amount of TNF-a in the BAL¯uid of calves killed post inoculation day (PID) 2 and 4 was at the same very low level as in the uninfected control animals. Large amounts of TNF-a were detected on PID 6, maximum levels of TNF-a were reached on PID 7, and smaller amounts of TNF-a were seen on PID 8. The high levels of TNF-a appeared on the days where severe lung lesions and clinical signs were obvious and the amounts of BRSV-antigen were at their greatest. Although Pasteurellaceae were isolated from some of the BRSV-infected calves, calves treated with antibiotics before and through the whole period of the infection, as well as BRSV-infected calves free of bacteria reached the same level of TNF-a as animals from which bacteria were isolated from the lungs. It is concluded that signi®cant quantities of TNF-a are produced in the lungs of the calves Veterinary Immunology and Immunopathology 76 (2000) 199±214Abbreviations: PID, post inoculation day; BRSV, bovine respiratory syncytial virus; Mock, mock inoculum; BAL, broncho alveolar lung lavage; TNF-a, tumor necrosis factor-a Abstract Multiple sclerosis (MS) and a number of related distinctive diseases are characterized by the active degradation of central nervous system (CNS) myelin, an axonal sheath comprised essentially of proteins and lipids. These demyelinating diseases appear to arise from complex interactions of genetic, immunological, infective, and biochemical mechanisms. While circumstances of MS etiology remain hypothetical, one persistent theme involves recognition by the immune system of myelin-specific antigens derived from myelin basic protein (MBP), the most abundant extrinsic myelin membrane protein, and/or another equally susceptible myelin protein or lipid component. Knowledge of the biochemical and physical-chemical properties of myelin proteins and lipids, particularly their composition, organization, structure, and accessibility with respect to the compacted myelin multilayers, thus becomes central to the understanding of how and why these antigens become selected during the development of MS. This review focuses on current understanding of the molecular basis underlying demyelinating disease as it may relate to the impact of the various protein and lipid components on myelin morphology; the precise molecular architecture of this membrane as dictated by protein-lipid and lipid-lipid interactions; and the relationship, if any, between the protein/lipid components and the destruction of myelin in pathological situations. Abstract The emergence of new infectious bronchitis virus (IBV) genotypes or serotypes along with the poor crossprotection observed among IBV serotypes have complicated the avian infectious bronchitis (IB) control programs in different geographic regions. In Cuba, the lack of genetic information regarding IBV and the increasing epidemiological importance of this virus in Cuban chicken flocks demand further characterization of IBV isolates. In the present work, studies of genetic diversity and phylogenetic relationships among recent IBV isolates from Cuban chicken flocks showing respiratory disorders were performed. Two putative genotypes genetically different to the Massachusetts genotype H120 strain used in the Cuban vaccination program were found in the flocks assessed. In addition, a potential nephropathogenic IBV isolate was found by first time in Cuba. Abstract Aspartame The synthetic low-calorie sweetener produced from L-phenylalanine and L-aspartic acid, having the structure L-phe-L-asp-methyl ester. BOTOX, BoNT, botulinum toxin The neurotoxin produced by Clostridium botulinum, responsible for food poisoning, which includes a zinc-dependent metalloprotease. Chymosin The predominant milk-clotting enzyme from the true stomach or abomasum of the suckling calf. Directed evolution Method used in protein engineering to harness the power of Darwinian selection to evolve proteins with desirable properties. Endoproteases Proteases that attack internal peptide bonds in the peptide chain remote from the C-or N-terminal. GRAS list A list of microorganisms used in fermentation and food processes, which are 'generally regarded as safe' by the US Food and Drug Administration. Heterologous protein production Synthesis of foreign proteins in a host organism following transformation of that organism by a vector carrying genes from a different organism. k-Casein in cheese coagulation k-Casein serves to maintain the micelles in milk in suspension. The cheese-making protease, rennet, hydrolyzes a single specific peptide bond in the k-casein fraction of milk resulting in destabilization of the milk micelles and coagulation. Protease engineering Techniques for creation of proteases with new or artificial amino acid sequences. Proteasome A large multicomponent barrel-like protease complex that digests a variety of proteins into short polypeptides and amino acids. Subtilisin A serine endopeptidase initially characterized from B. subtilis, but secreted in large amounts from many Bacillus species.Proteases represent a large and diverse group of hydrolytic enzymes that are classified by their site of action, enzyme active site structure, and specific reaction mechanisms. Proteases are ubiquitous in biology where they have a biochemical and/or physiological involvement in many aspects of cell and organism function, including nutrition, protein turnover, growth, adaptation, regulation,Comprehensive Biotechnology, 3rd edition, Volume 3 sporulation and germination, disease, and death. Proteases regulate much of what occurs in the human body including the essential cellular functions of differentiation, motility, division, and cell death. Proteases participate physiologically in intracellular protein-degradation processes using several systems, including lysosomes (membrane-bound organelles containing proteases) and the more recently characterized proteasome system, consisting of very large protein barrel-like stacked four-ringed complexes, the outer rings of which function as gates to proteins entering the central pore, while the interior surfaces of the inner rings contain the proteolytic active sites. 9 Proteasome proteolytic mechanisms involve an active site threonine, and involve three different chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing substrate specificities. Proteolysis is exploited physiologically for covalent activation, regulation, and inhibition of enzymes and other protein-based or linked effectors, for example, in the biochemical cascade leading to blood clotting and in production of insulin. Proteases remove signal sequences of peptides following their transport/secretion through membranes and they remove N-terminal methionine residues after translation.Animal, plant, and, especially, microbial proteases represent the largest and most important segment of an industrial enzyme market where they are used in detergents, in food processing, in the leather industry, as biocatalysts in organic synthesis, and as therapeutics. Because proteases also function as causative agents of some diseases, protease inhibitors have also been developed as a class of therapeutics. Commercial fermentation processes for production of microbial proteases were developed from about the 1950s. Some of the workhorse protease-producing strains, including Aspergillus, Rhizopus, and Bacillus species, were the same species as were found in traditional fermented food fermentations where their proteases participated in the degradation of proteins and associated development of distinct flavors in solid mash substrates containing soy beans, cereal grains, and other plant-based food materials. The protease production fermentation processes and conditions were optimized through characterization and control of factors affecting microbial growth and enzyme production. Overproducing strains were isolated through extensive screening processes and were further improved through application of elaborate mutation/selection procedures. Developments in genetic engineering, including the ability to express recombinant proteins in different host organisms and the ability to manipulate and enhance transcription, translation, secretion, and other processes, have been exploited to enhance protease production and underlining the technoeconomics of their use. The ability to engineer proteins to modify properties, such as kinetics, specificity, and stability, has been applied to improve or expand on protease applications. Random and site-directed mutagenesis and other techniques have been particularly useful in designing, constructing, and characterizing the biocatalytic and stability properties of wholly new protease structures.The estimated size of the industrial enzyme market is about $2.5-3 billion per year, which is made up of enzymes used in food processing ($$800 million), enzymes for animal feed ($$400 million), and nonfood/feed enzyme applications ($1.4-1.7 billion). Companies reported to have the largest share of the enzyme market are Novozymes ), with respective approximate market shares of 41%-44%, 21%, and 8%. Some Japanese Companies including Shin Nihon continue to produce and commercialize enzymes using the solid culture (koji) process (http://www.aichi-brand.jp/corporate/type/chemical/shin-nihon-e. html). The Amano Enzyme Group produces a more specialized range of enzymes with major applications in biotransformations, diagnostics, and as dietary supplements (http://www.amano-enzyme.co.jp/aeu/product/presentation.html). The indicated internet sites and others provide valuable information on the principal applications of proteases as well as on new research, technology, and links to technical reports and publications. Other companies mainly based in Asia, Europe, and North America account for the residual approximately 27%-30%. Proteases are thought to account for about 60% of the total enzyme market or $1.5-1.8 billion per year. Among protease applications, the most dominant use is in the detergents accounting for sales of alkaline protease of approximately $1 billion per year.Evidence of the importance of proteases in industry may also be gleaned from a simple search of granted US patents (1976-July 2010) and patent applications (2001-July 2010) on the US Patent and Trademark Office (USPTO) site (http://patft.uspto.gov/ netahtml/PTO/search-bool.html) as is indicated in Table 1 . It should be noted that there is some overlap in the data since patents applied for and granted since 2001 will be counted under both headers. 'Protease' was used as the initial 'term' in the search, which was separately applied to the fields 'abstract' and 'claims'. In a second search, the search term 'protease inhibitor' was used. The presumption is that the difference between these two numbers, that is, proteases but not 'protease inhibitors', provides a good indication of numbers of granted patents and applications involving proteases. In the past 10 years, nearly 6000 patents have been filed with claims dealing with protease.Examples of some selected recent patents (2008-10) related to proteases and protease inhibitors issued by the US Patent Office are listed in Tables 2 and 3, respectively. Notable among the protease patents are strain-and detergent-related proteases, animal-feed Abstract Investigations into the suspected airborne transmission of pathogens in healthcare environments have posed a challenge to researchers for more than a century. With each pathogen demonstrating a unique response to environmental conditions and the mechanical stresses it experiences, the choice of sampling device is not obvious. Our aim was to review bioaerosol sampling, sampling equipment, and methodology. A comprehensive literature search was performed, using electronic databases to retrieve English language papers on bioaerosol sampling. The review describes the mechanisms of popular bioaerosol sampling devices such as impingers, cyclones, impactors, and filters, explaining both their strengths and weaknesses, and the consequences for microbial bioefficiency. Numerous successful studies are described that point to best practice in bioaerosol sampling, from the use of small personal samplers to monitor workers' pathogen exposure through to large static samplers collecting airborne microbes in various healthcare settings. Of primary importance is the requirement that studies should commence by determining the bioefficiency of the chosen sampler and the pathogen under investigation within laboratory conditions. From such foundations, sampling for bioaerosol material in the complexity of the field holds greater certainty of successful capture of lowconcentration airborne pathogens. From the laboratory to use in the field, this review enables the investigator to make informed decisions about the choice of bioaerosol sampler and its application. ª Abstract the cerebrospinal fluid (CSF) were < 10 % of plasma levels.After oral administration AM concentrations were considerably lower than after i.m. administration. The concentration ofDHA was high on day 1 but almost nil on day 7 indicating its fast inactivation in dogs. Two hours after the 8 th oral administration neither AM nor DHA was detected in CSF which may explain the absence of neurotoxicity in dogs after oral administration of AM. Abstract Infection of cell monolayers by murine coronavirus A59 at pH 6 rather than 7 yielded a ten-fold increase in the infectious titer and a remarkable enhancement of the reactivities of monoclonal and polyclonal antibodies against the spike glycoprotein in immunoblotting, immunoprecipitation and enzyme-linked immunosorbent assays. These observations are very useful for detecting antibodies against the S glycoprotein of coronaviruses and enhancing infectious titers. Abstract Eight accessory proteins have been identified in severe acute respiratory syndrome-associated coronavirus (SARS-CoV). They are believed to play roles in the viral life cycle and may contribute to the pathogenesis and virulence. ORF9b as one of these accessory proteins is located in subgenomic mRNA9 and encodes a 98 amino acid protein. However, whether 9b protein is a structural component of SARS-CoV particles remains unknown. In this study, we demonstrate that 9b protein is translated from bicistronic mRNA9 via leaky ribosome scanning and it is incorporated into both virus-like particles (VLPs) and purified SARS-CoV virions. Further analysis shows that sufficient incorporation of 9b protein into VLPs is dependent upon the coexpression of E and M proteins, but not upon the presence of either S or N protein. Our data indicate that 9b protein of SARS-CoV is another virion-associated accessory protein. This finding will lead to a better understanding of the properties of the SARS-CoV 9b protein. Abstract Immunocompetent, but not RAG1 −/− mice infected with MHV-JHM develop demyelination. Transferred CD8 T cell-enriched splenocytes reconstitute demyelination, and this ability is dependent on donor IFN-γ. We used IFN-γR1 −/− mice to examine the target of IFN-γ in CD8 T cellmediated demyelination. In IFN-γR1 −/− RAG1 −/− recipients, demyelination is decreased, but not eliminated, while viral titers are significantly increased when compared to IFN-γR1 +/+ RAG1 −/− recipients. IFN-γR1 −/− CD8 T cells retain virus-specific effector function regardless of IFN-γR1 expression. Although IFN-γR1 responsiveness is critical for maximal demyelination, increased levels of infectious virus coupled with adoptive transfer of CD8 T cells may result in myelin destruction independent of IFN-γR1 expression. Abstract Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by porcine epidemic diarrhea virus (PEDV). However, when PEDV, TGEV and porcine pseudorabies virus were incubated with peptide H (HVTTTFAPPPPR), only infection of Vero cells by PEDV was inhibited. Immunofluoresence assays indicated that inhibition of PEDV infection by peptide H was independent of pAPN. Western blots demonstrated that peptide H interacted with PEDV spike protein and that pretreatment of PEDV with peptide H led to a higher inhibition than synchronous incubation with cells.These results indicate direct interaction with the virus is necessary to inhibit infectivity. Temperature shift assays demonstrated that peptide H inhibited pre-attachment of the virus to the cells. Abstract Background: Organizations are stockpiling respirators to prepare for an influenza pandemic. To understand better the effects of prolonged storage, this investigation evaluated the filtration efficiency of 21 different models of National Institute for Occupational Safety and Health (NIOSH)-certified disposable N95 filtering face piece respirators. These respirators had been stored in their original packaging for a period of at least 6 years in research laboratories and dry warehouse facilities, ranging in temperature between 158C and 328C and relative humidity between 20% and 80%. Methods: Filter penetration was measured using an abbreviated version of the NIOSH respirator certification test incorporating a polydisperse sodium chloride aerosol at 85 L/min. Results: Of the 21 respirator models tested, 19 models had both average penetration results of less than 5%. Mean initial penetration values ranged from 0.39% to 5.83%, whereas mean maximum penetration values ranged from 0.95% to 5.83%. There did not appear to be any correlation between the length of storage and failure to pass the filtration test. Conclusion: Results indicate that most N95 filtering face piece respirators stored for up to 10 years at warehouse conditions will likely have expected levels of filtration performance and that the degree of filtration efficiency degradation is likely model specific. Abstract Hepatitis C is a serious health issue and cause liver disorders in millions of people. Available therapeutic agents require long term administration with numerous side effects. Therefore, there is a dire need to find alternative treatment options for this disease. Since ancient times, medicinal plants are widely used to cure various diseases with no or less harmful effects. Therefore, this study was designed to find out phytochemicals and investigate antiviral activity of methanol extract of Ajuga bracteosa, Ajuga parviflora, Berberis lycium and Citrus lemon against Hepatitis C Virus (HCV infection). Phytochemical analysis of the plant extract was performed using various chemical tests. Toxicity of the plant extract was determined against using trypan blue exclusion method. Antiviral activity of the selected plant extract was find out against HCV infected HepG2 cells. For this purpose, HepG2 cells were seeded with HCV positive and negative serum and nontoxic doses of plant extract for 24 and 48 h. After this RNA was extracted and viral load was determined using Real-time PCR. Phytochemical analysis showed the presence of flavonoids and phenols in all plant extracts while amino acids, alkaloids and tannins were present in B. lycium and saponins were detected in C. lemon. Toxicity assay showed that all plant extracts were nontoxic at maximum concentration of 200 μg/ml except B. lycium, which showed mild toxicity at 40 μg/ml and were extremely toxic at 60 μg/ml and above doses. Real-time PCR quantitation result revealed that after 24 h treatments A. parviflora showed highest antiviral activity, followed by A. bracteosa, while B. lycium extract had low (35%) and C. lemon has no antiviral effects. The 48 h treatments showed an increase antiviral activity by A. bracteosa followed by A. parviflora and B. lycium while C. lemon showed negative effect. Our results depicted that mentioned plants might be used as an alternative therapeutic regime or in combination with existing treatments against HCV. Abstract Enzyme inhibition Molecular target Structure activity relationship a b s t r a c tThe emergence of disease and dearth of effective pharmacological agents on most therapeutic fronts, constitutes a major threat to global public health and man's existence. Consequently, this has created an exigency in the search for new drugs with improved clinical utility or means of potentiating available ones. To this end, accumulating empirical evidence supports molecular target therapy as a plausible egress and, b-glucuronidase (bGLU) e a lysosomal acid hydrolase responsible for the catalytic deconjugation of b-D-glucuronides has emerged as a viable molecular target for several therapeutic applications. The enzyme's activity level in body fluids is also deemed a potential biomarker for the diagnosis of some pathological conditions. Moreover, due to its role in colon carcinogenesis and certain drug-induced dose-limiting toxicities, the development of potent inhibitors of bGLU in human intestinal microbiota has aroused increased attention over the years. Nevertheless, although our literature survey revealed both natural products and synthetic scaffolds as potential inhibitors of the enzyme, only few of these have found clinical utility, albeit with moderate to poor pharmacokinetic profile. Hence, in this review we present a compendium of exploits in the present millennium directed towards the inhibition of bGLU. The aim is to proffer a platform on which new scaffolds can be modelled for improved bGLU inhibitory potency and the development of new therapeutic agents in consequential. Abstract a b s t r a c t Climate change and peak oil are likely to have a significant impact on future tourism growth in the Asia Pacific region. Dealing with these issues and the policies and strategies required for mitigation and adaptation need to be given far greater attention by the tourism industry and the public sector than has hitherto been the case. Existing approaches based on crisis and disaster management may be inadequate and a new approach to deal with shocks of this nature is required. This regional spotlight suggests a new approach based on the concept of disruption which is defined as an event that causes substantial and long-term change in the structure of the tourism industry. Abstract Coronavirus 3C-like protease (3CLpro) is responsible for the cleavage of coronaviral polyprotein 1a/1ab (pp1a/1ab) to produce the mature non-structural proteins (nsps) of nsp4-16. The nsp5 of the newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as 3CLpro and its canonical cleavage sites (between nsps) were predicted based on sequence alignment, but the cleavability of these cleavage sites remains to be experimentally confirmed and putative non-canonical cleavage sites (inside one nsp) within the pp1a/1ab awaits further analysis. Here, we proposed a method for predicting coronaviral 3CLpro cleavage sites which balances the prediction accuracy and false positive outcomes. By applying this method to MERS-CoV, the 11 canonical cleavage sites were readily identified and verified by the biochemical assays. The Michaelis constant of the canonical cleavage sites of MERS-CoV showed that the substrate specificity of MERS-CoV 3CLpro is relatively conserved. Interestingly, nine putative non-canonical cleavage sites were predicted and three of them could be cleaved by MERS-CoV nsp5. These results pave the way for identification and functional characterization of new nsp products of coronaviruses. Abstract . The green turtle, Chelonia mydas. Images of uninfected green turtles (courtesy of Jack Lenz). Abstract Research on respiratory protection against biologic agents is important to address major concerns such as occupational safety and terrorist attack. This review describes the literature on respiratory protection against bioaerosols and identifies research gaps. Respiratory protection is a complex field involving a number of factors, such as the efficiency of respirator filter material; facepiece fitting; and maintenance, storage, and reuse of respirators. Several studies used nonpathogenic microorganisms having physical characteristics similar to that of Mycobacterium tuberculosis to analyze microbial penetration through respirators. Some studies showed that high-efficiency particulate air (HEPA) and N95 filters provided a higher level of protection than dust/mist (DM) and dust/mist/fume (DMF) filters. Flow rate and relative humidity appear to alter the level of penetration of microorganisms through respirator filters. The relationship between microbial penetration through respirator filters and the aerodynamic diameter, length, or other physical characteristics of microorganisms remains controversial. Whether reaerosolization of bioaerosol particles should be a concern is unclear, given the fact that one study has demonstrated significant reaerosolization of 1-to 5-mm particles loaded onto respirator filters. Respirator maintenance, storage, and decontamination are important factors to be considered when reusing respirators. The respiratory protection against biologic warfare agents such as anthrax in military and civilian situations is described. (Am J Infect Control 2004;32:345-54.) Abstract Rotavirus (RV) is an important cause of gastrointestinal disease in animals and humans. In this study, we developed an RT-PCR to detect RV group B (RVB) and characterized the VP7 (G) gene segment detected in porcine samples. One hundred seventy three samples were tested for RV group A (RVA), RVB, and C (RVC) by RT-PCR and examined for RV-like lesion using histopathology. A majority (86.4%) of the samples had mixed RV infections and co-infections of RVA/RVB/RVC were detected at a higher rate (24.3%) than previously reported. RVB was identified in 46.8% of the 173 samples. An adapted VP7 classification was developed using previously published (n ¼ 57) and newly sequenced (n ¼68) RVB strains, resulting in 20 G genotypes based on an 80% nucleotide identity cutoff value. Our results revealed a broad genetic diversity of porcine RVB strains, suggesting RVB has been the cause of common/pre-existing, yet undiagnosed, disease in pigs. Abstract We previously developed a respiratory tract vaccine candidate against Ebola virus (EBOV) based on human parainfluenza virus type 3 (HPIV3), a respiratory paramyxovirus, expressing the EBOV GP envelope protein (HPIV3/GP) from an added gene. Two doses of this vaccine candidate delivered by the intranasal and intratracheal route protected monkeys against intraperitoneal challenge with EBOV; however, concerns exist that the vaccine candidate may have reduced immunogenicity in the adult human population due to pre-existing immunity against HPIV3. Here we developed a new vaccine candidate (NDV/GP) based on Newcastle disease virus (NDV), an avian paramyxovirus that is antigenically distinct from human viral pathogens and is highly attenuated in monkeys. Following one intranasal and intratracheal inoculation of Rhesus monkeys with NDV/GP, titers of EBOV-specific antibodies in respiratory tract secretions and serum samples determined by ELISA, as well as serum EBOV-neutralizing antibodies, were undetectable or low compared to those induced by HPIV3/GP. A second immunization resulted in a substantial boost in serum IgG ELISA titers, yet the titers remained lower than those induced by a second dose of HPIV3/GP. In contrast, the ELISA IgA titers in respiratory tract secretions and, more importantly, the serum EBOVneutralizing antibody titers were equal to those induced after the second dose of HPIV3/GP. These data suggest that NDV/GP can be effective for immunization against EBOV alone, or in combination with either HPIV3/GP or another vaccine platform in a heterologous prime-boost regimen. Abstract We report here the identification of a 24-kDa polypeptide in IBV-infected Vero cells by immunoprecipitation with a region-specific antiserum raised in rabbits against the IBV sequence encoded between nucleotides 10928 and 11493. Coexpression, deletion, and mutagenesis studies have demonstrated that this protein is encoded by ORF 1a from nucleotide 10915 to 11544 and is released from the 1a polyprotein by the 3C-like proteinase-mediated proteolysis. A previously predicted Q-S (Q 3462 S 3463 ) dipeptide bond encoded by the IBV sequence from nucleotide 10912 to 10917 is identified as the N-terminal cleavage site, and a Q-N (Q 3672 N 3673 ) dipeptide bond encoded by the IBV sequence between nucleotides 11542 and 11547 is identified as the C-terminal cleavage site of the 24-kDa polypeptide. Abstract Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R 2 ) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections. Abstract Dengue virus (DENV) emerged from the sylvatic environment and colonized urban settings, being sustained in a human-Aedes-human transmission chain, mainly by the bites of females of the anthropophilic species Aedes aegypti. Herein, we sought evidence for fine-tuning in viral codon usage, possibly due to viral adaptation to human transmission. We compared the codon adaptation of DENV serotype 2 (DENV-2) genotypes from urban and sylvatic habitats and tried to correlate the findings with key evolutionary determinants. We found that DENV-2 codons of urban and sylvatic genotypes had a higher CAI to humans than to Ae. aegypti. Remarkably, we found no significant differences in codon adaptation to human between urban American/Asian and sylvatic DENV-2 genotypes. Moreover, CAI values were significantly different, when comparing all genotypes to Ae. aegypti codon preferences, with lower values for sylvatic than urban genotypes. In summary, our findings suggest the presence of a molecular signature among the genotypes that circulate in sylvatic and urban environments, and may help explain the trafficking of DENV-2 strains to an urban cycle. Abstract e Abstract-Mycoplasma pneumoniae is one of the most common known bacterial pathogens of the respiratory tract, especially in patients between 5 and 30 years of age. It may be encountered at a relatively high rate in the non-life-threatened fraction of Emergency Department (ED) patients presenting with upper respiratory symptoms or cough. Yet its hallmarks are very non-specific, including a great variety of presentations from mild pharyngitis to potentially life-threatening complications such as the Stevens-Johnson Syndrome. Here, we describe a typical case of pneumonia due to Mycoplasma pneumoniae in a young adult with mild pharyngitis as the leading symptom. Disease presentation, complications, diagnostic means, therapeutic options, and suspicious clinical settings are discussed to provide a review on the clinical aspects of the disease that are important in the ED setting. © 2006 Elsevier Inc. e Keywords-atypical pneumonia; upper airway infection; pharyngitis; Mycoplasma pneumoniae Clinical Communications (Adults) is coordinated by Ron Walls, MD, Abstract The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Idris AM, Briddon RW, Bull SE, and Brown JK (2005) Cotton leaf curl Gezira virus-satellite DNAs represent a divergent, geographically isolated Nile Basin lineage: Predictive identification of a satDNA REP-binding motif. Virus Research 109: 19-32. Mansoor S, Briddon RW, Bull SE, et al. (2003) Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNA b. Archives of Virology 148: 1969-1986. Mansoor S, Khan SH, Bashir A, et al. (1999) Identification of a novel circular single-stranded DNA associated with cotton leaf curl disease in Pakistan. Virology 259: 190-199. Sanjaya VVS, Prasad V, Kirthi N, Maiya SP, Savithri HS, and Sita GL (2005) Development of cotton transgenics with antisense AV2 gene for resistance against cotton leaf curl virus (CLCuD) via Agrobacterium tumefaciens. Abstract Environmental disinfection with sodium hypochlorite and hydrogen peroxide vapor was performed after a hospital-associated outbreak of Middle East respiratory syndrome. Although only 11% of total beds were disinfected, the isolation and vancomycin-resistance rates of Enterococcus spp significantly decreased for 2 months, whereas other multidrug-resistant organisms did not. Abstract A 2-year-old neutered female Shiba dog exhibited laboured breathing for 1 month. Computed tomography of the thoracic cavity revealed multiple nodules (2e5 mm diameter) in the lungs. Grossly, the lungs were firm and normal in shape. The nodules were greyewhite in colour. Microscopically, the nodules were non-encapsulated and exhibited an irregular shape. They were composed of polygonal or spindle cells with indistinct cell borders arranged in sheets. The cells had large, round, hyperchromatic nuclei and abundant pale eosinophilic cytoplasm with no atypia. Intrapulmonary arterial emboli and infiltration into the bronchioles were observed. Immunohistochemically, the cells were positive for vimentin and negative for cytokeratin, glial fibrillary acidic protein and a-smooth muscle actin. Ultrastructurally, the cells displayed cytoplasmic processes, desmosomes and intermediate filaments. These findings led to a diagnosis of diffuse pulmonary meningotheliomatosis with sarcomatous transformation. To the best of our knowledge, this is the first report of diffuse pulmonary meningotheliomatosis in a dog. Abstract The amyloid precursor protein (APP), that plays a critical role in the development of senile plaques in Alzheimer disease (AD), and the gp41 envelope protein of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), are single-spanning type-1 transmembrane (TM) glycoproteins with the ability to form homo-oligomers. In this review we describe similarities, both in structural terms and sequence determinants of their TM and juxtamembrane regions. The TM domains are essential not only for anchoring the proteins in membranes but also have functional roles. Both TM segments contain GxxxG motifs that drive TM associations within the lipid bilayer. They also each possess similar sequence motifs, positioned at the membrane interface preceding their TM domains. These domains are known as cholesterol recognition/interaction amino acid consensus (CRAC) motif in gp41 and CRAC-like motif in APP. Moreover, in the cytoplasmic domain of both proteins other α-helical membranotropic regions with functional implications have been identified. Recent drug developments targeting both diseases are reviewed and the potential use of TM interaction modulators as therapeutic targets is discussed. Abstract A cascade reaction that involves a unique CeC bond cleavage has been discovered. This protocol affords an unusual and facile method for the synthesis of 1,3-oxazin derivatives under mild conditions. Abstract Alrstraet--Monoclonal antibodies have been produced against the 81/36F strain of rotavirus. One of them, was chosen as diagnostic reagent: it showed high ELISA reactivity with all the bovine, human and porcine rotavirus strains tested and reacted with VP6, structural protein product known to support the common rotavirus antigen.A sandwich ELISA procedure using the chosen monoclonal as "capture and detecting" antibody was performed to detect rotavirus in faecal samples from experimentally inoculated newborn calves: it always gave a negative response with meconium and a positive response for the stool specimens which rotavirus have been isolated. This assay was compared with Enzygnost and Slidex Rota Kit tests and with a non-commercial sandwich ELISA test using polyclonal antibodies: it showed more sensitivity than the agglutination test and was as sensitive as the other two tests to detect rotavirus in routine diagnostic material. The test evaluated showed no equivocal results. Abstract The mosquito-borne New World alphavirus, Venezuelan equine encephalitis virus (VEEV) is a Category B select agent with no approved vaccines or therapies to treat infected humans. Therefore it is imperative to identify novel targets that can be targeted for effective therapeutic intervention. We aimed to identify and validate interactions of VEEV nonstructural protein 3 (nsP3) with host proteins and determine the consequences of these interactions to viral multiplication. We used a HA tagged nsP3 infectious clone (rTC-83-nsP3-HA) to identify and validate two RNA helicases: DDX1 and DDX3 that interacted with VEEV-nsP3. In addition, DDX1 and DDX3 knockdown resulted in a decrease in infectious viral titers. Furthermore, we propose a functional model where the nsP3:DDX3 complex interacts with the host translational machinery and is essential in the viral life cycle. This study will lead to future investigations in understanding the importance of VEEV-nsP3 to viral multiplication and apply the information for the discovery of novel host targets as therapeutic options. Abstract Crimean-Congo hemorrhagic virus (CCHFV) causes hemorrhagic fever with high case mortality rates and is endemic in south-eastern Europe, Africa, and Asia. The limited catalog of specific treatment, highlight the necessity to look for additional therapeutic solutions.Previous experiments suggested that CCHFV enters the cells via a clathrin dependent pathway. Therefore, we have evaluated the potential anti-CCHFV activity of several molecules targeting this entry possibility. We identified two molecules chloroquine and chlorpromazine. Neutralization and virus yield reduction assays were tested in Vero E6 and Huh7 cells on two different CCHFV strains. Several combinations, including ribavirin, were assayed to test a potential synergistic effect.The two molecules inhibited CCHFV, and depending on the virus and the cell lines, the 50% inhibitory concentration (IC 50 ) values for chloroquine and chlorpromazine ranged from 28 to 43 and 10.8-15.7 lM, respectively. Time-of-addition studies demonstrated that these molecules had a direct effect on CCHFV infectivity and spread. The antiviral activity of the two molecules was still effective even when added up to 6 h post-infection and up to 24 h. The selectivity index ranging from 3 to 35 lead us to evaluate combinations with ribavirin. Combinations of ribavirin and chloroquine or chlorpromazine were synergistic against CCHFV. Though the low chlorpromazine selectivity index suggests the need for a chemical improvement, our present study highlights chloroquine as the main drug having the potential for drug repurposing. Abstract Background: Although the mini-clinical evaluation (mini-CEX) exercise has been adapted to a broad range of clinical situations, limited studies of the mini-CEX for postgraduate residency training in emergency medicine (EM) have been documented. Aim: The purpose of this study is to analyze the results of implementing the mini-CEX into the one-month postgraduate residency training in EM. Materials and methods: This study is a retrospective review of mini-CEXs completed by ED faculty members from August 2009 to December 2010. All PGY-1 residents enrolled in this study rotated through the one-month EM training. Each PGY-1 resident received one week of trauma training and three weeks of non-trauma training. The clinical competencies of each PGY-1 resident were evaluated with mini-CEXs, rated by a trauma surgeon and three emergency physicians (EPs). We analyzed the validity of weekly mini-CEX and the impact of seniority and specialty training of ED faculties on observation time, feedback time and rating scores. Results: Fifty-seven ED faculty members (42 EPs and 15 trauma surgeons) evaluated 183 PGY-1 residents during the 17 months of EM training. ED faculties with different specialty training provided similar assessment processes. Most competencies were rated significantly higher by trauma surgeons than by EPs. On the computerized mini-CEX rating, no data was missed and junior EPs rated all competencies significantly higher. The evaluators and PGY-1 residents were generally satisfied with the computerized format. As compared to the first assessment, only some competencies of PGY-1 residents were rated significantly higher in subsequent evaluations. Conclusion: The seniority and specialty training of ED faculty affected the mini-CEX ratings. The computer-based mini-CEX facilitated complete data gathering but showed differences for ED faculty with different levels of seniority. Further studies of the reliability and validity of the mini-CEX for PGY-1 EM training are needed. Abstract The features of the chemistry of 4-thiazolidinone and pyrazole/pyrazolines as pharmacologically attractive scaffolds were described in a number of reviews in which the main approaches to the synthesis of mentioned heterocycles and their biological activity were analyzed. However, the pyrazole/pyrazoline ethiazolidine-based hybrids as biologically active compounds is poorly discussed in the context of pharmacophore hybrid approach. Therefore, the purpose of this review is to summarize the data about the synthesis and modification of heterocyclic systems with thiazolidine and pyrazoline or pyrazole fragments in molecules as promising objects of modern bioorganic and medicinal chemistry. The description of biological activity was focused on SAR analysis and mechanistic insights of mentioned hybrids. Abstract Improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (RSV) are needed to aid the development and evaluation of novel intervention strategies. A quantitative real-time RT-PCR using specific locked nucleic acid (LNA) probes was developed to identify RSV and to distinguish RSV subgroups A and B (RSV LNA assay). RSV subgroup diversity and the relationship between viral load and disease severity in confirmed RSV infections were also explored. 264 archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex Seeplex TM RV detection kit (Seegene) and the novel RSV LNA assay. The LNA assay demonstrated a significantly higher sensitivity than Seeplex, improving overall detection rates from 24% (64/264) to 32% (84/264). Detection limits of 9.0 × 10 1 and 6.0 × 10 2 copies/mL were observed for RSV A and B, respectively. RSV A was detected in 53/84 (63%) cases, and 31/84 (37%) were positive for RSV B. This novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of RSV. Abstract Endoribonuclease-prepared siRNA (esiRNA) is an alternative tool to chemical synthetic siRNA for gene silencing. Since esiRNAs are directed against long target sequences, the genetic variations in the target sequences will have little influence on their effectiveness. The ability of esiRNAs to inhibit hepatitis B virus (HBV) gene expression and replication was tested. EsiRNAs targeting the coding region of HBV surface antigen (HBsAg) and the nucleocapsid (HBcAg) inhibited specifically the expression of HBsAg and HBcAg when cotransfected with the respective expression plasmids. Both esiRNAs reduced the HBV transcripts and replication intermediates in transient transfected cells and cells with HBV genomes integrated stably. Compared with synthetic siRNA, esiRNA targeting HBsAg was less effective than the selected synthetic siRNA in terms of the inhibition of HBV gene expression and replication. However, while the ability of synthetic siRNAs for specific gene silencing was impaired strongly by the nucleotide substitutions within the target sequences. The efficiency of gene silencing by esiRNAs was not influenced by sequence variation. The transfection of esiRNA did not induce interferon-stimulated genes (ISGs) like STAT1 and ISG15, indicating the absence of off-target effects. In general, esiRNAs strongly inhibited HBV gene expression and replication and may have an advantage against HBV strains which are variable genetically. Abstract Global sourcing strategy has been one of the most hotly debated management trends in the last 20 years. In its early years, global sourcing was examined mostly from ''in-house'' development and procurement perspectives; and in the last several years, research focus has shifted to ''outsourcing'' activities. Along with this shift from internal to external focus on global sourcing, many researchers and business practitioners have applied a core competency argument to justify increased levels of outsourcing activities on a global basis. Although the beneficial aspects of outsourcing are assumed in most cases, no consensus exists in reality as to the effect of outsourcing. Furthermore, the increased instability of the exchange rate environment in the last several years has also led to increased difficulties in managing globally scattered operations that were once fashionable in the 1980s-90s under the rubric of global strategy. In this article, the authors explore potential limitations and negative consequences of outsourcing strategy on a global scale. D Abstract http://creativecommons.org/licenses/by-nc-nd/4.0/ ) B. Poller, A. Tunbridge and S. Hall et al. / Journal of Infection 77 (2018) 496-502 497 made. At a workshop with an expert stakeholder group, the data were examined and a unified PPE ensemble agreed. This ensemble was then tested in the same simulation exercise and no evidence of any HCW contamination was seen after doffing. Following further review by the working group, a consensus agreement has been reached and a unified 'HCID assessment PPE' ensemble, with accompanying donning and doffing protocols, is presented here.Crown Abstract The pro-apoptotic properties of severe acute respiratory syndrome coronavirus (SARS-CoV) structural proteins were studied in vitro. By monitoring apoptosis indicators including chromatin condensation, cellular DNA fragmentation and cell membrane asymmetry, we demonstrated that the adenovirus-mediated over-expression of SARS-CoV spike (S) protein and its C-terminal domain (S2) induce apoptosis in Vero E6 cells in a time-and dosage-dependent manner, whereas the expression of its N-terminal domain (S1) and other structural proteins, including envelope (E), membrane (M) and nucleocapsid (N) protein do not. These findings suggest a possible role of S and S2 protein in SARS-CoV induced apoptosis and the molecular pathogenesis of SARS. Abstract The leukocyte-common antigen (L-CA) is a family of large molecular weight glycoproteins uniquely expressed on the surface of all nucleated cells of hematopoietic origin. The glycoprotein consists of a heavily glycosylated exterior domain, a single membrane spanning region, and a large cytoplasmic domain that contains tyrosine phosphatase activity. To investigate the function of this family, we generated T cell clones that lacked L-CA (L-CA-). The expression of the ap T cell receptor, CD3, CD4, IL-2 receptor (~55) LFA-1, Thy-l, and Pgp-1 (CD44) was normal. The L-CA T cell clones failed to proliferate in response to antigen or cross-linked CD3; however, they could still proliferate in response to 11-2. An L-CA+ revertant was obtained and the ability to proliferate in response to antigen and cross-linked CD3 was restored. These data indicate that L-CA is required for T cells to enter into cell cycle in response to antigen. Abstract This article reviews quantitative methods to estimate the basic reproduction number of pandemic influenza, a key threshold quantity to help determine the intensity of interventions required to control the disease. Although it is difficult to assess the transmission potential of a probable future pandemic, historical epidemiologic data is readily available from previous pandemics, and as a reference quantity for future pandemic planning, mathematical and statistical analyses of historical data are crucial. In particular, because many historical records tend to document only the temporal distribution of cases or deaths (i.e. epidemic curve), our review focuses on methods to maximize the utility of time-evolution data and to clarify the detailed mechanisms of the spread of influenza.First, we highlight structured epidemic models and their parameter estimation method which can quantify the detailed disease dynamics including those we cannot observe directly. Duration-structured epidemic systems are subsequently presented, offering firm understanding of the definition of the basic and effective reproduction numbers. When the initial growth phase of an epidemic is investigated, the distribution of the generation time is key statistical information to appropriately estimate the transmission potential using the intrinsic growth rate. Applications of stochastic processes are also highlighted to estimate the transmission potential using similar data. Critically important characteristics of influenza data are subsequently summarized, followed by our conclusions to suggest potential future methodological improvements. Abstract Traditional Chinese Medicine (TCM) is consisting of many practices that are common in East Asia. These practices have been around for more than 2000 years. Many of these methods are becoming increasingly popular in the Western world, where they are classified as alternative or complementary medicine. Most of these techniques are untested and some are even considered dangerous in the modern day world, however, they are sometimes able to help, where modern medicine can not. Therefore, it would be wrong to totally reject them. The SINOPOLL project is a joint R&D effort between Thor Medical Systems LLC and their Chinese partner Shenzhen Anke. Its goal is to combine the vast traditional knowledge in TCM with modern day medical system technology. This paper gives an overview of this research, as well, as proposes several possible solutions for common problems in this field.Keywords: Rehabilitation engineering and healthcare delivery, Decision support systems for the control of physiological and clinical variables, Pharmacokinetics and drug delivery Abstract Taiwan is a small, densely populated island with unique experiences in the construction and operation of incinerators. In such a small area, Taiwan has built 22 incinerators over a short span of time, combusting large amount of municipal solid waste as much as 23,250 tons per day. This study focuses on the history of construction and development of incinerators in Taiwan as well as the characteristics of pollutants, such as heavy metals (Pb, Cd, and Hg), acid gases (NO x , SO x , CO, and HCl), and dioxins emitted from the incinerators. Furthermore, the study also covers the generation and composition of municipal solid waste (MSW), and the production of energy in Taiwan. According to Taiwan's data on pollutant emissions, the emission level of pollutants is under control and meets the stringent regulations of Taiwan Environmental Protection Administration (TEPA). Researches have shown that using air pollution control devices (APCDs) in the operation of incinerators provides effective measures for air pollutant control in Taiwan. The main advantage of using incinerators is the generation of electricity (waste-to-energy) during the incineration of municipal solid waste, producing energy that can be consumed by the general public and the industry. Taiwan's extensive experience in incinerator construction and operation may serve as an example for developing countries in devising waste treatment technology, energy recovery, and the control of contagious viral diseases. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in a human with severe pneumonia in 2012. Since then, infections have been detected in >1500 individuals, with disease severity ranging from asymptomatic to severe, fatal pneumonia. To elucidate the pathogenesis of this virus and investigate mechanisms underlying disease severity variation in the absence of autopsy data, a rhesus macaque and common marmoset model of MERS-CoV disease were analyzed. Rhesus macaques developed mild disease, and common marmosets exhibited moderate to severe, potentially lethal, disease. Both nonhuman primate species exhibited respiratory clinical signs after inoculation, which were more severe and of longer duration in the marmosets, and developed bronchointerstitial pneumonia. In marmosets, the pneumonia was more extensive, with development of severe airway lesions. Quantitative analysis showed significantly higher levels of pulmonary neutrophil infiltration and higher amounts of pulmonary viral antigen in marmosets. Pulmonary expression of the MERS-CoV receptor, dipeptidyl peptidase 4, was similar in marmosets and macaques. These results suggest that increased virus replication and the local immune response to MERS-CoV infection likely play a role in pulmonary pathology severity. Together, the rhesus macaque and common marmoset models of MERS-CoV span the wide range of disease severity reported in MERS-CoVeinfected humans, which will aid in investigating MERS-CoV disease pathogenesis. (Am J Pathol 2016, 186: 630e638; http://dx. Abstract The humoral innate immune response consists of multiple components, including the naturally occurring antibodies (NAb), pentraxins and the complement and contact cascades. As soluble, plasma components, these innate proteins provide key elements in the prevention and control of disease. However, pathogens and cells with altered self proteins utilize multiple humoral components to evade destruction and promote pathogy. Many studies have examined the relationship between humoral immunity and autoimmune disorders. This review focuses on the interactions between the humoral components and their role in promoting the pathogenesis of bacterial and viral infections and chronic diseases such as atherosclerosis and cancer. Understanding the beneficial and detrimental aspects of the individual components and the interactions between proteins which regulate the innate and adaptive response will provide therapeutic targets for subsequent studies. Abstract Infectious HIV-1 requires gp160 cleavage by furin at the REKR 511 fl motif (site1) into the gp120/gp41 complex, whereas the KAKR 503 (site2) sequence remains uncleaved. We synthesized 41mer and 51mer peptides, comprising site1 and site2, to study their conformation and in vitro furin processing. We found that, while the previously reported 19mer and 13mer analogues represent excellent in vitro furin substrates, the present extended sequences require heparin for optimal processing. Our data support the hypothesis of a direct binding of heparin with site1 and site2, allowing selective exposure/accessibility of the REKR sequence, which is only then optimally cleaved by furin. Abstract Porcine epidemic diarrhea virus (PEDV) invades porcine intestinal epithelial cells (IECs) and causes diarrhea and dehydration in pigs. In the present study, we showed a suppression of PEDV infection in porcine jejunum intestinal epithelial cells (IPEC-J2) by an increase in autophagy. Autophagy was activated by rapamycin at a dose that does not affect cell viability and tight junction permeability. The induction of autophagy was examined by LC3I/LC3II conversion. To confirm the autophagic-flux (entire autophagy pathway), autophagolysosomes were examined by an immunofluorescence assay. Pretreatment with rapamycin significantly restricted not only a 1 h infection but also a longer infection (24 h) with PEDV, while this effect disappeared when autophagy was blocked. Co-localization of PEDV and autophagosomes suggests that PEDV could be a target of autophagy. Moreover, alleviation of PEDVinduced cell death in IPEC-J2 cells pretreated with rapamycin demonstrates a protective effect of rapamycin against PEDV-induced epithelial cell death. Collectively, the present study suggests an early prevention against PEDV infection in IPEC-J2 cells via autophagy that might be an effective strategy for the restriction of PEDV, and opens up the possibility of the use of rapamycin in vivo as an effective prophylactic and prevention treatment. Abstract The mammary glands represent one part of the mrrcosal immune system, a definable, subunit of humoral and cellular immune functions in man that appears to have developed particular qualities well suited to guard our inteflace with the environment. As our und~standing of secretory immunoglobulins and lymphocyte migration patterns continues to deveiop, the immunologic components found in breast milk appear increasingly hkely to play a spect& immunologic role in the protection of the nursing infant. The biologic basis for the observed protective effect of breast-feeding is reviewed with an emphasis on the mechanisms involved in the development and maintenance of mucosal immunity in general. (J .&LERGY C?trhi I&?MJhVL Abstract s u m m a r y Current explanations to the high 1918-1919 mortality involve either a higher pathogenicity of the virus or bacterial super-infection in the absence of adequate therapeutic resources. However, neither of these hypotheses accounts for the age-distribution of severe cases and deaths, or for the geographic and other variations in rates and explosiveness of mortality during the Pandemic. It will be shown here that, alternatively, the epidemiology of the influenza lethality could be completely explained by a combination of two determinants: (1) acquired immune-differentiation of birth-cohorts, within populations, through developmental epigenetic adaptation (and selection) secondary to maternal or early-life episodes of influenza infection and (2) a triggering context -emergence of a new sub-type/strain, and its co-circulation (competition?) with seasonal viruses immunologically related to ones that had circulated in the past and primed particular population birth-cohorts. This article (1) presents age, geographic, and temporal variations in 1918-1919 and 2009 influenza severity, (2) presents and discusses ecologic evidence in favor of the hypothesis to influenza lethality advanced here, (3) suggests biologic mechanisms capable of explaining it, (4) retrospectively, proposes co-circulation between the Pandemic and a 1918 seasonal (H3?) influenza virus as the context for the increased lethality during the second wave of the 1918 Pandemic, and (5) predicts an increase in influenza severity in the northern hemisphere as the 2009-2010 season advances and H3 circulation increases. Abstract Feline and canine coronaviruses (FCoV and CCoV, respectively) are common pathogens of cats and dogs sometimes leading to lethal infections named feline infectious peritonitis (FIP) and canine pantropic coronavirus infection. FCoV and CCoV are each subdivided into two serotypes, FCoV-I/II and CCoV-I/II. A phylogenetic relationship is evident between, on one hand, CCoV-I/FCoV-I, and on the other hand, CCoV-II/FCoV-II, suggesting that interspecies transmission can occur. The aim of the present study was to evaluate the prevalence of coronavirus (CoV)-infected cats according to their contact with dogs and to genetically analyse the CoV strains infecting cats. From 2003 to 2009, we collected 88 faecal samples from healthy cats and 11 ascitic fluids from FIP cats. We investigated the possible contact with dog in the household and collected dogs samples if appropriate. Out of 99 cat samples, 26 were coronavirus positive, with six cats living with at least one dog, thus showing that contact with dogs does not appear as a predisposing factor for cats CoV infections.Molecular and phylogenetic analyses of FCoV strains were conducted using partial N and S sequences. Six divergent strains were identified with the N gene clustering with CCoV-I whereas the 3 0 end of S was related to FCoV-I. Further analysis on those six samples was attempted by researching the presence of the ORF3 gene, the latter being peculiar to CCoV-I to date. We succeeded to amplify the ORF3 gene in five samples out of six. Thus, our data strongly suggest the circulation of atypical FCoV strains harbouring the CCoV-I ORF3 gene among cats. Moreover, the ORF3 genes recovered from the feline strains exhibited shared deletions, never described before, suggesting that these deletions could be critical in the adaptation of these strains to the feline host. Abstract We conducted a challenge/rechallenge trial in which 3 alpacas were infected with Middle East respiratory syndrome coronavirus. The alpacas shed virus at challenge but were refractory to further shedding at rechallenge on day 21. The trial indicates that alpacas may be suitable models for infection and shedding dynamics of this virus. Abstract Isolates of Escherichia coli which produce Vero cytotoxin (VTEC) were obtained during 1983-1989 from calves raised in 5 north-central states of the USA. All of the calves experienced intestinal epithelial colonization by VTEC, diarrhea or both; twelve of the calves had bloody diarrhea. Twenty one isolates were serogroup O111 and the others were O103, 069, 045, 026, 05, or non-typable (4 isolates ). All but one of the isolates hybridized with the CVD419 probe which identifies most VTEC strains. Thirty two isolates hybridized with the VTI probe, 3 with both the VT 1 and VT2 probes, and one with neither probe. The culture filtrate of the VT probe negative isolate was partially neutralized by SLT I monoclonal antibody. For the other isolates, the results of toxin neutralization by anti-SLT I and anti-SLT II monoclonal antibodies corresponded exactly with the VT 1 and VT2 probe hybridization results. Three of the strains adhered in a localized manner to HEp-2 cells and Intestine 407 cells. Abstract The ultimate goal of proteomics is to identify biologically active proteins and to produce them using biotechnology tools such as bacterial hosts. However, proteins produced by Escherichia coli must be refolded to their native state. Protein folding liquid chromatography (PFLC) is a new method developed in recent years, and it is widely used in molecular biology and biotechnology. In this paper, the new method, PFLC is introduced and its recent development is reviewed. In addition the paper includes definitions, advantages, principles, applications for both laboratory and large scales, apparatus, and effecting factors of PFLC. In addition, the role of this method in the future is examined. Abstract Infectious diseases due to microbes of high pathogenic potential remain a constant and variable threat for human and animal health. The emergence of new diseases or the re-emergence of diseases that were previously under control complicates the situation to date. Infectious disease research, which has undergone a dramatic progress in understanding disease mechanisms such as host-pathogen interactions, is now focusing increasingly on new strategies for prevention and therapy. Significant progress has been achieved in the development of delivery systems for protective heterologous protein antigens and in veterinary vaccinology. A landmark of infectious diseases research is the chemical synthesis of genomes, a major new field of research referred to as ''synthetic biology'', that to date has resulted in the chemical synthesis of the poliovirus and of phage fX174 genomes and their expression as infectious viruses. On the molecular level the evolution of pathogens and mechanisms of genome flexibility, which account for several pathogenic properties of infectious agents, have received increased attention. Bacterial toxins are an additional threat to human health and their interference with host cells and cellular functions is receiving more attention. Abstract Porcine reproductive and respiratory syndrome (PRRS) is one of the most challenging subjects of research in veterinary viral immunology, and the immune response against PRRS virus (PRRSV) still is poorly understood. Infected pigs develop a strong and rapid humoral response but these initial antibodies do not confer protection and can even be harmful by mediating an antibody-dependent enhancement of disease. In contrast, development of neutralising antibodies (NAs) is delayed and generation of cell-mediated immune responses, such as PRRSV-specific interferon (IFN)-c secreting cells, is initially erratic. In spite of this, induction of strong and rapid NAs and IFN-c responses seem to be required for effective vaccination. PRRSV strongly modulates the host's immune responses. The virus inhibits key cytokines, such as IFN-a, and may induce regulatory cytokines, such as interleukin (IL)-10. Development of NAs seems to be impaired by the existence of a decoy epitope close to the main neutralisation epitope in glycoprotein 5. This ability to modulate the host immune response probably varies among strains or isolates. The genetic diversity of the virus is very high and it has been shown that this diversity can have serious implications for the development of vaccines, since the immunity induced by one strain may be only partial against a different strain, even within the same genotype. With this panorama, the development of newer and universally efficacious PRRSV vaccines is challenging, but the present state of knowledge allows optimism if collaborative efforts are undertaken in the scientific community. Abstract Background: Long-term use of respiratory protection may be necessary, but compliance may be low, and physiologic effects have not been well evaluated. Methods: Ten nurses participated; physiologic effects, subjective symptoms, and compliance with wearing an N95 alone or with a surgical mask overlay were assessed. Longitudinal analysis based on multivariate linear regression models assessed changes in outcome variables (CO 2 , O 2 , heart rate, perceived comfort items, compliance measures, and others). Analyses compared changes over time, and compared wearing only an N95 to wearing an N95 with a surgical mask overlay. Results: Most nurses (90%, n ¼ 9) tolerated wearing respiratory protection for two 12-hour shifts. CO 2 levels increased significantly compared with baseline measures, especially when comparing an N95 with a surgical mask to only an N95, but changes were not clinically relevant. Perceived exertion; perceived shortness of air; and complaints of headache, lightheadedness, and difficulty communicating also increased over time. Almost one-quarter (22%) of respirator removals were due to reported discomfort. N95 adjustments increased over time, but other compliance measures did not vary by time. Compliance increased on day 2, except for adjustments, touching under the N95, and eye touches. Conclusion: Long-term use of respiratory protection did not result in any clinically relevant physiologic burden for health care personnel, although many subjective symptoms were reported. N95 compliance was fairly high. Abstract Lung diseases have shifted from infections e tuberculosis, pneumonia e to diseases of dirty air e chronic obstructive pulmonary disease, asthma and lung cancer. New diseases have emerged from industrial pollution and HIV, while better imaging has revealed others previously unrecognized. Scientific advances in microbiology, imaging and clinical measurement have improved diagnosis and allowed better targeted treatment. Advances in treatment have been dramatic, the most important being drugs (antibiotics, cortisone, b 2 -adrenoreceptor agonists), ventilatory support (from iron lung to nasal positive-pressure ventilation), inhaled therapy (metered dose inhalers, nebulizers) and lung surgery (resections, video-assisted thoracoscopic surgery, transplantation). Delivery of care has shifted from sanatoria for the rich but nothing at all for the poor, to hospitals and universal coverage. Generalists have turned into super-specialists and doctors have been joined by growing numbers of professions allied to medicine (PAMs). Management of lung disease has vastly improved but the impact of disease remains. Abstract Canine respiratory coronavirus (CRCoV) has been detected recently in dogs with canine infectious respiratory disease and is involved in the clinical disease complex. CRCoV is a group 2 coronavirus most closely related to bovine coronavirus and human coronavirus OC43. A real-time PCR assay was developed for the detection of CRCoV. The assay was validated against cell culture grown virus and shown to have a high level of sensitivity. A range of tissue samples were collected from dogs at a re-homing centre with a history of endemic respiratory disease. The samples were tested using a conventional nested PCR assay and CRCoV was quantitated by real-time PCR. CRCoV was detected most frequently in the nasal mucosa, nasal tonsil and trachea. It was also detected in the lung, and bronchial lymph node. Of the enteric tissues, only one mesenteric lymph node sample was positive. In addition two colon samples were positive for CRCoV by nested PCR only. In conclusion, CRCoV appears to infect the upper respiratory tract preferentially. The CRCoV real-time PCR assay has proved to be a highly specific and sensitive assay that can be applied for diagnostic purposes as well as to investigate further the tissue tropism of CRCoV. Abstract A. Pumfery et al.Proteomics of virusesTherefore, in this chapter we will discuss some recent findings on the proteomics of DNA and RNA viral infections that are associated with clinically important diseases in humans, including human cytomegalovirus (HCMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), hepatitis B and C (HBV and HCV, respectively), and adenovirus, as well as the coronavirus that causes severe acute respiratory syndrome (SARS).HCMV is the largest member of the human herpesviruses. After initial infection, HCMV remains in a persistent state with the host [2] . Immunity against the virus controls replication, although intermittent viral shedding can still take place in the seropositive immunocompetent person [2] . As replication of cytomegalovirus in the absence of an effective immune response is central to the pathogenesis of disease, complications are primarily seen in individuals whose immune system is immature or suppressed by drug treatment or coinfection with other pathogens [3] . Estimates of the coding capacity of HCMV range from 160 open reading frames (ORFs) to more than 200 ORFs [4] . Recent studies using MS to determine the viral proteome suggest that the number of viral proteins may be even greater than previous estimates [5] . Analysis of proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than the previously known 71 HCMV virion proteins. Twelve of the identified proteins were encoded by known viral ORFs previously not associated with virions, and 12 proteins were from novel viral ORFs [6] . Therefore, new protein markers including HCMV tegument and various cellular structural proteins, enzymes, and chaperones are now serving as biomarkers for HCMV infection and as possible drug targets.Other herpesvirus members have also been explored for the presence of possible biomarkers. EBV is a ubiquitous member of the herpesvirus family that is associated with a variety of lymphomas and lymphoproliferative diseases [7] . It encodes a multitude of genes that drive proliferation or confer resistance to cell death [8] . Infection of human B lymphocytes with EBV induces proliferative B-lymphoblastoid cell lines (LCLs). Recently, proteomic profiles of three LCLs were analyzed comparatively at the early and the late passages of cell culture. The phosphoprotein stathmin was identified, and expression significantly decreased with immortalization of LCLs [9]. Stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis [9] . In another study using standard matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methods, 20 EBNA2 target proteins were identified, 11 of which were c-myc dependent and therefore most probably associated with proliferation of the host cell [10]. These findings further stress the role of EBV viral proteins, namely EBNA and LMP, in disease pathogenesis. Interestingly, when EBV-infected cells were treated with the drug 5Ј-azacytidine (AZC)-a demethylating agent that induces the expression of silenced genes, i.e., the p16 tumor suppressor gene-21 polypeptides were down-regulated, while 312 A. Pumfery et al. Abstract Multiplex PCR has become the test of choice for the detection of multiple respiratory viruses in clinical specimens. However, there are few direct comparisons of different PCR assays. This study compares 4 different multiplex PCR assays for the recovery of common respiratory viruses. We tested 213 respiratory specimens using four different multiplex PCR assays: the xTAG respiratory viral panel fast (Abbott Molecular Laboratories), Fast-track Respiratory Pathogen assay (Fast-track Diagnostics), Easyplex respiratory pathogen 12 kit (Ausdiagnostics), and an in-house multiplex real-time PCR assay. The performance of the four assays was very similar, with 93-100% agreement for all comparisons. Other issues, such as through-put, technical requirements and cost, are likely to be as important for making a decision about which of these assays to use given their comparative performance. Abstract Foxp3 + regulatory T (Treg) cells limit inflammatory responses and maintain immune homeostasis. Although comprised of several phenotypically and functionally distinct subsets, the differentiation of specialized Treg cell populations within the periphery is poorly characterized. We demonstrate that the development of T-bet + Treg cells that potently inhibit T helper 1 (Th1) cell responses was dependent on the transcription factor STAT1 and occurred directly in response to interferon-g produced by effector T cells. Additionally, delayed induction of the IL-12Rb2 receptor component after STAT1 activation helped ensure that Treg cells do not readily complete STAT4-dependent Th1 cell development and lose their ability to suppress effector T cell proliferation. Thus, we define a pathway of abortive Th1 cell development that results in the specialization of peripheral Treg cells and demonstrate that impaired expression of a single cytokine receptor helps maintain Treg cell-suppressive function in the context of inflammatory Th1 cell responses.Immunity Abstract Fatigue is a symptom whose causes are protean and whose phenotype includes physical, mood, and behavioral components. Chronic fatigue syndrome (CFS) is an illness that has strong biological underpinnings and no definite etiology. Diagnostic criteria established by the Centers for Disease Control and Prevention have helped classify CFS as an overlap of mood, behavioral, and biological components. These include the presence of fatigue for more than 6 months associated with a diminution of functional activity and somatic symptoms, and pain not attributable to a specific diagnosis or disease. Four of the following criteria need to be present: sore throat, impaired memory or cognition, unrefreshing sleep, postexertional fatigue, tender glands, aching stiff muscles, joint pain, and headaches. Many researchers have observed that CFS shares features in common with other somatic syndromes, including irritable bowel syndrome, fibromyalgia, and temporomandibular joint dysfunction. Correlations between inflammation and infection, augmented sensory processing, abnormalities of neurotransmitters, nerve growth factors, low levels of serotonin and norepinephrine, abnormalities of homeostasis of the stress system, and autonomic dysfunction may be hallmarks of CFS. The relative contributions of each of these abnormalities to the profound fatigue associated with CFS need to be explored further to better evaluate and treat the syndrome. Abstract The stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-b receptor (LTbR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTbRsignaling in these cells was not essential for the formation of LNs. Although T cell zone reticular cells had lost podoplanin expression, they still formed a functional conduit system and showed enhanced expression of myofibroblastic markers. However, essential immune functions of FRCs, including homeostatic chemokine and interleukin-7 expression, were impaired. These changes in T cell zone reticular cell function were associated with increased susceptibility to viral infection. Thus, myofibroblasic FRC precursors are able to generate the basic T cell zone infrastructure, whereas LTbR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection.Immunity Abstract Over the past decade, microarrays have revolutionized the scientific world as dramatically as the internet has changed everyday life. From the initial applications of DNA microarrays to uncover gene expression patterns that are diagnostic and prognostic of cancer, understanding the interplay between immune responses and disease has been a prime application of this technology. More recent efforts have moved beyond genetic analysis to functional analysis of the molecules involved, including identification of immunodominant antigens and peptides as well as the role of post-translational glycosylation. Here, we focus on recent applications of microarray technology in understanding the detailed chemical biology of immune responses to disease in an effort to guide development of vaccines and other protective therapies. Abstract Healthcare-associated infection by meticillin-resistant Staphylococcus aureus (MRSA) is still a great concern in an intensive care unit (ICU). Our surveillance data in the ICU revealed that intubated patients were at eight times higher risk of acquiring MRSA than non-intubated patients, so we hypothesised that pre-emptive contact precautions for all intubated patients would prevent healthcare-associated infection by MRSA in the ICU. Patients staying in our ICU for >2 days were included in this study. The study period was divided into two periods. During 2004 (1st period), contact precautions were performed only for patients with MRSA. During 2005e2007 (2nd period), contact precautions were applied to all intubated patients regardless of MRSA infection status. Patients were defined as MRSA-positive on admission when MRSA was detected by surveillance or clinical culture on enrolment. Other MRSA-positive results were defined as healthcare-associated MRSA (HA-MRSA) transmission. HA-MRSA infection was diagnosed according to the National Nosocomial Infections Surveillance Manual. The 1st period comprised 415 patients, and the 2nd period comprised 1280 patients. In intubated patients, HA-MRSA infection rate decreased significantly in the 2nd period (1st period 12.2%, 2nd period 5.6%; P ¼ 0.015). HA-MRSA infection of all patients decreased from 3.6 to 2.3 incidents per 1000 patient-days (P < 0.05), despite a significant increase in the rate of patients MRSA positive on admission in the 2nd period (1st period 2.9%; 2nd period 6.1%). Preemptive contact precautions for intubated patients would be helpful in reducing HA-MRSA infection in ICU. Abstract In many hospitals, isolation rooms are used to contain patients who are highly infectious, and the spread of air and bacteria within the isolation room is closely relates to room air distribution. This article uses the computational fluid dynamics (CFD) method to investigate the effects of a moving person and the opening and closing of a sliding door on room air distribution, including velocity, pressure and contaminant fields. Dynamic meshes are employed to simulate the movement of the walking person and sliding door. According to numerical results, the impact of those moving objects on room air distribution is addressed in this study. r Abstract In tribute to Jules Dupuit, this study analyzes how the innovative financing tax for development has influenced the competition among airline companies and to what extent it affected their sales. To do so, we specify an econometric model where the representative consumer chooses the utility maximizing alternative among the differentiated products offered by the airlines which compete in terms of prices. It is a fairly reasonable and well accepted representation of the competition in the airline industry, assuming that the structure of their networks is given in the short run. The present solidarity tax has increased the average price for the European airlines by only 0:08% and could increase average prices by 1% if it were applied to all European airlines. Implemented by all countries it would lead to a 0:17% increase in Air France prices while the price increase would be 1:05% for the other airlines in our sample. These numbers compared to the growth rate of the air traffic show that the impact of the solidarity tax would be very small. Overall the air ticket levy applied by all European airlines would not affect significantly the degree of competition among airlines.Besides the various innovations that Jules Dupuit, this adept of the "laissez-faire," has offered to the economic science, in particular the concept marginal utility, his name is strongly associated with the need to ground taxes on solid economic analysis using the notion of surplus. A first key question is then how to estimate what he calls the "strength of desire" of consumers and he writes: "it is beyond doubt that a tax can add nothing to the utility of a product; but when we look at it from the consumer's point of view we can say that its existence brings to light undeniably that the product has a utility greater than the cost of production." (See Dupuit, 1884, p. 85.) A buyer pays a good on which a tax is imposed if he finds at least an equivalent utility in it. Dupuit adds: "for, in spite of the tax, he is at perfect liberty to buy it or not to buy it. It is not within the power of the state to make him pay, by means of the tax, anything more than the utility which he derives from this purchase." (See Dupuit, 1884, p. 85.) A second key question is to account for the differentiation of products which can trigger as many "strength of desire" than they are consumers. Jules Dupuit is one of the first economists to address this situation by business strategies based on price discrimination. In this sense, the market structure is really close to oligopolistic competition.The following study is an attempt along the lines of the Jules Dupuit's philosophy of economics. Abstract Isatis indigotica Fortune is a popular herb in traditional Chinese medicine, and various types of metabolites are the basis for its pharmacological efficacy. The biosynthesis and accumulation of these metabolites are closely linked to nitrogen availability; the benefits of low nitrogen application on the environment and herb quality are increasingly prominent. To analyze metabolic changes in the leaves and roots of I.indigotica in nitrogen deficiency conditions, and to identify the pathways and metabolites induced by low nitrogen availability, we used untargeted liquid chromatography coupled with mass spectrometry (UHPLC-TripleTOF) to obtain metabolomics profiling of I.indigotica under two N-deficiency treatments (0 kg/hm 2 ; 337.5 kg/hm 2 ) and normal nitrogen treatment (675 kg/hm 2 ). A total of 447 metabolites were annotated. Principal component analysis separated the three nitrogen treatments. A greater diversity of metabolites was observed in roots than in leaves under Ndeficiency treatments, suggesting that roots have a more important function in low N tolerance. Differential metabolites were mainly enriched in purine metabolism, phenylpropanoid biosynthesis, the shikimate pathway, tryptophan metabolism, and flavonoid biosynthesis that notably induced only in leaves in low nitrogen stress. Moderate N-deficiency benefits carbohydrate accumulation, whereas accumulation of most amino acids decreases. Uniquely, L-tryptophan was maintained at a high concentration in N-deficiency conditions. Low nitrogen stress induced the accumulation of some specialized metabolites (matairesinol, dictamnine, 5-hydroxyindoleacetate (serotonin) in roots and vitexin, xanthohumol, sinapyl alcohol in leaves). N-deficiency also increased the accumulation of adenosine and quality indicators of I.indigotica (indirubin-indigo, epigoitrin and anthranilic acid) in a certain degree. Our findings showed that nitrogen deficiency modified roots and leaves conditions of I.indigotica, affecting both the primary and secondary metabolism. Moderate nitrogen reduction was beneficial to the accumulation of active ingredients. Our methods and analysis are expected to provide an insight regarding the diversity of metabolites and regulation of their synthesis in low nitrogen application, and better investigate the nitrogen deficiency effect on I.indigotica. Abstract Chikungunya virus (CHIKV) is the aetiological agent of the mosquito-borne disease chikungunya fever, a debilitating arthritic disease that, during the past 7 years, has caused immeasurable morbidity and some mortality in humans, including newborn babies, following its emergence and dispersal out of Africa to the Indian Ocean islands and Asia. Since the first reports of its existence in Africa in the 1950s, more than 1500 scientific publications on the different aspects of the disease and its causative agent have been produced. Analysis of these publications shows that, following a number of studies in the 1960s and 1970s, and in the absence of autochthonous cases in developed countries, the interest of the scientific community remained low. However, in 2005 chikungunya fever unexpectedly re-emerged in the form of devastating epidemics in and around the Indian Ocean. These outbreaks were associated with mutations in the viral genome that facilitated the replication of the virus in Aedes albopictus mosquitoes. Since then, nearly 1000 publications on chikungunya fever have been referenced in the PubMed database. This article provides a comprehensive review of chikungunya fever and CHIKV, including clinical data, epidemiological reports, therapeutic aspects and data relating to animal models for in vivo laboratory studies. It includes Supplementary Tables of all WHO outbreak bulletins, ProMED Mail alerts, viral sequences available on GenBank, and PubMed reports of clinical cases and seroprevalence studies. Abstract Nosocomial infections are a major worldwide cause of death and disability, infection control programs are effective in limiting these infections, especially those acquired in the intensive care unit. The development of the world wide web has provided health care professionals with immediate access to continuously updated information in the field of infection control. We sought to identify websites that contain information on nosocomial infection control by using popular internet search engines, such as Google, Yahoo and AltaVista, and by reviewing relevant publications identified in the PubMed and Current Contents databases. Only those sites that were English language, open access, and developed by a government, academic institution, or national or international scientific association were eligible for inclusion. From a vast number of internet sites initially identified, we selected 49 that provide information on infection control for inclusion in our list of practical and relevant internet resources. Several sites provide general information on infection control practices, whereas others focus on one or a few specific infection(s). We provide health care professionals with a timely and succinct list of open access internet resources that contain information regarding the prevention and control of nosocomial infections in order to help in the dissemination of relevant information and so contribute to the limitation of such hazards. Good collection of preventive measures for infection control http://www.health.gov.au/internet/wcms/Publishing.nsf/content/ Infection control guidelines for the prevention of transmission of infectious icg-guidelines-index.htm diseases in the health care setting http://www.nice.org.uk/page.aspx?o=CG002Provides a set of measures to avoid infection that should be followed by anyone giving or receiving care at home or at a health centre http://www.cdc.gov/ncidod/dhqp/index.html Guidelines for keeping patients and health care workers in health care settings protected from infectious diseases http://www.nhsplus.nhs.uk/nhsstaff/infection.asp Limited information Abstract Chemical compounds studied in this article: Celastrol (PubChem CID: 122,724) triptocalline A (PubChem CID: 44,559,634) polpunonic acid (PubChem CID: 343,427) demethylzeylasteral (PubChem CID: 10,322,911) wilforol A (PubChem CID: 10,096,097) regelin D (PubChem CID: 129,520) triptotriterpenic acid B (PubChem CID: 195,563) wilforlide A (PubChem CID: 158,477) wilforlide B (PubChem CID: 174,362) triptocallic acid A (PubChem CID: 44,575,704) regelinol (PubChem CID: 163,809) regelin (PubChem CID: 163,808) dulcioic acid (PubChem CID: 101,051,955) tripterygic acid A (PubChem CID: 21,672,627) demethylregelin (PubChem CID: 44,559,663)Three new triterpenoids, triregelolides A, B (1, 2), and triregeloic acid (3), were isolated from the stems of Tripterygium regelii along with twenty known triterpene analogues (4-23). The structures of three new compounds were identified by analyzing their NMR spectroscopic and HRESIMS data. Compounds 4, 7, 8, 10, 13, 14, 17, [21] [22] [23] were isolated from T. regelii for the first time. Compounds 3, 5, 6, 8, 9, 10, 14 and 16 showed inhibitory effects on the proliferation of human breast cancer cells MCF-7 by 24.1%, 69.6%, 72.8%, 21.6%, 23.1%, 43.3%, 25.5% and 23.5% (p b 0.05) at a concentration of 10 μM, respectively. Abstract Infection of healthcare workers with the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is thought to occur primarily by either contact or large respiratory droplet transmission. However, infrequent healthcare worker infections occurred despite the use of contact and droplet precautions, particularly during certain aerosolgenerating medical procedures. We investigated a possible cluster of SARS-CoV infections in healthcare workers who used contact and droplet precautions during attempted cardiopulmonary resuscitation of a SARS patient. Unlike previously reported instances of transmission during aerosol-generating procedures, the index case-patient was unresponsive, and the intubation procedure was performed quickly and without difficulty. However, before intubation, the patient was ventilated with a bag-valve-mask that may have contributed to aerosolization of SARS-CoV. On the basis of the results of this investigation and previous reports of SARS transmission during aerosol-generating procedures, a systematic approach to the problem is outlined, including the use of the following: 1) administrative controls, 2) environmental engineering controls, 3) personal protective equipment, and 4) quality control. Abstract a b s t r a c t NK cells are innate lymphocytes capable of killing malignant or infected cells and to produce a wide array of cytokines and chemokines following activation.Chemokines, play critical roles in the regulation of NK cell tissue distribution in normal conditions as well as their rapid recruitment to the parenchyma of injured organs during inflammation, which is critical for NK cell ability to promote protective responses. In this regard, differences in chemokine receptor expression have been reported on specialized NK cell subsets with distinct effector functions and tissue distribution.Besides their role in the regulation of NK cell trafficking, chemotactic molecules can also affect NK cell effector functions by regulating their priming and their ability to kill and secrete cytokines. Abstract Two recombinant FIPV spike proteins were assessed for their immunogenic properties in 8week-old kittens, which were then challenged intranasally with FIPV 79-1146. Humoral responses were assessed by ELISA and serum neutralisation test. Changes in PBMC cytokine mRNA levels were detected by a reverse transcription, semiquantitative polymerase chain reaction assay (RT-sqPCR), assessing IL-2, IL-4, IL-6, IL-10, IL-12 and IFNg. All of the kittens developed clinical signs typical of FIP, which were confirmed on gross post mortem examination. The recombinant proteins induced little or no specific antibody response prior to challenge, and failed to alter the course of disease compared to controls. One week after virus challenge, the stimulated PBMCs showed small increases in the expression of IL-6 and IFNg mRNA, which correlated with a transient pyrexia. After this time expression of IL-6 mRNA remained unaltered but, as FIP developed, mRNA levels of IL-2, IL-4, IL-10, IL-12 and IFNg became markedly depressed. # Abstract a b s t r a c t 2 0 -b-D-Arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU L-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs. Abstract We have previously observed that all known HIV-1 broadly neutralizing antibodies (bnAbs) are highly divergent from germline antibodies in contrast to bnAbs against Hendra virus, Nipah virus and SARS coronavirus (SARS CoV). We have hypothesized that because the germline antibodies are so different from the mature HIV-1-specific bnAbs they may not bind the epitopes of the mature antibodies and provided the first evidence to support this hypothesis by using individual putative germline-like predecessor antibodies. To further validate the hypothesis and understand initial immune responses to different viruses, two phage-displayed human cord blood-derived IgM libraries were constructed which contained mostly germline antibodies or antibodies with very low level of somatic hypermutations. They were panned against different HIV-1 envelope glycoproteins (Envs), SARS CoV protein receptor-binding domain (RBD), and soluble Hendra virus G protein (sG). Despite a high sequence and combinatorial diversity observed in the cord blood-derived IgM antibody repertoire, no enrichment for binders of Envs was observed in contrast to considerable specific enrichments produced with panning against RBD and sG; one of the selected monoclonal antibodies (against the RBD) was of high (nM) affinity with only few somatic mutations. These results further support and expand our initial hypothesis for fundamental differences in immune responses leading to elicitation of bnAbs against HIV-1 compared to SARS CoV and Hendra virus. HIV-1 uses a strategy to minimize or eliminate strong binding of germline antibodies to its Env; in contrast, SARS CoV and Hendra virus, and perhaps other viruses causing acute infections, can bind germline antibody or minimally somatically mutated antibodies with relatively high affinity which could be one of the reasons for the success of sG and RBD as vaccine immunogens.Published by Elsevier Inc. Abstract Background : A variable rate of false-positive results may be observed with commercial assays for the detection of rotavirus and adenovirus antigen in stool specimens, depending on the quality of the reagents and the presence of potentially interfering substances in stool samples. Objective : The present report analyse the discrepant results that could be obtained by the commercially available diagnostic tests and that can mask the reliable viral diagnosis. Study design : One fecal sample was collected from a hospitalized child aged 6 months with acute watery diarrhea and dehydration. The fecal specimen was processed the same day for the rotavirus and adenovirus antigen detection. Results : The sample was positive for rotavirus antigen by one-step membrane test based on immunochromatographic assays (ICA) and enzyme immunoassays (EIA) monoclonal test but it was negative by an EIA polyclonal test, polyacrylamide gel electrophoresis (PAGE) and RT-PCR assays. In the other hand, the sample was positive for adenovirus antigen by ICA and EIA adenovirus type 40/41. Finally, the sample showed by PAGE an electrophoretic profile resembled that of reovirus. Conclusion : The use of a wide repertory of diagnosis tests allowed to reach an unusual reovirus-adenovirus type 40/41 dual infection. This case also point out the potential participation of reovirus in the ethiology of the diarrhea illness. Abstract Respiratory viruses cause acute respiratory diseases with a broad and overlapping spectrum of symptoms. We examined the clinical symptoms and explored the patterns of various respiratory viral infections in children in Hong Kong. Among 2090 specimens collected from outpatient care (2007-2010), 1343 (64.3%) were positive for any virus by the xTAG assay, and 81 (3.9%) were positive for coinfection. The most frequently detected viruses among children aged 6-15 years were enterovirus/rhinovirus and influenza virus A, whereas most non-influenza viruses were more frequently detected in younger children. Higher body temperature was more common for illnesses associated with influenza viruses than for those associated with non-influenza viruses, but other symptoms were largely similar across all infections. The seasonality pattern varied among different viruses, with influenza virus A being the predominant virus detected in winter, and enterovirus/rhinovirus being more commonly detected than influenza virus A in the other three seasons, except for 2009. Clinical Microbiology and Infection Abstract Induction of pro-inflammatory response is a crucial cellular process that detects and controls the invading viruses at early stages of the infection. Along with other innate immunity, this nonspecific response would either clear the invading viruses or allow the adaptive immune system to establish an effective antiviral response at late stages of the infection. The objective of this study was to characterize cellular mechanisms exploited by coronavirus infectious bronchitis virus (IBV) to regulate the induction of two pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, at the transcriptional level. The results showed that IBV infection of cultured human and animal cells activated the p38 mitogen-activated protein kinase (MAPK) pathway and induced the expression of IL-6 and IL-8. Meanwhile, IBV has developed a strategy to counteract the induction of IL-6 and IL-8 by inducing the expression of dual-specificity phosphatase 1 (DUSP1), a negative regulator of the p38 MAPK, in order to limit the production of an excessive amount of IL-6 and IL-8 in the infected cells. As activation of the p38 MAPK pathway and induction of IL-6 and IL-8 may have multiple pathogenic effects on the whole host as well as on individual infected cells, regulation of the p38 MAPK and DUSP1 feedback loop by IBV may modulate the pathogenesis of the virus. Abstract The optimal virulence of a pathogen is determined by a trade-off between maximizing the rate of transmission and maximizing the duration of infectivity. Treatment measures such as curative therapy and case isolation exert selective pressure by reducing the duration of infectivity, reducing the value of duration-increasing strategies to the pathogen and favoring pathogen strategies that maximize the rate of transmission. We extend the trade-off models of previous authors, and represents the reproduction number of the pathogen as a function of the transmissibility, host contact rate, disease-induced mortality, recovery rate, and treatment rate, each of which may be influenced by the virulence. We find that when virulence is subject to a transmissibility-mortality trade-off, treatment can lead to an increase in optimal virulence, but that in other scenarios (such as the activity-recovery trade-off) treatment decreases the optimal virulence. Paradoxically, when levels of treatment rise with pathogen virulence, increasing control efforts may raise predicted levels of optimal virulence. Thus we show that conflict can arise between the epidemiological benefits of treatment and the evolutionary risks of heightened virulence. r Abstract Protein ubiquitylation has emerged as an important regulatory mechanism that impacts almost every aspect of the DNA damage response. In this review, we discuss how DNA repair and checkpoint pathways utilize the diversity offered by the ubiquitin conjugation system to modulate the response to genotoxic lesions in space and time. In particular, we will highlight recent work done on the regulation of DNA double-strand breaks signalling and repair by the RNF8/RNF168 E3 ubiquitin ligases, the Fanconi anemia pathway and the role of protein degradation in the enforcement and termination of checkpoint signalling. We also discuss the various functions of deubiquitylating enzymes in these processes along with potential avenues for exploiting the ubiquitin conjugation/deconjugation system for therapeutic purposes. Abstract A 1.5-month-old Kangal breed puppy from a dairy cattle farm died after showing severe diarrhoea and incoordination. Necropsy examination revealed multifocal pulmonary consolidation and necrosis and fibrinohaemorrhagic enteritis. Microscopically, there was necrotic and purulent bronchopneumonia, myocarditis and non-purulent encephalitis. In the jejunum and ileum there was villous atrophy and crypt hyperplasia with oocyst-like and schizont-like structures in the epithelia. Immunohistochemically, Neospora caninum antigen was detected in association with the intestinal protozoal structures, degenerative neurons and areas of necrosis in the lungs and heart. Polymerase chain reaction confirmed that the organism was N. caninum and not Toxoplasma gondii. The seroprevalence for N. caninum was 74.2% (49/66 animals) for the cattle and 57.1% (4/7 animals) for dogs on this farm. This report documents fatal systemic neosporosis and enteroepithelial stages of N. caninum in a naturally infected puppy. To the authors' knowledge, this is the first definition of intestinal neosporosis in a naturally infected dog as well as the first evidence of fatal canine neosporosis in Turkey. Abstract Infectious diseases are a threat to human health and a hindrance to societal development. Consequently, the spread of diseases in both time and space has been widely studied, revealing the different types of spatial patterns. Transitions between patterns are an emergent property in spatial epidemics that can serve as a potential trend indicator of disease spread. Despite the usefulness of such an indicator, attempts to systematize the topic of pattern transitions have been few and far between. We present a mini-review on pattern transitions in spatial epidemics, describing the types of transitions and their underlying mechanisms. We show that pattern transitions relate to the complexity of spatial epidemics by, for example, being accompanied with phenomena such as coherence resonance and cyclic evolution. The results presented herein provide valuable insights into disease prevention and control, and may even be applicable outside epidemiology, including other branches of medical science, ecology, quantitative finance, and elsewhere. Abstract In this study, we report the complete genome sequence of two contemporary human coronavirus OC43 (HCoV-OC43) strains detected in 2003 and 2004, respectively. Comparative genetic analyses of the circulating strains and the prototype HCoV-OC43 strain (ATCC VR759) were performed. Remarkably, a lower than expected similarity is found between the complete genomes and more in particular between the spike genes of the BE03 and BE04 strains. This finding suggests the existence of two genetically distinct HCoV-OC43 strains, circulating in Belgium in 2003 and 2004. Spike gene sequencing of three additional 2003 and two additional 2004 HCoV-OC43 strains, and subsequent phylogenetic analysis confirm this assumption. Compared to the ATCC prototype HCoV-OC43 strain, an important amino acid substitution is present in the potential cleavage site sequence of the spike protein of all contemporary strains, restoring the N-RRXRR-C motif, associated with increased spike protein cleavability in bovine coronaviruses. We here describe specific characteristics associated with circulating HCoV-OC43 strains, and we provide substantial evidence for the genetic variability of HCoV-OC43. D Abstract Purpose: The field of HIV-1 vaccinology has evolved during the last 30 years from the first viral vector HIV gene insert constructs to vaccination regimens using a myriad of strategies. These strategies now include germline-targeting, lineagebased, and structure-guided immunogen design. This narrative review outlines the historical context of HIV vaccinology and subsequently highlights the scientific discoveries during the last 6 years that promise to propel the field forward.Methods: We conducted a search of 2 electronic databases, PubMed and EMBASE, for experimental studies that involved new HIV immunogen designs between 2013 and 2019. During the title and abstract reviews, publications were excluded if they were written in language other than English and/or were a letter to the editor, a commentary, or a conference-only presentation. We then used ClinicalTrials.gov to identify completed and ongoing clinical trials using these strategies.Findings: The HIV vaccinology field has undergone periods of significant growth during the last 3 decades. Findings elucidated in preclinical studies have revealed the importance of the interaction between the cellular and humoral immune system. As a result, several new rationally designed vaccine strategies have been developed and explored in the last 6 years, including native-like envelope trimers, nanoparticle, and mRNA vaccine design strategies among others. Several of these strategies have shown enough promise in animal models to progress toward first-inhuman Phase I clinical trials.Implications: Rapid developments in preclinical and early-phase clinical studies suggest that a tolerable and effective HIV vaccine may be on the horizon. (Clin Ther. xxxx;xxx:xxx) Abstract Background. The global outbreak of the severe acute respiratory syndrome (SARS) in 2003 has been an international public health threat. Quick diagnostic tests and specific treatments for SARS are not yet available; thus, prevention is of paramount importance to contain its global spread. This study aimed to determine factors associating with individuals' practice of the target SARS preventive behavior (facemask wearing).Methods. A total of 1329 adult Chinese residing in Hong Kong were surveyed. The survey instrument included demographic data, measures on the five components of the Health Belief Model, and the practice of the target SARS preventive behavior. Logistic regression analyses were conducted to determine rates and predictors of facemask wearing.Results. Overall, 61.2% of the respondents reported consistent use of facemasks to prevent SARS. Women, the 50 -59 age group, and married respondents were more likely to wear facemasks. Three of the five components of the Health Belief Model, namely, perceived susceptibility, cues to action, and perceived benefits, were significant predictors of facemask-wearing even after considering effects of demographic characteristics.Conclusions. The Health Belief Model is useful in identifying determinants of facemask wearing. Findings have significant implications in enhancing the effectiveness of SARS prevention programs. Abstract Experimental demyelination induced by coronavirus JHM (MHV-4) : molecular identification of a viral determinant of paralytic disease . Microbial Pathogenesis 1987 ; 3 : 9-20 .The molecular basis for demyelination induced by the neurotropic murine coronavirus JHM (JHMV or MHV4) is unknown . We have attempted to explore this issue by using neutralizing monoclonal antibodies specific for the major JHMV glycoprotein (E2) to select sets of neutralization resistant (NR) antigenic variant viruses . Monoclonal antibodies J .7 .2 and J .2 .2 bind to topographically distinct sites on E2 . NR variants selected with J .7 .2, like parental JHMV, predominantly cause a fatal encephalitis when given intracerebrally to mice, while J .2 .2-selected NR variants cause a subacute disease characterized by paralysis and severe demyelination . We report here that consecutive selection with both J .2 .2 and J .7 .2 monoclonal antibodies results in NR variants which are markedly attenuated in both encephalitic potential and ability to induce demyelination . Analysis of the different variants suggests that the subregion of E2 bound by monoclonal antibody J .7 .2 may be a critical viral determinant of paralysis and demyelination in this model system . Abstract Recombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique, operating at 37e42 C, with minimal sample preparation and capable of amplifying as low as 1e10 DNA target copies in less than 20 min. It has been used to amplify diverse targets, including RNA, miRNA, ssDNA and dsDNA from a wide variety of organisms and samples. An ever increasing number of publications detailing the use of RPA are appearing and amplification has been carried out in solution phase, solid phase as well as in a bridge amplification format. Furthermore, RPA has been successfully integrated with different detection strategies, from end-point lateral flow strips to real-time fluorescent detection amongst others. This review focuses on the different methodologies and advances related to RPA technology, as well as highlighting some of the advantages and drawbacks of the technique. Abstract Dicer Dicer is an endoribonuclease member of RNase III family encoded by dicer gene, which has the function of cleaving long double-stranded RNA or pre-miRNA into siRNA or miRNA that are in 20-25 base pairs length with 2 nt overhangs at 3 0 ends. MicroRNA (miRNA) miRNA is a kind of 22 nt small non-coding RNA fragments discovered in viruses, plants, animals and human, which have the functions of post-transcriptional regulation in gene expression. p19 protein Plant tombusvirus 19 kDa protein (p19) is an RNA silencing suppressor protein. p19 selectively binds to exactly matched, double-stranded antiviral siRNAs of 21 nt, but not to imperfectly paired miRNAs, ssRNAs or other dsRNAs. Prokaryotic siRNA (pro-siRNA) Pro-siRNA are bacterial RNase III products that have chemical and functional properties similar to eukaryotic siRNAs. Ribonucleic acid (RNA) RNA is a linear molecule composed of four types of smaller molecules called ribonucleotide bases: adenine (A), cytosine (C), guanine (G), and uracil (U).Comprehensive Biotechnology, 3rd edition, Volume 5 Abstract The small passerines, canaries and finches, are social birds often bred and housed in flock aviaries. The aviary may be a mixed aviary housing different species or a breeding aviary concentrating on a single species. Multiple birds in contact with each other provide the means by which infectious disease can spread. Dietary and husbandry requirements vary for the species of passerines housed and can also influence disease outbreaks when they are less than optimal. Stress factors, including nutritional, husbandry (overcrowding, aviary maintenance), breeding, and the introduction of new birds, may play a significant role in disease outbreaks. An overview of viral, bacterial, fungal, and parasitic issues affecting passerines housed in aviaries will be addressed.Copyright 2003, Elsevier Science (USA). All rights reserved. Abstract The objectives of this study were to evaluate the effects of free-access acidified milk replacer feeding on the pre-and postweaning health of dairy and veal calves. Individually housed calves were systematically assigned at birth to 1 of 2 feeding programs: free-access feeding (ad libitum) of acidified milk replacer (ACD, n = 249) or traditional restricted feeding (3 L fed twice daily) of milk replacer (RES, n = 249). Calves were fed milk replacer containing 24% crude protein and 18% fat. Acidified milk replacer was prepared to a target pH between 4.0 and 4.5 using formic acid. Calves were weaned off milk replacer at approximately 6 wk of age. Weaning occurred over 5 d, and during this weaning period, ACD calves had access to milk replacer for 12 h/d and RES calves were offered only one feeding of milk replacer (3 L) daily. Calves were monitored daily for signs of disease. Fecal consistency scores were assigned each week from birth until weaning. A subset of calves was systematically selected for fecal sampling at 3 time points between 7 and 27 d of age. Fecal samples were analyzed for enterotoxigenic Escherichia coli F5, Cryptosporidium parvum, rotavirus, and coronavirus. Hip width, hip height, body length, heart girth, and body weight were measured at birth and weaning. Postweaning body weight measurements were collected from the heifers at approximately 8 mo of age. Postweaning body weight and carcass grading information was collected from the veal calves at slaughter once a live weight between 300 and 350 kg had been achieved. The odds of ACD calves being treated for a preweaning disease event tended to be lower than that of the RES calves (1.2 vs. 5.2%, respectively). Preweaning mortality, postweaning disease treatment, and postweaning mortality did not differ between feeding treatments. The ACD feeding treatment supported greater preweaning average daily gain (0.59 vs. 0.43 kg/d) and structural growth than RES feeding. Postweaning average daily gain and carcass characteristics were similar for ACD and RES calves. These results indicate that free-access acidified milk replacer feeding tended to support improved health, and greater body weight gain and structural growth during the preweaning period; these effects did not persist in the postweaning period. The growth advantage observed before weaning in the ACD calves likely disappeared due to the weaning methods used. Abstract Water is in short supply with countries vying for access to river heads. Hong Kong (HK) is no different. It relies on supply from Mainland China with other cities rivalling access. Yet there is still no great impetus for water conservation. This paper reports on progress in setting up a pilot biological wastewater treatment plant and a PV-powered UV-LED disinfection system plus a feasibility study carried out in conjunction with a local developer investigating the application of the treated water for irrigation. The objective is to incorporate the systems within a small-scale community for its contribution to water and energy conservation as well as establish the parameters for replication in other countries with potential up-scaling for urban application. r Abstract Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution -10 0.7 TCID 50 ). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains. Abstract Background There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods.Objectives First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections.Methods RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v.Results The ''perfect match'' M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a ''gold standard'', while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. ''Perfect match'' M gene RRT PCR had 100% sensitivity and 95AE2% specificity for swabs, 93AE6% and 98AE6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99AE1%, respectively, for the swabs, and 100% and 100% for the tissues.Conclusions Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated. Keywords H1N1v, Pandemic H1N1 2009 virus, RRT PCR, swine influenza virus (SIV), validation. Please cite this paper as: Slomka et al. (2010) Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs. Influenza and Other Respiratory Viruses 4(5), 277-293. Abstract The Kumbh Mela in India is the largest mass gathering in the world which witnessed close to 100 million visitors in 2013. An event of this magnitude presents challenges. Increased population density, reduced hygienic conditions and exposure to environmental pollutants pave the way for easy transmission of pathogens. Due to the possibility of epidemics, the primary focus should be on identifying the potential risk factors and implementing appropriate preventive measures. The context of religion and psychology of the pilgrims is also closely associated with the evolution of the risk factors and so forms an important part of the discussion. We provide a brief background to the Kumbh Mela with a description of the existing and potential risk factors that require our attention.Clinical Microbiology and Infection Abstract The Diachasmimorpha longicaudata entomopoxvirus (DlEPV) is the first symbiotic EPV described from a parasitic wasp. The DlEPV is introduced into the tephritid fruit fly larval host along with the wasp egg at oviposition. We sequenced a shotgun genomic library of the DlEPV DNA and analyzed and compared the predicted protein sequences of eight ORFs with those of selected poxviruses and other organisms. BlastP searches showed that five of these are homologous to poxvirus putative proteins such as metalloprotease, a putative membrane protein, late transcription factor-3, virion surface protein, and poly (A) polymerase (PAP) regulatory small subunit. Three of these are similar to those of other organisms such as the gamma-glutamyltransferase (GGT) of Arabidopsis thaliana, eukaryotic initiation factor 4A (eIF4A) of Caenorhabditis briggsae and lambda phage integrase (l-Int) of Enterococcus faecium. Transcription motifs for early (TGA,A/T,XXXXA) or late (TAAATG, TAAT, or TAAAT) gene expression conserved in poxviruses were identified with those ORFs. Phylogenetic analysis of multiple alignments of five ORFs and 20 poxvirus homologous sequences and of a concatenate of multiple alignments suggested that DlEPV probably diverged from the ancestral node between the fowlpox virus and the genus B, lepidopteran and orthopteran EPVs, to which Amsacta moorei and Melanoplus sanguinipes EPV, respectively, belong. The DlEPV putative GGT, eIF4A, and l-Int contained many conserved domains that typified these proteins. These homologues may be involved in either viral pathogenicity or enhancing parasitism via the gamma-glutamyl cycle and compensation of eIF4A levels in the parasitized fly, or via the integration of a portion of the viral genome into the wasp and/or parasitized fly. r Abstract This study was conducted to assess the knowledge of H1N1 among medical students, their perceptions, and behavioral intentions in the wake of the H1N1 pandemic influenza. There were significant gaps in important self-isolation protocols and preventive measures. Increased contact with both patients and colleagues can lead to unintentional transmission and contraction of influenza. Universities should introduce and encourage infection control guidelines into routine curriculum. Abstract Protein microarray that consists of virulence-associated proteins of Yersinia pestis is used to compare antibody profiles elicited by the wildtype and quorum sensing (QS) mutant strain of this bacterium to define the immunogens that are impacted by QS. The results will lead the way for future functional proteomics studies. The antibody profile that was induced by the QS mutant differed from that of the parent strain. Detailed comparison of the antibody profiles, according to the proteins' functional annotations, showed that QS affects the expression of many virulenceassociated proteins of Y. pestis. The antibodies to many virulence-associated proteins were not detected or lower titers of antibodies to many proteins were detected in the sera of rabbits immunized with the QS mutant, relative to those of the wild type, which indicated that these proteins were not expressed or expressed at relatively lower levels in the QS mutant. The results demonstrated that antibody profiling by protein microarrays is a promising high-throughput method for revealing the interactions between pathogens and the host immune system. Abstract Livestock production is an important source of animal protein worldwide. In the developed world meat consumption will remain steady but demand is forecast to grow enormously in developing countries. The use of genomics will speed genetic improvement and increase levels of production quickly in the developed world but might face problems in the developing world, including scientific, economic and political challenges. Considerable increases in public and private research funding will be required to develop and utilize novel tools and collections of detailed trait information on appropriate animals. The development of policies protecting the environment and managing all genetic resources will also be needed. Advances in livestock genomics have major implications for increasing food output as well as improving human health. Abstract Genotyping of single nucleotide polymorphisms (SNPs) in point-of-care (POC) settings could be further improved through simplifying the treatment of samples. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on direct loop-mediated isothermal amplification (LAMP) without DNA extraction, known as Direct-LAMP. Samples from various sources (including whole blood, dried blood spot, buccal swab and saliva), treated with NaOH, can be used directly in amplification. The turnaround time was about 30 min from sample collection to provision of results. The accuracy was evaluated by assessing the polymorphisms of methylenetetrahydrofolate reductase (MTHFR) C677T and aldehyde dehydrogenase-2 (ALDH2) Glu504Lys, which are better known for their critical role in folate and ethanol metabolism, respectively. Completely consistent genotyping results reveal that Direct-LAMP is generally concordant with sequencing. This system can serve as a very promising platform in the fields of disease predisposition, drug metabolism and personalized medicine. Abstract Porcine deltacoronavirus (PDCoV), an emerging animal coronavirus causing enteric disease in pigs, belongs to the newly identified Deltacoronavirus genus in the Coronaviridae family. Although extensive studies have been carried out to investigate the regulation of interferon (IFN) responses by alphacoronaviruses, betacoronaviruses, and gammacoronaviruses, little is known about this process during deltacoronavirus infection. In this study, we found that PDCoV infection fails to induce, and even remarkably inhibits, Sendai virus-or poly(I: C)-induced IFN-β production by impeding the activation of transcription factors NF-κB and IRF3. We also found that PDCoV infection significantly suppresses the activation of IFN-β promoter stimulated by IRF3 or its upstream molecules (RIG-I, MDA5, IPS-1, TBK1, IKKε) in the RIG-I signaling pathway, but does not counteract its activation by the constitutively active mutant of IRF3 (IRF3-5D). Taken together, our results demonstrate that PDCoV infection suppresses RIG-I-mediated IFN signaling pathway, providing a better understanding of the PDCoV immune evasion strategy. Abstract ROC h i g h l i g h t s < Hallmark sporting events may not generate consistent and substantial tourism demand. < Factors contribute to the soft hotel occupancy and revenue intakes during the 2009 World Games are explained. < Displacement effects of visitors and the imbalance power between stakeholders are considered as the major causes.Hosting mega-and hallmark-events is perceived as an important vehicle for tourism development. However, in the case of the 2009 World Games, the first international hallmark sporting event in Taiwan, the volume of hotel occupancies and revenue fell short of expectations, despite 4 years of planning and an investment of US$218 million. This study set out to investigate this phenomenon by adopting semistructured interviews to gather opinions from key stakeholders regarding their business operation and societal conditions. Factors contributing to the lower than expected revenue included inefficient collaboration among stakeholders, a substantial displacement effect to regional hotel competitors, and the unfriendly local atmosphere associated with the fear of H1N1, Typhoon Morakot, and controversial political events. To mitigate inefficiencies from stakeholder collaborations, the study results suggest greater equality in terms of responsibility, information distribution, and risk sharing is needed among the broader tourism industry, the event planning association, and the event supervising authority. In addition, business strategies should take into account Taiwan, an island destination, and current tourism characteristics to counter the displacement effects of loyal and potential customers. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.Sensitization to 1 or more of the common indoor allergens has been consistently associated with asthma among children and young adults (odds ratios for asthma, 3-18). For dust mite and cockroach allergens, there is a dose response relationship between domestic exposure and sensitization. Given that allergen provocation can induce many of the features of asthma, the findings strongly suggest that there is a causal relationship between allergen exposure in the home and asthma. However, it remains unclear at what time the critical exposure occurs (ie, in infancy or later) and what role allergen exposure has played in the increasing prevalence and severity of asthma. Objective evidence of an immune response to allergens is generally not present until after 2 years of age. Viral infections play several different roles in asthma in childhood. In infancy, respiratory syncytial virus infection can induce bronchiolitis and set up recurrent wheezing over the next few years. However, the risk factors for this are maternal smoking and small lungs at birth, rather than allergy. By contrast, the role of rhinovirus in precipitating attacks in children and young adults is strongly associated with allergy. Thus the likely scenario is that allergen exposure over the first few years of life induces sensitization (ie, T H2 cells and IgE antibodies). Continuing exposure can maintain inflammation in the nose and lungs. However, many other factors contribute to wheezing such that there is no simple relationship between allergen exposure and asthma. Nonetheless, it is clear that the changes that have increased asthma have acted on allergic children. (J Allergy Clin Immunol 2000;105:S503-8.) Abstract Rhesus macaque models have been instrumental for the development and testing of vaccines prior to human studies and have provided fundamental insights into the determinants of immune efficacy in a variety of infectious diseases. However, the characterization of antigen-specific T cell receptor (TCR) repertoires during adaptive immune responses in these models has previously relied on human TCR gene assignments. Here, we extracted and characterized TCR β-chain (TRB) genes from the recently sequenced rhesus macaque genome that are homologous to the human TRB genes. Comparison of the rhesus macaque TRB genes with the human TRB genes revealed an average best-match similarity of 92.9%. Furthermore, we confirmed the usage of most rhesus macaque TRB genes by expressed TCRβ sequences within epitope-specific TCR repertoires. This primary description of the rhesus macaque TRB genes will provide a standardized nomenclature and enable better characterization of TCR usage in studies that utilize this species.RESULTSA total of 72 TRBV genes were extracted from chromosome 3 of the rhesus macaque genome (Table 1 and Rhesus_macaque_TRBV.fsa in Supporting Information). The human Greenaway et al. Abstract Studies from Eastern European countries proved that porcine reproductive and respiratory syndrome virus Type 1 (PRRSV-1) harbours high genetic diversity and that genetically divergent subtypes 2-4 circulate in this area.In the present study, we compared the pathogenicity of two different PRRSV-1 subtype 2 strains and a strain representing PRRSV-1 subtype 1. Four groups of 8-week-old specific pathogen free pigs were either infected with subtype 2 strain ILI6, subtype 2 strain or BOR59, subtype 1 strain 18794, or mock inoculated.The most pronounced clinical signs were observed in pigs infected with BOR59. Pigs from both subtype 2 strain infected groups exhibited significantly elevated mean body temperatures on DPI 2 compared to the other two groups, the difference remaining significant up to DPI 13 for the BOR59 group, only. The pigs in the latter group also displayed significantly highest levels of early viremia together with the most rapid APP response.Overall, the results indicated that BOR59 strain can be considered a highly pathogenic strain, similarly to subtype 3 strains Lena and SU1-bel, while the virulence of the other subtype 2 strain ILI6 was intermediate between BOR59 and subtype 1 strain. Abstract The prototype coronavirus mouse hepatitis virus (MHV) exhibits highly selective packaging of its genomic positive-stranded RNA into assembled virions, despite the presence in infected cells of a large excess of subgenomic viral mRNAs. One component of this selectivity is the MHV packaging signal (PS), an RNA structure found only in genomic RNA and not in subgenomic RNAs. It was previously shown that a major determinant of PS recognition is the second of the two RNA-binding domains of the viral nucleocapsid (N) protein. We have now found that PS recognition additionally depends upon a segment of the carboxy-terminal tail (domain N3) of the N protein. Since domain N3 is also the region of N protein that interacts with the membrane (M) protein, this finding suggests a mechanism by which selective genome packaging is accomplished, through the coupling of genome encapsidation to virion assembly. Abstract Aynaud, J.M., Bernard, S., Bottreau, E., Lantier, I., Salmon, H. and Vannier, Ph., 1991. Induction of lactogenic immunity to transmissible gastroenteritis virus of swine using an attenuated coronavirus mutant able to survive in the physicochemical environment of the digestive tract. Vet. Microbiol., 26: 227-239.A transmissible gastroenteritis (TGE) coronavirus mutant (188-SG), selected as attenuated and resistant to acidity and proteases of the digestive tract of adult pigs, was used as vaccine ("Nouzilly strain") in sows to protect suckling piglets against a challenge exposure carried out with a highly virulent TGEV strain. The pregnant sows were immunized once (42-49 days before farrowing) or twice (42-49 and 7-15 days before farrowing) by the oral, intramuscular or conjunctival route with the 188-SG strain. Sows exposed to virulent TGEV in the field and experimentally infected sows (two oral inoculations during pregnancy) were used as positive controls leading to high protection. The neutralizing antibody response to vaccination and/or infection was studied in serum and milk. No protection against mortality was observed in the litters of ( 1 ) the nine seronegative, susceptible sows, with piglet mortality of 65/70, (2) the seven once orally vaccinated sows, with mortality of 44/54, (3) the seven sows vaccinated twice by the conjunctival route, with mortality of 55/76. Moderate protection was observed in ( 1 ) the eight sows vaccinated intramuscularly twice with piglet mortality of 36 / 90, ( 2 ) the seven orally and intramuscularly vaccinated sows with piglet mortality of 31 / 51. In contrast, improved protection was observed in ( 1 ) the 10 sows vaccinated twice orally, with piglet mortality of 23/95, (2) the four naturally infected sows with piglet mortality of 6/41, (3) the six sows experimentally infected with virulent TGEV with piglet mortality of 1/59. No correlation was found between neutralizing antibodies titers in serum and milk and protection rate of the piglets. The results indicate that relative protective lactogenic immunity against TGEV is induced only by repeated ingestion of the attenuated 188-SG strain of TGEV. Abstract While the transfusion-transmission (TT) risk associated with the major transfusion-relevant viruses such as HIV is now very low, during the last 20 years there has been a growing awareness of the threat to blood safety from emerging infectious diseases, a number of which are known to be, or are potentially, transfusion transmissible. Two published models for estimating the transfusion-transmission risk from EIDs, referred to as the Biggerstaff-Petersen model and the European Upfront Risk Assessment Tool (EUFRAT), respectively, have been applied to several EIDs in outbreak situations. We describe and compare the methodological principles of both models, highlighting their similarities and differences. We also discuss the appropriateness of comparing results from the two models. Quantitating the TT risk of EIDs can inform decisions about risk mitigation strategies and their cost-effectiveness. Finally, we present a qualitative risk assessment for Zika virus (ZIKV), an EID agent that has caused several outbreaks since 2007. In the latest and largest ever outbreak, several probable cases of transfusion-transmission ZIKV have been reported, indicating that it is transfusion-transmissible and therefore a risk to blood safety. We discuss why quantitative modeling the TT risk of ZIKV is currently problematic.Crown Abstract The intranasal (IN-) administration of substances is attracting attention from scientists as well as pharmaceutical companies. The effects are surprisingly fast and specific. The present review explores our current knowledge about the routes of access to the cranial cavity. 'Direct-access-pathways' from the nasal cavity have been described but many additional experiments are needed to answer a variety of open questions regarding anatomy and physiology.Among the IN-applied substances oxytocin (OT) has an extensive history. Originally applied in women for its physiological effects related to lactation and parturition, over the last decade most studies focused on their behavioral 'prosocial' effects: from social relations and 'trust' to treatment of 'autism'.Only very recently in a microdialysis study in rats and mice, the 'direct-nose-brain-pathways' of IN-OT have been investigated directly, implying that we are strongly dependent on results obtained from other IN-applied substances. Especially the possibility that IN-OT activates the 'intrinsic' OT-system in the hypothalamus as well needs further clarification.We conclude that IN-OT administration may be a promising approach to influence human communication but that the existing lack of information about the neural and physiological mechanisms involved is a serious problem for the proper understanding and interpretation of the observed effects. Abstract Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n = 33) and 41.1% (n = 23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control. Abstract Guidelines issued by the Centers for Disease Control and Prevention and the World Health Organization state that healthcare workers should wear N95 masks or higher-level protection during all contact with suspected cases of severe acute respiratory syndrome. Before use, the manufacturer recommends performing a user seal check to ensure that the mask is fitted correctly. This study aimed to test the ability of the user seal check to detect poorly fitting masks. This study is a retrospective review of a mask-fitting programme carried out in the intensive care unit of the Prince of Wales Hospital in Hong Kong. In this programme, all staff were tested with two types of N95 mask and one type of N100 mask. The results of the documented user seal check were then compared with the formal fit-test results from a PortaCount. Using a PortaCount reading of 100 as the criterion for a correctly fitted mask, the user seal check wrongly indicated that the mask fitted on 18-31% of occasions, and wrongly indicated that it did not fit on 21-40% of occasions. These data indicate that the user seal check should not be used as a surrogate fit test. Its usefulness as a pre-use test must also be questioned. Abstract Negative pressure isolation rooms are used to house patients with highly contagious diseases (e.g. with airborne diseases) and to contain emitted pathogens to reduce the risk for cross-infection in hospitals. Airflows induced by door opening motion and healthcare worker passage can, however, transport the potentially pathogen laden air across the doorway. In this study airflow patterns across the isolation room doorway induced by the operation of single hinged and sliding doors with simulated human passage were examined. Smoke visualizations demonstrated that the hinged door opening generated a greater flow across the doorway than the sliding door. Tracer gas measurements showed that the examined ventilation rates (6 and 12 air changes per hour) had only a small effect on the air volume exchange across the doorway with the hinged door. The results were more variable with the sliding door. Supply-exhaust flow rate differential reduced the door motion-induced air transfer significantly with both door types. The experiments showed that the passage induced substantial air volume transport through the doorway with both door types. However, overall, the sliding door performed better in all tested scenarios, because the door-opening motion itself generated relatively smaller air volume exchange across the doorway, and hence should be the preferred choice in the design of isolation rooms. Abstract Background: The annual Hajj to the Kingdom of Saudi Arabia attracts millions of pilgrims from around the world. International health community's attention goes towards this mass gathering and the possibility of the development of any respiratory tract infections due to the high risk of acquisition of respiratory viruses. Method: We searched MEDLINE/PubMed and Scopus databases for relevant papers describing the prevalence of respiratory viruses among Hajj pilgrims. Results: The retrieved articles were summarized based on the methodology of testing for these viruses. A total of 31 studies were included in the quantitative/qualitative analyses. The main methods used for the diagnosis of most common respiratory viruses were polymerase chain reaction (PCR), culture and enzyme-linked immunosorbent assay (ELISA). Influenza, rhinovirus and parainfluenza were the most common viruses detected among pilgrims. Coronaviruses other than MERS-CoV were also detected among pilgrims. The acquisition of MERS-CoV remains very limited and systematic screening of pilgrims showed no infections. Conclusions: Well conducted multinational follow-up studies using the same methodology of testing are necessary for accurate surveillance of respiratory viral infections among Hajj pilgrims. Post-Hajj cohort studies would further evaluate the impact of the Hajj on the acquisition of respiratory viruses. * Corresponding author. Ministry of Health, Abstract DNA array High-throughput sequencing Virus identification Porcine reproductive and respiratory syndrome virus PRRSV Propidium monoazide PMA Ethidium bromide monoazide EMA a b s t r a c t Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membraneimpermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses. Abstract Epizootic rabbit enteropathy (ERE), a highly lethal (30-80% mortality) disease of broiler rabbits aged 6-14 weeks, first appeared in 1997 in French intensive enclosed rabbitries and is of unknown aetiology. Bacteriological, virological and parasitical examination of the intestinal contents of rabbits that had died either in spontaneous field cases or after experimental reproduction of ERE, were undertaken in an attempt to identify infectious agents that may play a role in the disease. Two bacterial strains, Clostridium perfringens and non-enteropathogenic Escherichia coli, were repeatedly isolated at high faecal counts from naturally infected animals. In field cases, a correlation between typical gross lesions of epizootic enteropathy and the presence of the alpha toxin of Cl. perfringens was observed (P < 0.0001; Chi-squared test). Although attempts to reproduce the disease by inoculation with different pools of cultivable bacterial strains failed, the disease was successfully reproduced by inoculation with one French and two Belgian samples of caecal contents. Abstract Meticillin was introduced in 1959 to treat infections caused by penicillin-resistant Staphylococcus aureus. In 1961 there were reports from the UK of S aureus isolates that had acquired resistance to meticillin (meticillin-resistant S aureus, MRSA). Similar MRSA isolates were soon found in other European countries, and later from Japan, Australia, and the USA. Today MRSA is a major cause of hospital-acquired infections, and a serious public-health concern. In this forum, we present different perspectives from across the globe to better understand the complexity of the problem, and examine the challenges that individual countries face in trying to control the spread of MRSA. Abstract The Japanese encephalitis virus (JE) structural glycoprotein (E) and two nonstructural glycoproteins (NSl and NSl') were processed differently by JE-infected vertebrate and invertebrate cell lines. All three proteins were released slowly (tllP > 6 hr) from JE-infected monkey cells (Vera cells). Mosquito cell lines released E at a similar rate (tl12 > 8 hr), while NSl and NSl' were retained in an undegraded form in the cell layer. The proteolytic processing of the three proteins appeared identical in both cell types, but some differences in N-linked glycosylation were observed. E, NSl, and NSl' found within the infected cells of both types contained high-mannose oligosaccharide groups for more than 8 hr after synthesis. Additional sugar residues were added to the single E protein oligosaccharide group prior to release from Vero cells, while sugar residues were trimmed from the E protein oligosaccharide group prior to release from mosquito cells. The forms of NSl and NSl'found in the culture fluid of infected Vero cells contained one complex and one highmannose oligosaccharide.All three glycoproteins released from JE-infected Vero cells were associated with extracellular particles, the virion in the case of E and a low density particle in the case of NSl and NSl'. Furthermore, E, NSl, and NSl' exhibited amphipathic properties in Triton X-l 14 extraction experiments. Taken together, these results suggest that both the structural (E) and nonstructural (NSl and NSl') glycoproteins were accumulated within the secretory pathway of the infected Vero cells, assembled into particles, and then released into the extracellular fluid. o tsss Academic Press, Inc. Abstract The last 2 decades have witnessed enormous changes in our understanding of allergic rhinitis. As we have begun to unravel the complex underlying immunologic and inflammatory pathophysiology of the disease, new therapeutic strategies as well as specific molecular and cellular constituents have emerged as potential targets for clinical intervention. These efforts also have shed light on the mechanisms by which current antiallergy medications act-or sometimes fail to be effe~tive.~, 31* 51*89 The similar pathophysiologic basis for allergic rhinitis and the often comorbid condition, asthma, was underscored in the recently published American Thoracic Society Workshop Summary on the Immunobiology of Asthma and Rhinitis: Pathogenic Factors and Therapeutic 0ptions.l8 In his conclusion, workshop chair, Thomas Casale,18 counsels readers to consider that ". . .allergic asthma and rhinitis represent a systemic disease affecting two organs, the lung and the nose. Asthma and allergic rhinitis share many of the same pathogenic factors, but they operate in different parts of the airway. Inflammatory cells and mediators are often the same, and there may be common alterations that occur in the immune system." Thus, therapeutic strategies and potential therapeutic agents found to be beneficial in the treatment of one airway target may show similar effects in the other. For this reason, and because many of the therapies now being developed are at early stages in their evolution, Abstract Recu le 6 octobre 1997, accept6 le 9 novembre 1997) RCsumC -Les virus representent une forme de pollution biologique vehiculee par les eaux udes. Dans une station d'epuration naturelle ou un pilote de laboratoire, le traitement biologique d'un effluent contamine entraine une reduction de la charge virale. Celle-ci est considerable ?I l'etape du traitement secondaire. Les bacteriophages sont considerees comme indicateurs de contamination f&ale. Ce sont Cgalement des modeles de virus endriques. Dans une etude experimentale, deux pilotes ont Ct.5 utilises (l'un a lagunage naturel, l'autre a boues activees) pour suivre la cinetique et Cvaluer le taux d'elimination du coliphage somatique 4X-174 et celui a ARN F-sp&ifique MS2. L'Climination des deux phages, qui atteint 99 % dans les deux systemes, est comparable a celle rapportee dans les etudes anterieures sur les virus enteriques. L'influence de plusieurs facteurs sur le retrait et l'inactivation virale (adsorption a la mat&e solide, action des microorganismes, effet des rayonnements solaires et des composes dissous) a CtC abordee. RevClCe dans des travaux anterieurs, l'importance de l'adsorption a Ctt montree dans le cas des pilotes et aussi en mettant en contact le $X-174 et le MS2 avec des mineraux argileux (la montmorillonite et le kaolin). L'effet des rayons solaires est connu dans le milieu marin. Dans une etude in vitro, une exposition de 12 h au soleil a cause l'inactivation quasi-totale des suspensions phagiques. Enfin, une baisse de la concentration phagique est enregistree aprbs un contact prolong& dans une eau de lagunage filtree. En revanche, la phagocytose des phages par Tetrahymena pyriformis n'a pas CtC Cvidente. 0 1998, Elsevier, Paris Climination virale 1 boues actides / lagunage / pilote * Correspondance et tires a part Abstract were denied by insurance. Of 41 patients initially screened, 10% lacked an available haploidentical donor, 29% had elevated donor-specific antibodies (DSAs), and 34% declined to proceed to HSCT. All 10 patients who were transplanted received peripheral blood stem cells. The initial 2 were conditioned with alemtuzumab/total body irradiation (TBI) 3 Gy followed by post-transplant cyclophosphamide and failed to engraft. The next 8 patients received the regimen developed at Johns Hopkins University with TBI 3 Gy. Granulocyte colony-stimulating factor was administered from day +12 in those with HbS < 30%. All 8 patients engrafted with a median time to neutrophil >.5 × 10 9 /L of 22 days (range, 18 to 23). One patient subsequently lost the graft, and 7 (87.5%) maintained >95% donor cell chimerism at 1-year post-HSCT. Two patients developed acute graft-versus-host disease (GVHD) of at least grade II. One had chronic GVHD and died >1 year after HSCT of unknown causes. With a median follow-up of 16 months (range, 11 to 29), 7 patients (87.5%) are alive. Our findings suggest that limited insurance coverage, high rate of DSAs, and patient declining HSCT may limit the availability of haploidentical HSCT in adult SCD patients. The modified Hopkins regimen used here demonstrates high engraftment and low morbidity rates and should be tested in larger, multicenter, prospective clinical trials. Abstract The 2014-2016 Ebola virus disease epidemic in West Africa highlighted challenges faced by the global response to a large public health emergency. Consequently, the US Centers for Disease Control and Prevention established the Global Rapid Response Team (GRRT) to strengthen emergency response capacity to global health threats, thereby ensuring global health security. Dedicated GRRT staff can be rapidly mobilized for extended missions, improving partner coordination and the continuity of response operations. A large, agencywide roster of surge staff enables rapid mobilization of qualified responders with wide-ranging experience and expertise. Team members are offered emergency response training, technical training, foreign language training, and responder readiness support. Recent response missions illustrate the breadth of support the team provides. GRRT serves as a model for other countries and is committed to strengthening emergency response capacity to respond to outbreaks and emergencies worldwide, thereby enhancing global health security. Abstract In Spain, human bocavirus (HBoV) was detected in 48 (9.1%) of 527 children with gastroenteritis at similar frequency as for children with respiratory illness (40/520, 7.7%). Fecal excretion adds new concern about the transmission of HBoV. To our knowledge, this report is the first to document HBoV in human feces. Abstract The authors report on a modified "3-day rule" for limiting low-yield stool cultures obtained from hospitalized patients 1054World Literature Review Abstract Background: Methods for the longitudinal study of respiratory virus infections are cumbersome and limit our understanding of the natural history of these infections in solid organ transplant (SOT) recipients. Objectives: To assess the feasibility and patient acceptability of self-collected foam nasal swabs for detection of respiratory viruses in SOT recipients and to define the virologic and clinical course. Study design: We prospectively monitored the course of symptomatic respiratory virus infection in 18 SOT patients (14 lung, 3 liver, and 1 kidney) using patient self-collected swabs. Results: The initial study sample was positive in 15 patients with the following respiratory viruses: rhinovirus (6), metapneumovirus (1), coronavirus (2), respiratory syncytial virus (2), parainfluenza virus (2), and influenza A virus (2). One hundred four weekly self-collected nasal swabs were obtained, with a median of 4 samples per patient (range 1-17). Median duration of viral detection was 21 days (range 4-77 days). Additional new respiratory viruses detected during follow-up of these 15 patients included rhinovirus (3), metapneumovirus (2), coronavirus (1), respiratory syncytial virus (1), parainfluenza virus (1), and adenovirus (1). Specimen collection compliance was good; 16/18 (89%) patients collected all required specimens and 79/86 (92%) follow-up specimens were obtained within the 7 ± 3 day protocoldefined window. All participants agreed or strongly agreed that the procedure was comfortable, simple, and 13/14 (93%) were willing to participate in future studies using this procedure. Conclusion: Self-collected nasal swabs provide a convenient, feasible, and patient-acceptable methodology for longitudinal monitoring of upper respiratory virus infection in SOT recipients. Abstract Lower respiratory infections remain one of the top global causes of death and the emergence of new diseases continues to be a concern. In the first two decades of the 21st century, we have born witness to the emergence of newly recognized coronaviruses that have rapidly spread around the globe, including severe acute respiratory syndrome virus (SARS) and Middle Eastern respiratory syndrome virus (MERS). We have also experienced the emergence of a novel H1N1 pandemic influenza strain in 2009 that caused substantial morbidity and mortality around the world and has transitioned into a seasonal strain. Although we perhaps most frequently think of viruses when discussing emerging respiratory infections, bacteria have not been left out of the mix, as we have witnessed an increase in the number of infections from Legionella spp. since the organisms' initial discovery in 1976. Here, we explore the basic epidemiology, clinical presentation, histopathology, and clinical laboratory diagnosis of these four pathogens and emphasize themes in humans' evolving relationship with our natural environment that have contributed to the infectious burden. Histology alone is rarely diagnostic for these infections, but has been crucial to bettering our understanding of these diseases. Together, we rely on the diagnostic acumen of pathologists to identify the clinicopathologic features that raise the suspicion of these diseases and lead to the early control of the spread in our populations. (A. Bryan).Pathology 36 (2019) 152-159 0740-2570/ Abstract One of the most important anthropic missions is preventing the global spread of infectious diseases. Vaccination is the only available preventive treatment for infectious diseases, but the availability of vaccines in developing countries is not adequate. We report a simple, easy-to-use, noninvasive hydrogel patch transcutaneous vaccination system. Antigen (Ag)-specific IgG production was induced by applying an Agimmersed patch to non-pretreated mouse auricle or hairless rat back skin. Immunofluorescence histochemical analysis revealed that Langerhans cells resident in the epidermal layer captured the antigenic proteins delivered by the hydrogel patch, which promoted the penetration of antigenic proteins through the stratum corneum, and that Ag-capturing Langerhans cells migrated into draining lymph nodes. Humoral immunity elicited by our transcutaneous vaccination system demonstrated neutralizing activity in both adenoviral infection and passive-challenge tetanus toxin experiments. The use of this hydrogel patch transcutaneous vaccination system will facilitate the global distribution of effective and convenient vaccines. Abstract Experimental infection studies with bluetongue virus (BTV) in the mammalian host have a history that stretches back to the late 18th century. Studies in a wide range of ruminant and camelid species as well as mice have been instrumental in understanding BTV transmission, bluetongue (BT) pathogenicity/pathogenesis, viral virulence, the induced immune response, as well as reproductive failures associated with BTV infection. These studies have in many cases been complemented by in vitro studies with BTV in different cell types in tissue culture. Together these studies have formed the basis for the understanding of BTV-host interaction and have contributed to the design of successful control strategies, including the development of effective vaccines. This review describes some of the fundamental and contemporary infection studies that have been conducted with BTV in the mammalian host and provides an overview of the principal animal welfare issues that should be considered when designing experimental infection studies with BTV in in vivo infection models. Examples are provided from the authors' own laboratory where the three Rs (replacement, reduction and refinement) have been implemented in the design of experimental infection studies with BTV in mice and goats. The use of the ARRIVE guidelines for the reporting of data from animal infection studies is emphasized. Abstract Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGNR are C-type lectins that serve both as cell adhesion and pathogen recognition receptors. Because of the essential role of the these molecules in the immune response, the implication of their alleles in human disease states, and the possible genetic variation at these loci among ethnically diverse populations, we undertook a study to analyze the full extent of DC-SIGN and DC-SIGNR polymorphisms in Caucasian Canadian and indigenous African populations. We report several novel nucleotide variants within regulatory 5=-and 3=-untranslated regions of the genes that could affect their transcription and translation. There were significant differences in the distribution of DC-SIGN and DC-SIGNR alleles among African and non-African populations. Finally, our study clearly demonstrates that Africans show greater genetic diversity at these two closely-related immune loci than observed in other major population groups. The differences may reflect evolutionary pressures generated by environmental factors, such as prevalent pathogens in these geographically distinct regions. Further studies will be needed to determine the net impact of DC-SIGN and DC-SIGNR genetic variants on the expression, translation, and function of the proteins and to understand how these functional polymorphisms may affect immune responses or immune escape. Abstract Background China has undergone rapid demographic and epidemiological changes in the past few decades, including striking declines in fertility and child mortality and increases in life expectancy at birth. Popular discontent with the health system has led to major reforms. To help inform these reforms, we did a comprehensive assessment of disease burden in China, how it changed between 1990 and 2010, and how China's health burden compares with other nations.We assessed relative performance of China against G20 countries (signifi cantly better, worse, or indistinguishable from the G20 mean) with age-standardised rates and 95% uncertainty intervals.Findings The leading causes of death in China in 2010 were stroke (1·7 million deaths, 95% UI 1·5-1·8 million), ischaemic heart disease (948 700 deaths, 774 500-1 024 600), and chronic obstructive pulmonary disease (934 000 deaths, 846 600-1 032 300). Age-standardised YLLs in China were lower in 2010 than all emerging economies in the G20, and only slightly higher than noted in the USA. China had the lowest age-standardised YLD rate in the G20 in 2010. China also ranked tenth (95% UI eighth to tenth) for HALE and 12th (11th to 13th) for life expectancy. YLLs from neonatal causes, infectious diseases, and injuries in children declined substantially between 1990 and 2010. Mental and behavioural disorders, substance use disorders, and musculoskeletal disorders were responsible for almost half of all YLDs. The fraction of DALYs from YLDs rose from 28·1% (95% UI 24·2-32·5) in 1990 to 39·4% (34·9-43·8) in 2010. Leading causes of DALYs in 2010 were cardiovascular diseases (stroke and ischaemic heart disease), cancers (lung and liver cancer), low back pain, and depression. Dietary risk factors, high blood pressure, and tobacco exposure are the risk factors that constituted the largest number of attributable DALYs in China. Ambient air pollution ranked fourth (third to fi fth; the second highest in the G20) and household air pollution ranked fi fth (fourth to sixth; the third highest in the G20) in terms of the age-standardised DALY rate in 2010.Interpretation The rapid rise of non-communicable diseases driven by urbanisation, rising incomes, and ageing poses major challenges for China's health system, as does a shift to chronic disability. Reduction of population exposures from poor diet, high blood pressure, tobacco use, cholesterol, and fasting blood glucose are public policy priorities for China, as are the control of ambient and household air pollution. These changes will require an integrated government response to improve primary care and undertake required multisectoral action to tackle key risks. Analyses of disease burden provide a useful framework to guide policy responses to the changing disease spectrum in China. Abstract At present more than 250 FDA approved chlorine containing drugs were available in the market and many pharmaceutically important drug candidates in pre-clinical trials. Thus, it is quite obvious to expect that in coming decades there will be an even greater number of new chlorine-containing pharmaceuticals in market. Chlorinated compounds represent the family of compounds promising for use in medicinal chemistry. This review describes the recent advances in the synthesis of chlorine containing heterocyclic compounds as diverse biological agents and drugs in the pharmaceutical industries for the inspiration of the discovery and development of more potent and effective chlorinated drugs against numerous death-causing diseases. Abstract Bovine herpesvirus-1 (BHV-1) is a major cause of respiratory tract diseases in cattle. Vaccination of cattle against BHV-1 is a high priority. A major concern of currently modified live BHV-1 vaccines is their ability to cause latent infection and subsequent reactivation resulting in many outbreaks. Thus, there is a need for alternative strategies. We generated two recombinant Newcastle disease viruses (NDVs) expressing the glycoprotein D (gD) of BHV-1 from an added gene. One recombinant, rLaSota/gDFL, expressed gD without any modification. The other recombinant, rLaSota/gDF, expressed a chimeric gD in which the ectodomain of gD was fused with the transmembrane domain and cytoplasmic tail of the NDV fusion F glycoprotein. Remarkably, the native gD expressed by rLaSota/gDFL virus was incorporated into the NDV virion 2.5-fold more efficiently than the native NDV proteins, whereas the chimeric gD was not detectably incorporated even though it was abundantly expressed on the infected cell surface. The expression of gD did not increase the virulence of the rNDV vectors in chickens. A single intranasal and intratracheal inoculation of calves with either recombinant NDV elicited mucosal and systemic antibodies specific to BHV-1, with the responses to rLaSota/gDFL being higher than those to rLaSota/gDF. Following challenge with BHV-1, calves immunized with the recombinant NDVs had lower titers and earlier clearance of challenge virus compared to the empty vector control, and reduced disease was observed with rLaSota/gDFL. Following challenge, the titers of serum antibodies specific to BHV-1 were higher in the animals immunized with the rNDV vaccines compared to the rNDV parent virus, indicating that the vaccines primed for secondary responses. Our data suggest that NDV can be used as a vaccine vector in bovines and that BHV-1 gD may be useful in mucosal vaccine against BHV-1 infection, but might require augmentation by a second dose or the inclusion of additional BHV-1 antigens. Abstract Nature is the most abundant source for novel drug discovery. Lycorine is a natural alkaloid with immense therapeutic potential. Lycorine is active in a very low concentration and with high specificity against a number of cancers both in vivo and in vitro and against various drug-resistant cancer cells. This review summarized the therapeutic effect and the anticancer mechanisms of lycorine. At the same time, we have discussed the pharmacology and comparative structure-activity relationship for the anticancer activity of this compound. The researches outlined in this paper serve as a foundation to explain lycorine as an important lead compound for new generation anticancer drug design and provide the principle for the development of biological strategies to utilize lycorine in the treatment of cancers. Abstract Although the history of genomics research can be traced back to the 1970s, with the development of DNA sequencing technology, Reviews GENE TO SCREEN Corresponding author: Rappuoli, R. (rino.rappuoli@novartis.com) 252 www.drugdiscoverytoday.com Abstract The human immunodeficiency virus (HIV) pandemic is driving the re-emergence of tuberculosis (TB) as a global health threat, both by increasing the susceptibility of HIV-infected people to infection with Mycobacterium tuberculosis (Mtb), and increasing the rate of emergence of drug-resistant Mtb. There are several other clinical challenges for treatment of co-infected patients including: expense, pill burden, toxicity, and malabsorption that further necessitate the search for new drugs that may be effective against both pathogens simultaneously. The anti-helminthic niclosamide has been shown to have activity against a laboratory strain of Mtb in liquid culture while bacteriostatic activity against non-replicating M. abscessus was also recently described. Here we extend these findings to further demonstrate that niclosamide inhibits mycobacterial growth in infected human macrophages and mediates potent bacteriostatic activity against the virulent Mtb Beijing strain. Importantly, we provide the first evidence that niclosamide inhibits HIV replication in human macrophages and Jurkat T cells through post-integration effects on pro-virus transcription. The dual antiviral and anti-mycobacterial activity was further observed in an in vitro model of HIV and Mtb co-infection using human primary monocyte-derived macrophages. These results support further investigation of niclosamide and derivatives as anti-retroviral/antimycobacterial agents that may reduce clinical challenges associated with multi-drug regimens and drug resistance. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Asthma, rhinitis, other respiratory diseases Background: Although known as common causes of upper respiratory infections, rhinoviruses, enteroviruses, and coronaviruses are poorly studied as inducers of wheezing in infants, and their possible role in the development of childhood asthma has not been investigated. Objective: The purposes of this study were to assess the occurrence of RV, enterovirus, and coronavirus infections in wheezing infants and to evaluate the association of these viral findings with early school-age asthma. Methods: In 1999, outcome in relation to asthma was studied in 82 of 100 initially recruited children who had been hospitalized for wheezing in infancy during the period 1992-1993. In 2000, etiologic viral studies regarding the index episode of wheezing were supplemented by rhinovirus, enterovirus, and coronavirus detection by RT-PCR from frozen nasopharyngeal aspirates in 81 of the children for whom adequate samples were available. Of these children, 66 had participated in the follow-up in 1999. Results: Rhinoviruses were identified in 27 (33%) of the 81 children, enteroviruses in 10 (12%), and coronaviruses in none. Rhinoviruses were present as single viral findings in 22 (81%) of the 27 rhinovirus-positive cases, and rhinovirus infections were associated with the presence of atopic dermatitis in infancy. Enteroviruses were commonly encountered in mixed infections and had no association with atopy. As single viral findings, rhinoviruses were associated with the development of asthma (P = .047; odds ratio, 4.14; 95% CI, 1.02-16.77 versus rhinovirus-negative cases [by logistic regression adjusted for age, sex, and atopic dermatitis on entry)]. Conclusion: Our results present rhinoviruses as important inducers of wheezing even in infancy. The association with atopy and subsequent asthma calls for reevaluation of the role of rhinoviruses in the development of asthma. (J Allergy Clin Immunol 2003;111:66-71.) Abstract Of evolutionary biology's many practical applications, those in medicine are the most obvious and potentially the most important. So far, however, medicine, nursing and public health have made use of only a fraction of what evolution has to offer. The magnitude of the gap is impressive. Studies of medical education found that most medical schools in the UK and the USA have not one evolutionary biologist on the faculty (Nesse and SchiffmanEvolutionary biology is an essential basic science for medicine, but few doctors and medical researchers are familiar with its most relevant principles. Most medical schools have geneticists who understand evolution, but few have even one evolutionary biologist to suggest other possible applications. The canyon between evolutionary biology and medicine is wide. The question is whether they offer each other enough to make bridge building worthwhile. What benefits could be expected if evolution were brought fully to bear on the problems of medicine? How would studying medical problems advance evolutionary research? Do doctors need to learn evolution, or is it valuable mainly for researchers? What practical steps will promote the application of evolutionary biology in the areas of medicine where it offers the most?To address these questions, we review current and potential applications of evolutionary biology to medicine and public health. Some evolutionary technologies, such as population genetics, serial transfer production of live vaccines, and phylogenetic analysis, have been widely applied. Other areas, such as infectious disease and aging research, illustrate the dramatic recent progress made possible by evolutionary insights. In still other areas, such as epidemiology, psychiatry, and understanding the regulation of bodily defenses, applying evolutionary principles remains an open opportunity. In addition to the utility of specific applications, an evolutionary perspective fundamentally challenges the prevalent but fundamentally incorrect metaphor of the body as a machine designed by an engineer. Bodies are vulnerable to disease -and remarkably resilient -precisely because they are not machines built from a plan. They are, instead, bundles of compromises shaped by natural selection in small increments to maximize reproduction, not health. Understanding the body as a product of natural selection, not design, offers new research questions and a framework for making medical education more coherent. We conclude with recommendations for actions that would better connect evolutionary biology and medicine in ways that will benefit public health. It is our hope that faculty and students will send this article to their undergraduate and medical school Deans, and that this will initiate discussions about the gap, the great opportunity, and action plans to bring the full power of evolutionary biology to bear on human health problems. Abstract This chapter describes how the impact of psychological trauma and posttraumatic stress disorder (PTSD) differ, depending on individual differences and the social and cultural context and culture-specific teachings and resources available to individuals, families, and communities. We use the framework (Kira et al., 2011) formulated to differentiate the impact on the individual's or group's (i) personal, unique physical characteristics, including skin color, racial background, gender, and sexual orientation; and (ii) family, ethnocultural, and community membership, including majority or minority group status, religious beliefs and practices, socioeconomic resources, and political and civic affiliations. While personal and social factors can be a positive resource contributing to safety and well-being, they also can be a basis for placing the person or group in harm's way.Disadvantaged persons and communities-such as those in poverty or who face stigma and discrimination, disabilities, homelessness, political repression, communal/ societal violence (including paramilitary, military, and gang warfare), forced immigration (refugees and asylum seekers), interrogation and torture, terrorism, genocide, and catastrophic disasters-are particularly vulnerable both to being exposed to traumatic stressors and to developing PTSD and associated psychiatric disorders and psychosocial problems (Betancourt et al.This vulnerability largely is explained by exposure to chronic and cumulative stressors (such as stigma, discrimination, and poverty), ongoing interpersonal and group victimization, intergroup conflict, systems of belief and tradition, and exposure to chronic or particularly horrific traumatic stressors (including the often lengthy aftermath of catastrophic disasters, and societal conflicts), rather than being due solely to ethnic or cultural differences (Kira et al., 2011) .Racially or ethnoculturally based discrimination is widely recognized as having been a scourge on the human race for millennia. Genocide-the attempt to eradicate an entire racial, ethnic, or cultural group-has caused untold suffering and cost the lives of hundreds of millions of people across the planet throughout human history. The term historical trauma has been coined to describe the traumatic impact that racially or ethnoculturally based genocide has on not only the immediate targets but moreover on their progeny for generations afterward. The living memory of traumatic assaults on the survival and human dignity of entire groups or societies can persist as a form of vigilance against any sign of a potential recurrence of the genocide.Posttraumatic Stress Disorder 504 Posttraumatic Stress Disorder 506 5. PTSD therapists must avoid stereotypic assumptions in order to ensure that assessment and treatment are genuinely collaborative and sensitive to each client's ethnocultural and other expectations, goals, and preferences. A culturally competent approach to treating PTSD begins with a collaborative discussion in which the therapist adopts the stance of a respectful visitor to the client's outer and inner world-clarifying the client's expectations and preferences, and the meaning of sensitive interpersonal communication modalities (such as spatial proximity, gaze, choice of names, private versus public topics, synchronizing of talk and listening, use of colloquialisms, providing advice or education). 6. A systematic assessment of trauma history and PTSD thus should include not only a listing of events and symptomatic or resilient responses in the aftermath but how the person interpreted these events and reactions based on his or her cultural framework, beliefs, and values. 7. In contrast to the general pattern of stigma-related violence being directed toward girls and women, gay and bisexual boys and men were more likely to report violent victimization or threats than lesbian or bisexual women or girls. The findings from a survey of gay, lesbian, and bisexual (GLB) adolescents suggest that stigma and victimization begin early in life, with physical and sexual attacks occurring as early as ages 8-9 years old. One in 11 GLB adolescents have been found to have PTSD, as much as 20 times more than other children and adolescents. 8. Poverty puts people at risk for traumatic violence, and not having a stable residence (homelessness) compounds this risk and further increases the likelihood of developing PTSD and associated mental health distress and health conditions. 9. Political repression, genocide, and mass armed conflicts involve psychological (and often physical as well) assaults by the people and institutions in power on people, families, communities, and organizations who are deprived of access to political power and socioeconomic resources-and therefore also on their fundamental freedoms and values. Without access to self-determination and the resources necessary to sustain independence, people are vulnerable not just to traumatic exploitation and violence but also to the traumatic loss of their intimate relationships, their families, their way of life, and their values. 10. Refugees and "internally displaced" or "stateless" persons who have been displaced from or lost their families, homes, and communities due to armed conflicts or mass disasters (such as tsunamis, hurricanes, or earthquakes) are at least 10 times (and as much as 30 times) more likely than other children or adults to develop PTSD, with as many as one in three refugees or disaster survivors developing Acute Stress Disorder (ASD) or PTSD. Abstract An anti-influenza compound, DP2392-E10 based on inhibition of the nuclear export function of the viral nucleoprotein-nuclear export signal 3 (NP-NES3) domain was successfully identified by our previous highthroughput screening system. Here, we demonstrated that DP2392-E10 exerts its antiviral effect by inhibiting replication of a broad range of influenza A subtypes. In regard to the molecular mechanism, we revealed that DP2392-E10 inhibits nuclear export of both viral NP and nuclear export protein (NEP). More specifically, in vitro pull-down assays revealed that DP2392-E10 directly binds cellular CRM1, which mediates nuclear export of NP and NEP. In silico docking suggested that DP2392-E10 binds at a region close to the HEAT9 and HEAT10 domains of CRM1. Together, these results indicate that the CRM1-mediated nuclear export function of influenza virus represents a new potential target for antiviral drug development, and also provide a core structure for a novel class of inhibitors that target this function. Abstract The use of vaccines is an effective and relatively inexpensive means of controlling infectious diseases, which cause heavy economic losses to the livestock industry through animal loss, decreased productivity, treatment expenses and decreased carcass quality. However, some vaccines produced by conventional means are imperfect in many respects including virulence, safety and efficacy. Moreover, there are no vaccines for some animal diseases. Although genetic engineering has provided new ways of producing effective vaccines, the cost of production for veterinary use is a critical criterion for selecting the method of production and delivery of vaccines. The cost effective production and intrinsic ability to enter cells has made adenovirus vectors a highly efficient tool for delivery of vaccine antigens. Moreover, adenoviruses induce both humoral and cellular immune responses to expressed vaccine antigens. Since nonhuman adenoviruses are species specific, the development of animal specific adenoviruses as vaccine delivery vectors is being evaluated. This review summarizes the work related to the development of bovine adenovirus-3 as a vaccine delivery vehicle in animals, particularly cattle. Abstract Passenger throughput at Hong Kong International Airport (HKIA) has shown steady growth since its opening. Various aspects relating to HKIA have been studied in prior literature. This paper investigated changes in HKIA's passenger network for the period of 2001e2012 and used the gravity model to examine the key factors explaining its passenger traffic flows. The findings suggested that HKIA's passenger network has changed significantly and expanded to many new different destinations. Two regions (East Asia and Southeast Asia) were the most important markets for HKIA, and most key destinations connected by Hong Kong showed healthy growth. Nine factors could explain passenger traffic flows between Hong Kong and its key destinations: Hong Kong GDP per capita, GDP per capita of destinations connected by HKIA, distance, airport hub status of the destination airport, tourist destinations connected by HKIA, numbers of passenger airlines in service, bilateral trade flows, speaking a common language and having strong cultural/colonial links with Hong Kong, and the route presence of Cathay Pacific.j o u rn a l h o m e p a g e : w w w . e ls e v i e r . c o m / l o c a t e / j a i r t r a m a n http://dx. Abstract Provide cut-off times for processing test requests and turnaround times for reporting resultsProvide guidelines for specimen collection and transportMaintain an effective computerized system for acknowledging receipt of specimens, testing in-progress, and reporting of results; immediate notification to the physician of critical results should be a component of an overall communication systemPeriodic publication of antimicrobial susceptibility patterns for the most commonly isolated bacteria in the institution Maintain a program of quality control that ensures the accuracy of all offered tests Establish a laboratory that conforms to regulatory standards A system of short-term storage of all specimens and long-term storage of important isolates should be established to facilitate additional testing if requiredMaintain knowledge of the laboratory test menu and specimen collection and transport guidelinesAlert the laboratory when a specific organism is sought (e.g., a fastidious or highly pathogenic organism)Establish an open communication with the laboratory director when testing needs are not satisfied by the available test menu or special handling of a specimen is requiredAspirate of pus or fluid in anaerobic transport vial is preferred; swabs usually have insufficient material for stains and culture. Clean surface of closed abscess with 70% alcohol; collect specimens at margins of abscess. Aspirates in anaerobic transport tubes are acceptable for aerobic and anaerobic bacterial, fungal, and mycobacterial cultures.Specimen sent immediately in capped syringe after the air is excluded is also acceptable. Specify location of abscess for optimal processing; provide all other pertinent information (e.g., surgical infection, trauma, bite wound).Routine aerobic and anaerobic bacteria Disinfect bottle tops with 70% isopropyl alcohol; disinfect phlebotomy site with alcohol, followed by tincture of iodine or chlorhexidine; allow the disinfectants to dry. Collect 10-20 mL/adults and 1-3 mL/child for each blood culture; divide blood into two blood culture bottles, preferably an aerobic and an anaerobic bottle; collect two to three cultures per 24-hr period. Abstract In support of accelerated development of Ebola vaccines from preclinical research to clinical trials, in November, 2014, the Wellcome Trust and the Center for Infectious Disease Research and Policy (CIDRAP) at the University of Minnesota established the Wellcome Trust-CIDRAP Ebola Vaccine Team B initiative. This ongoing initiative includes experts with global experience in various phases of bringing new vaccines to market, such as funding, research and development, manufacturing, determination of safety and effi cacy, regulatory approval, and vaccination delivery. It also includes experts in community engagement strategies and ethical issues germane to vaccination policies, including eight African scientists with direct experience in developing and implementing vaccination policies in Africa. Ebola Vaccine Team B members have worked on a range of vaccination programmes, such as polio eradication (Africa and globally), development of meningococcal A disease vaccination campaigns in Africa, and malaria and HIV/AIDS vaccine research. We also provide perspective on how this experience can inform future situations where urgent development of vaccines is needed, and we comment on the role that an independent, expert group such as Team B can have in support of national and international public health authorities toward addressing a public health crisis. Abstract In recent years, there has been increased interest in topic-focused multi-document summarization. In this task, automatic summaries are produced in response to a specific information request, or topic, stated by the user. The system we have designed to accomplish this task comprises four main components: a generic extractive summarization system, a topic-focusing component, sentence simplification, and lexical expansion of topic words. This paper details each of these components, together with experiments designed to quantify their individual contributions. We include an analysis of our results on two large datasets commonly used to evaluate task-focused summarization, the DUC2005 and DUC2006 datasets, using automatic metrics. Additionally, we include an analysis of our results on the DUC2006 task according to human evaluation metrics. In the human evaluation of system summaries compared to human summaries, i.e., the Pyramid method, our system ranked first out of 22 systems in terms of overall mean Pyramid score; and in the human evaluation of summary responsiveness to the topic, our system ranked third out of 35 systems. Abstract N orovirus infection is a major cause of endemic and epidemic acute gastroenteritis. These viruses have been classified into 7 genogroups on the basis of the major capsid protein, VP1. Noroviruses Abstract Human rhinoviruses (HRV), members of the Picornaviridae family, are comprised of over 100 different virus serotypes. HRV represent the single most important etiological agents of the common cold [Arruda, E., Pitkaranta, A., Witek Jr., T.J., Doyle, C.A., Hayden, F.G., 1997. Frequency and natural history of rhinovirus infections in adults during autumn. Abstract This paper critically examines the ways that tuberculosis (TB) has been represented in the print media in New Zealand over recent years (2002)(2003)(2004). Our broad contention is that, notwithstanding its biomedical reality, TB is socially constructed by, and through, human experience. Further, public health practitioners depend, to a large extent, on the media to alert the public to threats of disease and opportunities for protection. However, the messages conveyed are sometimes neither helpful nor accurate. In our analysis of TB coverage in three major daily newspapers in New Zealand, we enumerate and classify references to the disease, as well as undertake a discursive analysis of the revealed themes. Of the 366 texts we retrieved in the database search, we selected 120 for in-depth analysis. Our examination indicated the importance of bovine TB within the national consciousness, the stigmatised character of TB and the association between TB and immigrants. We observe that newspaper 'stories' in general, and commentaries by public health officials in particular, are invariably offered on a 'case by case' basis. We conclude that this specificity in time and place avoids more challenging discourses linking TB with deeply embedded determinants of health such as the strong link between TB and poverty. r Abstract We describe the use of epidural analgesia in a 39-year-old G2P1 parturient presenting at 38 +6 weeks estimated gestation with confirmed influenza A H1N1 and superimposed bilateral pneumonia. Although the patient had an uncomplicated intra-and post-partum course, little is known about the safety of performing neuraxial analgesia or anesthesia in patients with influenza. The prevalence of viremia and possible translocation of blood-borne virus to the central nervous system are discussed.Magnesium is commonly used in the prevention of eclampsia. Reports of acute toxicity are rare and we are not aware of detailed management algorithms. We present a case of acute magnesium toxicity presenting as ventilatory impairment and failure to rouse adequately from general anaesthesia. The patient was managed with controlled ventilation, further sedation, intravenous calcium gluconate, forced diuresis and dextrose-insulin infusion. We present a guideline for the management of life-threatening magnesium toxicity and discuss measures that may prevent future similar occurrences.Crown Abstract Human serum albumin (HSA) Niclosamide SterneVolmer equationThermodynamic parameters a b s t r a c t A study of the binding of niclosamide (NC) to serum proteins such as human serum albumin, hemoglobin, and globulin was carried out using fluorescence and UV-visible spectroscopy. Interactions between NC and these proteins were estimated by Stern eVolmer and van't Hoff equations. The binding constants and the thermodynamic parameters, DH, DS, and DG at different temperatures were also determined by using these equations. Data showed that NC may exhibit a static quenching mechanism with all proteins. The thermodynamic parameters were calculated. Data showed that van der Waals interactions and hydrogen bonds are the main forces for human serum albumin and hemoglobin. Globulin, however, bound to NC via hydrophobic interaction. The spectral changes of synchronous fluorescence suggested that both the microenvironment of NC and the conformation of the proteins changed in relation to their concentrations during NC's binding. Abstract Histophilus somni (formerly Haemophilus somnus) is a Gram-negative pleomorphic coccobacillus that causes respiratory, reproductive, cardiac and neuronal diseases in cattle. H. somni is a member of the bovine respiratory disease complex that causes severe bronchopneumonia in cattle. Previously, it has been reported that bovine neutrophils and macrophages have limited ability to phagocytose and kill H. somni. Recently, it was discovered that bovine neutrophils and macrophages produce extracellular traps in response to Mannheimia haemolytica, another member of the bovine respiratory disease complex. In this study, we demonstrate that H. somni also causes extracellular trap production by bovine neutrophils in a dose-and time-dependent manner, which did not coincide with the release of lactate dehydrogenase, a marker for necrosis. Neutrophil extracellular traps were produced in response to outer membrane vesicles, but not lipooligosacchride alone. Using scanning electron microscopy and confocal microscopy, we observed H. somni cells trapped within a web-like structure. Further analyses demonstrated that bovine neutrophils trapped and killed H. somni in a DNA-dependent manner. Treatment of DNA extracellular traps with DNase I freed H. somni cells and diminished bacterial death. Treatment of bovine monocyte-derived macrophages with H. somni cells also caused macrophage extracellular trap formation. These findings suggest that extracellular traps may play a role in the host response to H. somni infection in cattle. Abstract The 3 untranslated region (UTR) of porcine arterivirus genome plays a pivotal role for virus replication, yet the properties of 3 UTR remain largely undefined. We conducted site-directed mutagenesis to the 3 UTR of the type II porcine reproductive and respiratory syndrome virus (PRRSV). Serial deletions of the 3 UTR showed that at least 40 nucleotides immediately following the ORF7 stop codon were dispensable for the viability of PRRSV in cultured cells. We then constructed a chimeric PRRSV cDNA clone using type II PRRSV as the backbone containing the 3 UTR from the type I PRRSV. The chimeric virus was viable and shared similar properties with the parental virus. Our results provided the first description of the 40nt dispensable region in type I PRRSV 3 UTR, and further predicted structure demonstrated that the high-order structure of 3 UTR might play significant roles in its function. Abstract Background: The emergence of novel respiratory viruses such as avian influenza A(H7N9) virus and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) highlights the importance of understanding determinants of transmission to healthcare workers (HCWs) and the public. Objectives: We aim to determine the viral content of the air emitted by symptomatic inpatients or longterm care residents with laboratory-confirmed influenza virus infection (emitters), and in the breathing zones of healthcare workers who attend to them. Design: A prospective pilot study of patients with laboratory-confirmed influenza virus infection was undertaken. Air within 1 m of the patient was sampled using a high volume air sampler. In addition, a lower volume air sampler was placed <1 m from the patient, with another >1 m from the patient. Viral RNA was recovered from the samplers and submitted for quantitative real time PCR. In addition, personal button samplers were provided to HCWs. Results: The air emitted by 15 participants with laboratory-confirmed influenza virus infection was sampled. Of the patients infected with influenza A, viral RNA was recovered from the air emitted by 9/12 patients using the low-volume sampler; no viral RNA was detected from air emitted by patients with influenza B (n = 3). Influenza virus RNA was recovered from one HCW's sampler. Conclusions: Patients with respiratory virus infection emit virus into the air which disperses to >1 m and may reach the breathing zone of a HCW. This pilot study highlights the feasibility and importance of conducting a larger-scale study to identify determinants of exposure and transmission from patient to HCW. Abstract Eukaryotic cellular and most viral RNAs carry a 5 0 -terminal cap structure, a 5 0 -5 0 triphosphate linkage between the 5 0 end of the RNA and a guanosine nucleotide (cap-0). SARS coronavirus (SARS-CoV) nonstructural protein nsp16 functions as a methyltransferase, to methylate mRNA cap-0 structure at the ribose 2 0 -O position of the first nucleotide to form cap-1 structures. However, whether there is interplay between nsp16 and host proteins was not yet clear. In this report, we identified several potential cellular nsp16-interacting proteins from a human thymus cDNA library by yeast two-hybrid screening. VHL, one of these proteins, was proven to interact with nsp16 both in vitro and in vivo. Further studies showed that VHL can inhibit SARS-CoV replication by regulating nsp16 ubiquitination and promoting its degradation. Our results have revealed the role of cellular VHL in the regulation of SARS-CoV replication. Abstract P53, a vital anticancer gene, controls the transcription and translation of a series of genes, and implement the cell cycle arrest and cell apoptosis by regulating their complicated signal pathways. Under radiotherapy, cell can trigger internal self-defense mechanisms in fighting against genome stresses induced by acute ion radiation (IR). To simulate the investigating of cellular responding acute IR at single cell level further, we propose a model of P53 gene regulatory networks under radiotherapy. Our model can successfully implement the kinetics of double strand breaks (DSBs) generating and their repair, ataxia telangiectasia mutated (ATM) activation, as well as P53-MDM2 feedback regulating. By comparing simulations under different IR dose, we can try to find the optimal strategy in controlling of IR dose and therapy time, and provide some theoretical analysis to obtain much better outcome of radiotherapy further. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. J. Dairy Sci. 96 :981-992 http://dx.Farmers attempting to reduce first-calving age (FCA) need to understand which rearing management factors influence FCA and first-lactation milk production (FLP). Reduced FCA might be associated with lower FLP. This study describes the association between herd FCA, FLP, and several herd-level health and rearing management variables and describes the association between FCA and FLP at the cow level. It uses data from a 2010 survey of 100 Dutch dairy farms about general management, colostrum and milk feeding, housing, cleanliness, healthcare, disease, and breeding. It also used available data on FCA and 305-d FLP at both cow and herd level. The associations between median FCA and median FLP of the herd and herd-level health and rearing management variables were determined using multivariate regression analysis. The median FCA was associated with minimum age of first insemination, feeding of waste milk, and the amount of milk given preweaning. The median FLP was associated with median FCA and vaccination status for bovine respiratory syncytial virus. The association between FCA and FLP (based on 8,454 heifers) was analyzed with a single-effect linear mixed model, where the dependent variable was either FCA or relative FCA (defined as the difference between FCA of the heifer and median FCA of the herd to which they belonged). Heifers having an FCA of 24 mo produced, on average, 7,164 kg of milk per 305 d, and calving 1 mo earlier gave 143 kg less milk per 305 d. When FCA did not deviate from the median herd FCA, heifers produced, on average, 7,272 kg of milk per 305 d. From the median FCA of the herd, heifers calving 1 mo earlier produced 90 kg of milk per 305 d less, and heifers calving 1 mo later produced 86 kg per 305 d more. This is the first study that explained FLP using relative FCA. It assumes that heifers raised within the same farm have similar development because they are similarly managed. Similar management is reflected by the median FCA of the herd, with a deviation of the heifer's FCA from median FCA reflecting the heifer's development relative to the herd's average. The advantage of using relative FCA was that it accounts for between-farm differences in rearing management. It showed that earlier insemination without adjusting management to ensure sufficient development lowers FLP. An economic optimum exists between rearing costs, FCA, and FLP and, as a consequence, decisions with regard to young stock management should be made with care. Abstract Streptococcus mutans is a bacterial cause of dental caries that is resistant to bacitracin. The aim of this study was to elucidate the mbrABCD-related bacitracin resistance mechanism of S. mutans. Transcriptome data demonstrated that the expression levels of 33 genes were induced more than twofold by bacitracin. Fourteen genes were selected from the upregulated genes, and defective mutants of these genes were constructed for measurement of their sensitivity to bacitracin. Among the mutants, only the mbrA-or mbrB-deficient mutants exhibited 100-to 121-fold greater sensitivity to bacitracin when compared with the wild-type strain. Moreover, knockout of the mbrC and mbrD genes abolished the bacitracin-induced mbrAB upregulation. These results suggest that both mbrC and mbrD are required for mbrAB upregulation that confers the bacitracinresistant phenotype on S. mutans. Abstract This study compares some etiological factors involved in the naturally occurrence of lamb scours in 18 Merino sheep farms randomly selected in the area of "La Serena" (Southwest Spain).A lack of influence of some variables (flock size, type of facilities, type of breeding, lambing percentage, isolation of Campylobacter jejuni, Rotavirus spp., Coronavirus spp. and Salmonella spp.) on lamb mortality rate was detected. The opposite was true for cleaning of the lambing areas, organization of lambing periods, accumulation of lambs in the pens, high content of fat, protein and lactose in milk and low serum gamma globulin and total protein in lambs and ewes. Cryptosporidium spp. and Escherichia coli isolation showed a trend to be linked to the presence of diarrhoea. A positive correlation was found between serum total protein in ewes and serum gamma globulin in lambs. Thus, the measure of serum total protein in ewes before lambing would be a valuable indicator of the risk of lamb scouring and it would be useful to establish an adequate programme to prevent this condition in the farms of this geographical area. Abstract The study explores the characteristics of China's independent research articles published from 1980 to 2011, based on the database of Science Citation Index Expanded. The publication outputs of seven major industrialized countries including Canada, France, Japan, Germany, Italy, the UK, and the USA were compared with China. Annual production, field performance, research emphases and trends, top articles, as well as main institutional and individual contributors by its top cited articles were analyzed. Some newly developed indicators related to words in title, author keywords, KeyWords Plus, first author, corresponding author, and Y-index were employed to provide in-depth information on topic and author contributions. Results showed that China has been closing the gap with the USA with the greatest growth, and has stood the second since 2006. Most top cited articles were published in 2000s, made up approximately seven tenths of total articles. Pronounced activities were found in chemistry and physics related categories. The core categories included multidisciplinary chemistry, physical chemistry, multidisciplinary materials science, and applied physics. Moreover, China's performance of nanotechnology and science, especially carbon nanotubes, nanoparticles, nanowires, and nanostructures showed dramatic growth. Six top articles with at least 1000 citations were examined, and were observed to concern medicine, nanotube, and adsorption. In addition, main contributing institutions and authors were also revealed and evaluated. Chinese Academy of Sciences played a dominant role, and Tsinghua University, Peking University and five universities in Hong Kong showed good scientific performance. Abstract Severity assessment s u m m a r y Pandemic (H1N1) 2009 poses a serious global health threat. However, the global impact of this new pandemic remains uncertain. Past pandemics had different impacts on mortality which varied between countries. Several countries in South-east Asia have already developed their national pandemic preparedness plans. However, these plans have focused on surveillance for and response to the highly pathogenic avian influenza (H5N1), including the rapid containment of H5N1. The newly emerged pandemic (H1N1) 2009 is different from H5N1 in terms of severity and requires different approaches. There are several factors that can potentially affect the severity of pandemic (H1N1) 2009, including a population's vulnerability and response capacity. The pattern of severity appears to be changing with the spread of pandemic (H1N1) 2009, which can be conceptualized in a stepwise manner based on observation of the current situation. The overall impact of pandemic (H1N1) 2009 remains unknown and it is difficult to assess its severity. However,there is an urgent need to assess its potential severity based on the available data so that appropriate responses can be provided in order to mitigate its impact. Abstract An 9.4-kDa antifungal peptide designated as campesin was isolated from seeds of the cabbage Brassica campestris. The isolation procedure involved affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Q-Sepharose and Mono S, and gel filtration on Superdex 75 and Superdex Peptide. The peptide was adsorbed on the first three chromatographic media. It exerted an inhibitory action on mycelial growth including Fusarium oxysporum and Mycosphaerella arachidicola, with an IC 50 of 5.1 μM and 4.4 μM, respectively. The peptide was characterized by remarkable thermostability and pH stability. It inhibited proliferation of HepG2 and MCF cancer cells with an IC 50 of 6.4 μM and 1.8 μM, and the activity of HIV-1 reverse transcriptase with an IC 50 of 3.2 μM. It demonstrated lysolecithin binding activity. Abstract 2009 H1N1 Non-pharmaceutical intervention Model of goal-directed behavior Travel intention a b s t r a c t Theoretically, in the tourism context this study introduced a new concept of non-pharmaceutical intervention (NPI) for influenza, and tested the impact of NPI on the behavioral intention of potential international tourists. This study also extended the model of goal-directed behavior (MGB) by incorporating the new concepts of NPI, and the perception of 2009 H1N1. The model found that desire, perceived behavioral control, frequency of past behavior, and non-pharmaceutical interventions predicted tourists' intention but perceptions of 2009 H1N1 had nil effect on desire and intention. Personal non-pharmaceutical interventions were theorized as adaptive behavior of tourists intending to travel during a pandemic which should be supported by tourism operators on a system-wide basis. Practically, this study dealt with the issue of influenza 2009 H1N1 with the study findings and implications providing government agencies, tourism marketers, policy-makers, transport systems, and hospitality services with important suggestions for NPI and international tourism during pandemics. Abstract We study an efficient, bio-inspired immunization strategy for network epidemiology. Inspiration stems from a single-celled, ameba-like organism, Physarum polycephalum. Our strategy goes beyond the node degree in selecting targets for immunization. The strategy performs considerably better than several well-known competitors.Well-known immunization strategies, based on degree centrality, betweenness centrality, or closeness centrality, either neglect the structural significance of a node or require global information about the network. We propose a biologically inspired immunization strategy that circumvents both of these problems by considering the number of links of a focal node and the way the neighbors are connected among themselves. The strategy thus measures the dependence of the neighbors on the focal node, identifying the ability of this node to spread the disease. Nodes with the highest ability in the network are the first to be immunized. To test the performance of our method, we conduct numerical simulations on several computer-generated and empirical networks, using the susceptible-infected-recovered (SIR) model. The results show that the proposed strategy largely outperforms the existing well-known strategies. Abstract Alternative jet fuels are one of the four mechanisms by the United Nations International Civil Aviation Organization (ICAO) to limit and reduce carbon emissions from international aviation. By using Carol Bacchi's what's the problem represented to be? method of discourse analysis, the objective of this paper was to identify and understand the premises and effects of the problem-solving paradigm underlying ICAO's alternative jet fuel strategy. As a result, three problem representations were identified, from which two out of four underlying assumptions have reinforced ICAO's weak sustainability approach to international aviation's growth and have led to a number of discursive, subjectification and lived effects. The selected method also allowed the authors to identify several options to disrupt those premises in favor of the implementation of more aggressive mitigation and adaptation strategies without constraining air travel demand, including: (i) raising awareness of the environmental impacts of aviation beyond the tailpipe emissions, (ii) improving the understanding of the effects of climate change on the air transport sector, and (iii) reassessing the sectoral approach to the Sustainable Development Goals so as to gain consistency with the aims of the UN 2030 Agenda for Sustainable Development. Abstract Infection of mice with the JHM strain of mouse hepatitis virus (MHV) results in an acute encephalomyelitis associated with primary demyelination of the central nervous system. Efforts at understanding the components of the immune response in the development of chronic MHV-induced demyelination have implicated the antibody response and both the CD4' and CD8+ T cell responses. In this report, we demonstrate that Balbk (H-2d) mice immunized with the JHM (JHMV) strain of MHV develop a CD8+ cytotoxic T lymphocyte (CTL) response. One population of these virus-specific CTL recognize the nucleocapsid (N) protein. Recombinant vaccinia viruses expressing either the entire N protein or carboxy-terminal deletions were used to determine the number and location of the epitope(s) recognized. The CTLs were found to recognize a peptide contained within the carboxy-terminal 149 amino acids of the N protein. Analysis of infected cell lines expressing transfected major histocompatibility genes demonstrated that the anti-N protein CTLs were restricted exclusively to the Ld molecule. These data provide the first definition of a MHV-specific CTL response directed to a viral protein and suggest that the anti-N protein CTL response is one potential mechanism used by the host to clear JHMV from the central nervous system. o 1992 Academic PESS. I~C. Abstract In most current air ventilation assessment (AVA) studies, a simple neutral assumption that does not consider thermal effects is adopted, particularly for numerical simulation practices. With statistics of daytime observations during summer in Hong Kong as an example, this study demonstrates that neutral atmospheric boundary conditions occur with a very low probability, which implies that current practices are indeed far away from reality. This study is devoted to addressing this knowledge gap by cross-comparisons of field measurements, wind tunnel tests, and large-eddy simulations (LES) under neutral and unstable conditions. It is found that LEScomputed velocity ratios under unstable conditions are in line with field measurements, while results of simulations under neutral conditions are close to those of wind tunnel tests. Enhanced vertical mixing due to surface heating produces improved ventilation performance in the unstable case. The neutral assumption tends to underestimate pedestrian-level velocity ratios compared to a diabatic condition; hence it is deemed conservative when it is adopted in AVA practices. Moreover, stronger wind direction variance under unstable conditions results in weaker correlation between velocity ratios and frontal area indices than neutral conditions, which implies that street orientations become less important in ventilation under unstable conditions. Abstract Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly using helper dependent expression systems and it will now be possible to validate them using single genomes. The genome of coronaviruses has been engineered by modification of the infectious cDNA leading to an efficient ( \ 20 mg ml − 1 ) and stable ( \20 passages) expression of the foreign gene. The possibility of engineering the tissue and species tropism to target expression to different organs and animal species, including humans, increases the potential of coronaviruses as vectors. Thus, coronaviruses are promising virus vectors for vaccine development and, possibly, for gene therapy. Abstract Background: Viral and bacterial pathogens may be present in the air after being released from infected individuals and animals. Filters are installed in the heating, ventilation, and air-conditioning (HVAC) systems of buildings to protect ventilation equipment and maintain healthy indoor air quality. These filters process enormous volumes of air. This study was undertaken to determine the utility of sampling used ventilation filters to assess the types and concentrations of virus aerosols present in buildings. Methods: The HVAC filters from 2 large public buildings in Minneapolis and Seattle were sampled to determine the presence of human respiratory viruses and viruses with bioterrorism potential. Four air-handling units were selected from each building, and a total of 64 prefilters and final filters were tested for the presence of influenza A, influenza B, respiratory syncytial, corona, parainfluenza 1-3, adeno, orthopox, entero, Ebola, Marburg, Lassa fever, Machupo, eastern equine encephalitis, western equine encephalitis, and Venezuelan equine encephalitis viruses. Representative pieces of each filter were cut and eluted with a buffer solution.Results: Attempts were made to detect viruses by inoculation of these eluates in cell cultures (Vero, MDCK, and RK-13) and specific pathogen-free embryonated chicken eggs. Two passages of eluates in cell cultures or these eggs did not reveal the presence of any live virus. The eluates were also examined by polymerase chain reaction or reverse-transcription polymerase chain reaction to detect the presence of viral DNA or RNA, respectively. Nine of the 64 filters tested were positive for influenza A virus, 2 filters were positive for influenza B virus, and 1 filter was positive for parainfluenza virus 1. Conclusion: These findings indicate that existing building HVAC filters may be used as a method of detection for airborne viruses. As integrated long-term bioaerosol sampling devices, they may yield valuable information on the epidemiology and aerobiology of viruses in air that can inform the development of methods to prevent airborne transmission of viruses and possible deterrents against the spread of bioterrorism agents. Abstract We studied the viral etiology of acute expiratory wheezing (bronchiolitis, acute asthma) in 293 hospitalized children in a 2-year prospective study in Finland. A potential causative viral agent was detected in 88% of the cases. Eleven different viruses were represented. Respiratory syncytial virus (RSV) (27%), enteroviruses (25%), rhinovirus (24%), and nontypable rhino/enterovirus (16%) were found most frequently. In infants, RSV was found in 54% and respiratory picornaviruses (rhinovirus and enteroviruses) in 42% of the cases. In older children, respiratory picornaviruses dominated (65% of children ages 1-2 years and 82% of children ages >3 years). Human metapneumovirus was detected in 4% of all children and in 11% of infants. To prevent and treat acute expiratory wheezing illnesses in children, efforts should be focused on RSV, enterovirus, and rhinovirus infections. Abstract This overview describes the main categories of specific occupational disorders and it covers also how work exposures are potential determinants of common respiratory conditions. Acute inhalation injuries may present as inhalation fever or as acute tracheobronchitis and pneumonitis. Occupational asthma is the most frequent work-related respiratory disease. It may be caused by allergic sensitization to macromolecules of biologic origin or to chemicals of low molecular weight, as well as by (heavy) exposure to workplace irritants. The pneumoconioses are caused by the accumulation of dust particles or fibres in the lungs. They include silicosis, coal workers' pneumoconioses, asbestosis and other less common pneumoconioses. Chronic beryllium disease is caused by a cell-mediated sensitization to beryllium and resembles sarcoidosis. Hard-metal lung disease is caused by sensitivity to cobalt and resembles hypersensitivity pneumonitis. Extrinsic allergic alveolitis or hypersensitivity pneumonitis is generally caused by sensitization to aerosolized biological antigens. Several types of infections may be related more or less specifically to work. Chronic obstructive lung disease is mainly caused by cigarette smoking, but exposure to dusts and gases contribute to its incidence. Similarly, bronchopulmonary cancer is not only caused by smoking, but also by occupational agents, most notably asbestos. Asbestos is also a cause of nonmalignant and malignant pleural disease. Abstract This paper characterises a virulent strain (CB/05) of canine coronavirus (CCoV) isolated from the internal organs of pups that had died of a systemic disease without evidence of other common canine pathogens. High viral RNA titres were detected in the internal organs by a real-time RT-PCR assay specific for CCoV type II. Sequence analysis of the 3 end (8.7 kb) of the genomic RNA of strain CB/05 revealed conserved structural as well as non-structural proteins, with the exception of a truncated form of non-structural protein 3b. The exceptional form was due to a 38-nucleotide deletion and a frame shift in ORF3b that introduced an early stop codon. By phylogenetic analysis of the structural proteins, the spike (S) protein was found to cluster with feline coronavirus type II strain 79-1683, whereas, the envelope (E), membrane (M) and nucleocapsid (N) proteins segregated together with the reference strain Purdue of transmissible gastroenteritis virus of swine. Abstract The aim of this study was to assess the possible effect of the fly repellent deltamethrin on the full microbiological profile of the intramammary infections as well as on the somatic cell count in one Greek dairy cattle farm under intensive management, during peak fly season. Fifty five multiparous cows, stabled in the same farm, were randomly divided in three groups; cows of Group A were dressed on their back with deltamethrin, cows of Group B remained untreated within the same pen and cows of Group C remained untreated in a separate pen. Clinical records of the herd showed a history of clinical and subclinical mastitis (especially during spring and early summer) and fly infestation. Deltamethrin reduced fly population, landing on the cows of Group A, facilitating a significant decrease of S. aureus, coagulase negative staplylococci, E. coli intramammary infections and somatic cell count, throughout the study period. Consequently, there is a relationship between fly abundance and transmission of bacteria resulting in intramammary infections and mastitis in dairy cows. The use of the pyrethroid deltamethrin reduced fly abundance and therefore, the possibility of transmitting pathogenic bacteria in the mammary gland. Given the impact of intramammary infections in dairy cows on the https://doi. Abstract SARS-CoV 3a protein was a unique protein of SARS coronavirus (SARS-CoV), which was identified in SARS-CoV infected cells and SARS patients' specimen. Recent studies revealed that 3a could interact specifically with many SARS-CoV structural proteins, such as M, E and S protein. Expressed 3a protein was reported to localize to Golgi complex in SARS-CoV infected cells. In this study, it was shown that 3a protein was mainly located in Golgi apparatus with different tags at N-or C-terminus. The localization pattern was similar in different transfected cells. With the assay of truncated 3a protein, it was shown that 3a might contain three transmembrane regions, and the second or third region was properly responsible for Golgi localization. By ultra-centrifugation experiment with different extraction buffers, it was confirmed that 3a was an integral membrane protein and embedded in the phospholipid bilayer. Immunofluorescence assay indicated that 3a was co-localized with M protein in Golgi complex in co-transfected cells. These results provide a new insight for further study of the 3a protein on the pathogenesis of SARS-CoV. Abstract The aim of this study was to examine the incidence of Clostridioides (previously Clostridium) difficile and Clostridium perfringens in the feces of diarrheic and non-diarrheic dogs. Also, the presence of other common canine enteropathogens was examined. Toxigenic C. difficile and C. perfringens positive for the NetF-encoding gene (netF) were detected in 11 (11.9%) and seven (7.6%) diarrheic dogs, respectively. Three dogs were diagnosed simultaneously with toxigenic C. difficile and netF-positive C. perfringens. Among other enteropathogens, Giardia sp. was the most common agent detected in dogs positive for toxigenic C. difficile or netF-positive C. perfringens. The results suggest that C. difficile and C. perfringens occur more frequently as a primary cause of diarrhea. Abstract a b s t r a c t a r t i c l e i n f o Zika virus (ZIKAV) is classically described as causing minor symptoms in adult patients, however neurologic complications have been recognized. The recent outbreak in Central and South America has resulted in serious illness in some adult patients. We report adult patients in Latin America diagnosed with ZIKAV infection admitted to Intensive Care Units (ICUs). Methods: Multicenter, prospective case series of adult patients with laboratory diagnosis of ZIKAV in 16 ICUs in 8 countries. Abstract Ethnopharmacological relevance: Licorice (Glycyrrhiza uralensis Fisch., Leguminosae) has been used in herbal medicine and food supplement worldwide for centuries. Licorice is a common ingredient of several prescriptions of traditional Chinese medicine which have been proved to inhibit infection of human respiratory syncytial virus (HRSV). There are two preparations of licorice, Radix Glycyrrhizae and Radix Glycyrrhizae Preparata. However, it is unknown whether licorice or which preparation of licorice is effective against HRSV, nor is its active constituent. Aim of the study: We tested the hypothesis that Radix Glycyrrhizae can effectively decrease HRSVinduced plaque formation in respiratory mucosal cell lines. We also tried to find out the active constituent. Materials and methods: Anti-HRSV activities of hot water extracts of preparations of licorice, glycyrrhizin and 18β-glycyrrhetinic acid (18β-GA), the active constituents of licorice, were examined by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines. Abilities of crude licorice to inhibit viral replication and to stimulate IFN-β were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Results: Radix Glycyrrhizae and Radix Glycyrrhizae Preparata dose-dependently inhibited HRSV-induced plaque formation in both HEp-2 and A549 cell lines (po 0.0001). The effect of Radix Glycyrrhizae was better than that of Radix Glycyrrhizae Preparata on HEp-2 cells. However, there was no difference of their anti-HRSV effects on A549 cells. Besides, glycyrrhizin was ineffective at all. Nevertheless, 18β-GA showed a potent anti-HRSV activity. Radix Glycyrrhizae was more effective when given before viral inoculation (po 0.0001) which may be due to its inhibition of viral attachment on (po 0.0001) and penetration (po 0.0001) into the host cells. The anti-HRSV activity of Radix Glycyrrhizae was further confirmed by RT-PCR and qRT-PCR. 300 μg/ml Radix Glycyrrhizae markedly decreased the viral amounts within the cells and in the suspension. Radix Glycyrrhizae might further stimulate mucosal cells to secrete IFN-β to counteract viral infection. Conclusions: Both Radix Glycyrrhizae and Radix Glycyrrhizae Preparata are effective against HRSV infection on airway epithelial cells. Radix Glycyrrhizae inhibited HRSV mainly by preventing viral attachment, internalization, and by stimulating IFN secretion. 18β-GA may be one of its active constituents. Abstract Respiratory viruses, particularly influenza viruses, respiratory syncytial virus (RSV), parainfluenza viruses, and adenoviruses, are ubiquitous pathogens among humans, especially among young children. However, relatively little is known about the impact of these common infections on individuals with the human immunodeficiency virus (HIV). A review of the literature identifies three key areas that need further exploration. First, moderate-to-severe and even fatal lower respiratory viral illnesses ininfected adults and children. The frequency of prolonged shedding in this population has not been well defined, but data from a small newborn cohort study suggest that, at least for RSV, prolonged shedding is common. Prolonged respiratory viral shedding has implications for infection control in medical facilities where HIVinfected individuals are treated and in nursing homes, child care centers, and group foster homes that provide care for HIV-infected individuals. Therapies to help eliminate these chronic viral infections should be explored. Finally, indirect evidence suggests that respiratory viral infection may result in changes in HIV replication and, theoretically, HIV disease Abstract The objective of this study was to evaluate under field conditions, whether daily administration of oregano essential oil is effective in preventing and/or diminishing the severity of neonatal diarrhea syndrome in calves aged less than 15 days. Ninety-one newborn calves from three dairy farms were assigned into two groups; "Eco" group (n = 46) calves were drenched with Greek oregano (Origanum vulgare ssp. Hirtum) essential oil (ECODIAR® liquid 5%) at the dose of 12.5 mg/kg body weight once per day until the age of 10 days. "Conts" group (n = 45) calves were left untreated and served as controls. All animals were monitored daily for the incidence of diarrhea until the age of 15 days and their fecal score was recorded. Fecal samples were collected on days 3, 6 and 10 for microbiological and parasitological evaluation. Average fecal score throughout the experiment, incidence of diarrhea, duration and severity of diarrhea episodes were significantly lower in Eco group compared to the controls. Daily administration of oregano essential oil in calves for the first 10 days of their life effectively diminishes the severity of naturally acquired diarrhea under field conditions and, under certain hygiene practices, possess a preventive effect against neonatal diarrhea syndrome. Abstract Patients were given oral arbidol and LPV/r in the combination group and oral LPV/r only in the monotherapy group for 5-21 days Our study shows that oral arbidol and LPV/r in the combination group is associated with a significant elevated negative conversion rate of coronavirus' test in 7-day and 14-day, compared with LPV/r only in the monotherapy group. Combination therapy is associated with a significantly improved the chest CT scans in 7-day. We suppose that reducing the viral load as soon as possible could benefit the delay of the progression of lung lesions.Corona Virus Disease 2019 (COVID-19) due to the 2019 novel coronavirus (SARS-CoV-2) emerged in Wuhan city and rapidly spread throughout China. We aimed to compare arbidol and lopinavir/ritonavir(LPV/r) treatment for patients with COVID-19 with LPV/r only.In this retrospective cohort study, we included adults (age≥18years) with laboratory-confirmed COVID-19 without Invasive ventilation, diagnosed between Jan 17, 2020, and Feb 13, 2020. Patients, diagnosed after Jan 17, 2020, were given oral arbidol and LPV/r in the combination group and oral LPV/r only in the monotherapy group for 5-21 days. The primary endpoint was a negative conversion rate of coronavirus from the date of COVID-19 diagnosis(day7, day14), and assessed whether the pneumonia was progressing or improving by chest CT (day7).We analyzed 16 patients who received oral arbidol and LPV/r in the combination group and 17 who oral LPV/r only in the monotherapy group, and both initiated after diagnosis. Baseline clinical, laboratory, and chest CT characteristics were similar between groups. The SARS-CoV-2 could not be detected for 12(75%) of 16 patients' nasopharyngeal specimens in the combination group after seven days, compared with 6 (35%) of 17 in the monotherapy group (p<0·05). After 14 days, 15 (94%) of 16 and 9 (52·9%) of 17, respectively, SARS-CoV-2 could not be detected (p<0·05). The chest CT scans were improving for 11(69%) of 16 patients in the combination group after seven days, compared with 5(29%) of 17 in the monotherapy group (p<0·05).In patients with COVID-19, the apparent favorable clinical response with arbidol and LPV/r supports further LPV/r only. Abstract Parasitic nematodes of livestock have major economic impact worldwide. Despite the diseases caused by these nematodes, some advances towards the development of new therapeutic agents and attempts to develop effective vaccines against some of them, there has been limited progress in the development of practical diagnostic methods. The specific and sensitive diagnosis of parasitic nematode infections of livestock underpins effective disease control, which is now particularly important given the problems associated with anthelmintic resistance in parasite populations. Traditional diagnostic methods have major limitations, in terms of sensitivity and specificity. This chapter provides an account of the significance of parasitic nematodes (order Strongylida), reviews conventional diagnostic techniques that are presently used routinely and describes advances in polymerase chain reaction (PCR)-based methods for the specific diagnosis of nematode infections. A particular emphasis is placed on the recent development of a robotic PCR-based platform for high-throughput diagnosis, and its significance and implications for epidemiological investigations and for use in control programmes. Abstract Graphical Abstract Highlights d MERS-CoV infected cells expressing DPP4 from 16 bat species d MERS-CoV spike rapidly adapted to species variation in DPP4 d Viral adaptations modified the surface charge of viral spike d Different routes of spike adaptation enhanced entry with the same DPP4 variant Correspondence michael.letko@nih.gov (M.L.), vincent.munster@nih.gov (V.M.) SUMMARY Middle East Respiratory Syndrome Coronavirus (MERS-CoV) likely originated in bats and passed to humans through dromedary camels; however, the genetic mechanisms underlying cross-species adaptation remain poorly understood. Variation in the host receptor, dipeptidyl peptidase 4 (DPP4), can block the interaction with the MERS-CoV spike protein and form a species barrier to infection. To better understand the species adaptability of MERS-CoV, we identified a suboptimal speciesderived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication. Parallel passages revealed distinct paths of viral adaptation to the same DPP4 variant. Structural analysis and functional assays showed that these mutations enhanced viral entry with suboptimal DPP4 by altering the surface charge of spike. These findings demonstrate that MERS-CoV spike can utilize multiple paths to rapidly adapt to novel species variation in DPP4. Abstract Ethnopharmacological relevance: Peperomia sui Lin and Lu (Peperomia sui), a well-known Taiwanese folk medicine, has a broad range of biological effects, especially in treatment of upper respiratory tract diseases. However, no previous study has explored the activity of Peperomia sui against influenza virus infections. This study was carried out to evaluate the anti-influenza virus activity and the potential virucidal effect of the ethanolic extract of Peperomia sui (PSE). Methods: The anti-H6N1 avian influenza viral activity of PSE against the influenza virus A/Chicken/TW/ 0518/2011 (H6N1) in chicken fibroblast DF-1 cells was evaluated by cell viability assay, hemagglutination assay, neuraminidase activity assay, indirect immunofluorescence assay and quantitative RT-PCR assay. Results: PSE significantly increased the viability of cells that were infected by the H6N1 virus. PSE also suppressed the synthesis of viral nucleoprotein (NP), and inhibited the growth of the virus in DF-1 cells. Further, PSE inhibited the neuraminidase activity of H6N1 virus. Conclusions: The findings of this study provide important information for the exploitation and utilization of Peperomia sui in treatment of influenza infection. Abstract Nosocomial respiratory infections cause significant morbidity and mortality, especially among the extremely susceptible neonatal population. Human rhinovirus C is a common viral respiratory illness that causes significant complications in children <2 years old. We describe a nosocomial outbreak of human rhinovirus C in a level II-III neonatal intensive care unit in an urban public safety net hospital. Abstract Ribosomes are specialized entities that participate in regulation of gene expression through their rRNAs carrying ribozyme activity. Ribosome biogenesis is overactivated in p53-inactivated cancer cells, although involvement of p53 on ribosome quality is unknown. Here, we show that p53 represses expression of the rRNA methyl-transferase fibrillarin (FBL) by binding directly to FBL. High levels of FBL are accompanied by modifications of the rRNA methylation pattern, impairment of translational fidelity, and an increase of internal ribosome entry site (IRES)-dependent translation initiation of key cancer genes. FBL overexpression contributes to tumorigenesis and is associated with poor survival in patients with breast cancer. Thus, p53 acts as a safeguard of protein synthesis by regulating FBL and the subsequent quality and intrinsic activity of ribosomes. Abstract Chikungunya virus (CHIKV) has emerged as one of the most significant arboviral threats in many parts of the world. In spite of large scale morbidity, and long lasting polyarthralgia, no licensed vaccine or antivirals are available for the clinical management of CHIKV infection. In this study, a novel RNA interference based strategy has been adopted for effective inhibition of CHIKV. Four artificial microRNAs (amiRNAs) were designed to target different regions of CHIKV genome. These amiRNAs significantly inhibited CHIKV replication in Vero cells at both RNA and protein levels as assessed by qRT-PCR, immunoblotting and immunofluorescence techniques. Further inhibition of the infectious CHIKV up to 99.8% was demonstrated by plaque reduction assay. Concatemerization of amiRNA resulted in higher inhibition of CHIKV than individual amiRNAs. In addition, we studied the effect of combination of RNAi based therapy with other classical antivirals like chloroquine, ribavirin and mycophenolic acid, that helped in understanding the rational selection of RNAi based combination therapy. These findings provide a promising avenue for the development of novel amiRNA or combination based therapeutics against emerging CHIKV. Abstract Diarrhea and deaths in new-born camel calves were noticed by veterinary investigators and pastoralist in Saudi Arabia to be very high. Hence, it is thought to be necessary to investigate this problem from the virological and bacteriological point of view. The role of pathogenic bacteria and viruses in six different towns of North Province (Al-Assafia, Arar, Domat Aljandal, Hail, Skaka and Khoa) in Saudi Arabia was studied. Survey was conducted in diarrheic camel calves aged 12 months or younger. In our study calf diarrhea was reported in 184 out of 2308 camels examined clinically during one year, the prevalence of diarrhea was found to be 8.0% in calves ranging from one month to one year. In the present study group A rotavirus and Brucella abortus were detected in 14.7% and 8.98%, respectively, using ELISA technique. Escherichia coli was isolated from diarrheic calf camel (58.2%) 99/170 samples during dry and wet season. Salmonella spp. and Enterococcus spp. were detected in 12% and 8.8% of the specimens, respectively. In this study enterotoxogenic E. coli (ET E. coli) was isolated from 7% of diarrheic camel, which indicates the strong correlation between the camel calf diarrhea and the detection of enterotoxogenic E. coli. This study represented the first report for the detection of group A rotavirus and B. abortus antigen and antibodies in calf Abstract In this article, we introduce three 3D graphical representations of DNA primary sequences, which we call RY-curve, MK-curve and SW-curve, based on three classifications of the DNA bases. The advantages of our representations are that (i) these 3D curves are strictly non-degenerate and there is no loss of information when transferring a DNA sequence to its mathematical representation and (ii) the coordinates of every node on these 3D curves have clear biological implication. Two applications of these 3D curves are presented: (a) a simple formula is derived to calculate the content of the four bases (A, G, C and T) from the coordinates of nodes on the curves; and (b) a 12-component characteristic vector is constructed to compare similarity among DNA sequences from different species based on the geometrical centers of the 3D curves. As examples, we examine similarity among the coding sequences of the first exon of betaglobin gene from eleven species and validate similarity of cDNA sequences of beta-globin gene from eight species. Abstract . Application of radioimmunological methods for checking the quality of class-specific antibodies against bovine and porcine immunoglobulins, vet. Immunol,lmmunopathol,, 5: 377-387, class-specific antibodies against bovine IgOl, IgG2, IgM and IgA and porcine IgG, IgM and IgA immunoglobulins were prepared. Their class specificity was assessed by two radioimmunological methods, nameIy, radioimmunoelectrophoresis and double antibody sandwich radioimmunoassay. The methods are highIy specific and sensitive and do not require the use of purified immunoglobulins, but can be performed with normai serum or colostrum. It was confirmed that antibodies found satisfactory in these tests were suitable for a wide range of use including radioimmunoassay and enzyme linked immunosorbent assay. Abstract Manure application is a source of pathogens to the environment. Through overland runoff and tile drainage, zoonotic pathogens can contaminate surface water and streambed sediment and could affect both wildlife and human health. This study examined the environmental occurrence of gene markers for livestock-related bacterial, protozoan, and viral pathogens and antibiotic resistance in surface waters within the South Fork Iowa River basin before and after periods of swine manure application on agricultural land. Increased concentrations of indicator bacteria after manure application exceeding Iowa's state bacteria water quality standards suggest that swine manure contributes to diminished water quality and may pose a risk to human health. Additionally, the occurrence of HEV and numerous bacterial pathogen genes for Escherichia coli, Enterococcus spp., Salmonella sp., and Staphylococcus aureus in both manure samples and in corresponding surface water following periods of manure application suggests a potential role for swine in the spreading of zoonotic pathogens to the surrounding environment. During this study, several zoonotic pathogens were detected including Shiga-toxin producing E. coli, Campylobacter jejuni, pathogenic enterococci, and S. aureus; all of which can pose mild to serious health risks to swine, humans, and other wildlife. This research provides the foundational understanding required for future assessment of the risk to environmental health from livestock-related zoonotic pathogen exposures in this j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / s c i t o t e n v region. This information could also be important for maintaining swine herd biosecurity and protecting the health of wildlife near swine facilities.Published by Elsevier B.V. Abstract An antifungal peptide with a defensin-like sequence and exhibiting a molecular mass of 7.3 kDa was purified from dried seeds of Phaseolus vulgaris 'Cloud Bean'. The isolation procedure entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography an Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Although the antifungal peptide was unadsorbed on DEAE-cellulose, it was adsorbed on both Affi-gel blue gel and SP-Sepharose. The antifungal peptide exerted antifungal activity against Mycosphaerella arachidicola with an IC 50 value of 1.8 M. It was also active against Fusarium oxysporum with an IC 50 value of 2.2 M. It had no inhibitory effect on HIV-1 reverse transcriptase when tested up to 100 M. Proliferation of L1210 mouse leukemia cells and MBL2 lymphoma cells was inhibited by the antifungal peptide with an IC 50 of 10 M and 40 M, respectively. Abstract This study contributes to the fields of hospitality and tourism by examining interactions between business conditions and financial performance of tourism firms in both China and Taiwan. We investigate whether the improvement of business conditions enhances financial performance of tourism firms and whether financial success of tourism firms matters to business development. In general, cointegration test results support a long-run equilibrium relationship between the two variables, business conditions and financial performance of tourism firms, and Granger causality test results show that these two factors reinforce each other in both China and Taiwan. This study therefore documents significant contributions of financial success of tourism firms to business development and strengthening of financial performance of tourism firms by business conditions. Some managerial implications are also discussed. r Abstract BACKGROUND: There is a need for more research on all forms of rhinosinusitis. Progress in this area has been hampered by a lack of consensus definitions and the limited number of published clinical trials.OBJECTIVES: To develop consensus definitions for rhinosinusitis and outline strategies useful in clinical trials.STUDY DESIGN: Five national societies, The can Rhinologic Society formed an expert panel from multiple disciplines. Over two days, the panel developed definitions for rhinosinusitis and outlined strategies for design of clinical trials.RESULTS: Committee members agreed to adopt the term "rhinosinusitis" and reached consensus on definitions and strategies for clinical research on acute presumed bacterial rhinosinusitis, chronic rhinosinusitis without polyposis, chronic rhinosinusitis with polyposis, and classic allergic fungal rhinosinusitis. Symptom and objective criteria, measures for monitoring research progress, and use of symptom scoring tools, quality-of-life instruments, radiologic studies, and rhinoscopic assessment were outlined for each condition.CONCLUSIONS: The recommendations from this conference should improve accuracy of clinical diagnosis and serve as a starting point for design of rhinosinusitis clinical trials. (Otolaryngol Head Neck Surg 2004;131:S1-S62.)Recognizing a need for evidence-based rhinosinusitis guidelines, 5 national societies, The American Abstract We have designed and utilized two in vivo assays of kinetochore integrity in S. cerevisiae. One assay detects relaxation of a transcription block formed at centromeres; the other detects an increase in the mitotic stability of a dicentric test chromosome.clf13-30 and cff74.42 were identified as putative kinetochore mutants by both assays. CTF14 is identical to NDC70I CBF2, a recently identified essential gene that encodes a 110 kd kinetochore component. CTF13 is an essential gene that encodes a predicted 478 amino acid protein with no homology to known proteins. cffl3 mutants missegregate chromosomes at permissive temperature and transiently arrest at nonpermissive temperature as large-budded cells with a 62 DNA content and a short spindle. Antibodies recognizing epitope-tagged CTF13 protein decrease the electrophoretic mobility of a CEN DNA-protein complex formed in vitro. Together, the genetic and biochemical data indicate that CTF13 is an essential kinetochore protein. Abstract a b s t r a c t H1N1 is an infectious virus which, when spread affects a large volume of the population. It is an airborne disease that spreads easily and has a high death rate. Development of healthcare support systems using cloud computing is emerging as an effective solution with the benefits of better quality of service, reduced costs and flexibility. In this paper, an effective cloud computing architecture is proposed which predicts H1N1 infected patients and provides preventions to control infection rate. It consists of four processing components along with secure cloud storage medical database. The random decision tree is used to initially assess the infection in any patient depending on his/her symptoms. Social Network Analysis (SNA) is used to present the state of the outbreak. The proposed architecture is tested on synthetic data generated for two million users. The system provided 94% accuracy for the classification and around 81% of the resource utilization on Amazon EC2 cloud. The key point of the paper is the use of SNA graphs to calculate role of an infected user in spreading the outbreak known as Outbreak Role Index (ORI). It will help government agencies and healthcare departments to present, analyze and prevent outbreak effectively. Abstract The relative lack of efficient methods for evaluating antiseptic antiviral activity, together with weaknesses in the existing European Standard (i.e. NF EN 14476+A1), underlines the need to seek a new method which could allow a more precise evaluation of the antiseptic antiviral activity of chemical agents.This protocol is based on an original gel-based filtration method, using "in-house" G-25 and G-10 Sephadex TM columns. This method allows the neutralization of both the activity and the cytotoxicity of a large range of molecules, according to their molecular size, in only 1 min.The viral model used was the human coronavirus (HCoV) 229E chosen for (i) its increasing medical interest, (ii) its potential resistance and (iii) its representing enveloped viruses mentioned in the European Standard.First, the protocol was validated and it was demonstrated that it was fully operational for evaluating antiviral antiseptic potentiality and useful to screen potentially antiseptic molecules.Second, chlorhexidine (CHX) and hexamidine (HXM) were assessed for their potential anti-HCoV 229E antiseptic activities. It was demonstrated clearly that (i) HXM had no activity on the HCoV 229E and (ii) CHX showed a moderate anti-HCoV 229E activity but insufficient to be antiseptic. Abstract Antisense antimicrobial therapeutics are synthetic oligomers that silence expression of specific genes. This specificity confers an advantage over broad-spectrum antibiotics by avoiding unintended effects on commensal bacteria. The sequencespecificity and short length of antisense antimicrobials also pose little risk to human gene expression. Because antisense antimicrobials are a platform technology, they can be rapidly designed and synthesized to target almost any microbe. This reduces drug discovery time, and provides flexibility and a rational approach to drug development. Recent work has shown that antisense technology has the potential to address the antibiotic-resistance crisis, since resistance mechanisms for standard antibiotics apparently have no effect on antisense antimicrobials. Here, we describe current reports of antisense antimicrobials targeted against viruses, parasites, and bacteria. Abstract A data base for a large diagnostic virology laboratory is described. The system uses a network of personal computers. It allows the entry, long-term storage, and subsequent retrieval of specimen and patient records (comprising personal identifiers and specimen and result information), and hard-copy results reporting.Sited entirely within the laboratory, the network is not connected to a modem. Within the laboratory there is restricted access to human immunodeficiency virus test results to guarantee patient confidentiality. Retention of a hard-copy of specimen request cards ensures the availability of the original clinical information. The data base is copied on a second file server to facilitate searches, and daily streaming onto magnetic tape provides system protection in the event of hard disc failure. Matching of old and new patient records is done by surname, date of birth, and sex, and therefore duplicate records accumulate when patient names are misspelt on specimen request forms. The system requires further development to speed searches of the data base and to achieve automatic generation of laboratory worksheets. Future goals are the replacement of hard-copy records of clinical information and hard-copy reporting with on-line access to hospital data bases and on-line requesting by and reporting to the clinician. Abstract Eucalyptus has become one of the world's most widely planted genera and E. camaldulensis (The River Red Gum) is a plantation species in many parts of the world. The plant traditional medical application indicates great antimicrobial properties, so E. camaldulensis essential oils and plant extracts have been widely examined. Essential oil of E. camaldulensis is active against many Gram positive (0.07-1.1%) and Gram negative bacteria (0.01-3.2%). The antibacterial effect is confirmed for bark and leaf extracts (conc. from 0.08 μg/mL to 200 mg/ mL), with significant variations depending on extraction procedure. Eucalyptus camaldulensis essential oil and extracts are among the most active against bacteria when compared with those from other species of genus Eucalyptus. The most fungal model organisms are sensitive to 0.125-1.0% of E. camaldulensis essential oil. The extracts are active against C. albicans (0.2-200 mg/mL leaf extracts and 0.5 mg/mL bark extracts), and against various dermatophytes. Of particular importance is considerable the extracts' antiviral activity against animal and human viruses (0.1-50 μg/mL). Although the antiprotozoal activity of E. camaldulensis essential oil and extracts is in the order of magnitude of concentration several hundred mg/mL, it is considerable when taking into account current therapy cost, toxicity, and protozoal growing resistance. Some studies show that essential oils' and extracts' antimicrobial activity can be further potentiated in combinations with antibiotics (beta-lactams, fluorochinolones, aminoglycosides, polymyxins), antivirals (acyclovir), and extracts of other plants (e.g. Annona senegalensis; Psidium guajava). The present data confirm the river red gum considerable antimicrobial properties, which should be further examined with particular attention to the mechanisms of antimicrobial activity.limited number of antiprotozoal and antiviral agents (El-Taweel, 2015; Irwin et al., 2016) . This highlights the necessity for examination of new antimicrobial agents and treatment strategies of infections caused by the mentioned microorganisms.The plant kingdom represents the source of various medicines. Indeed, since ancient times medicinal plants play an important role of health care population and could represent a significant source of new antimicrobial drugs for combating pan-and multi-drug resistant microorganisms. These new antimicrobial agents could be hidden in medicinal plant extracts and essential oils. One of the significant medicinal plants is Eucalyptus camaldulensis. Thus, this review represents the summary of previous researches data, regarding chemical composition, antimicrobial activity, and other significant effects of Eucalyptus camaldulensis. Abstract We have analyzed the ability of three molecular clones of feline immunodeficiency virus (FIV) and an ex vivo variant to infect nine distinct specific-pathogen-free feline cell lines in tissue culture. The purpose of these studies was to elucidate mechanisms by which host cells regulate the level of virus infection and expression and to assess host cell cytokine responses to virus infection. Cells used for the analyzes included four IL-2-dependent continuous T-cell lines (104-C1, 104-C7, MCH5-4 and DB FeTs) which arose from long-term passage, followed by limiting dilution cloning of peripheral blood mononuclear cells (PBMCs); two IL-2-independent T-cell lines (104-C1DL and MCH5-4DL) which originated from two of the IL-2-dependent lines, 104-C1 and MCH5-4 Abstract After decades of low personal risk for contracting lethal diseases, physicians are suddenly facing the possibility of a substantial increase in occupational risk during an influenza pandemic. If they are not confronted before the onset of an influenza pandemic, feelings of unease and fear or ignorance about physicians' professional obligations could profoundly hinder individual physicians in fulfilling their professional duties. Such feelings could therefore undermine institutional and societal preparations. In their review published in Critical Care, Anantham and coworkers outline the ethical framework that forms the basis of the professional obligations of physicians who respond to health care emergencies, such as an influenza pandemic. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.Corresponding author: Agol, V.I. (agol@belozersky.msu.ru). Abstract 1969) Use of enzymelabelled antibody for electron microscope localization of lymphocytic choriomeningitis virus antigens in infected cell cultures. Abstract Cholesterol 25-hydroxylase Porcine epidemic diarrhea virus 25-hydroxycholesterol Porcine transmissible gastroenteritis virus A B S T R A C TCholesterol 25-hydroxylase (CH25H) has been shown lately to be a host restriction factor that encodes an enzyme, which catalyzes the oxidized form of cholesterol to 25-hydroxycholesterol (25HC). A series of studies have shown that 25HC activity in hosts plays a vital role in inhibiting viral infection. In this study, we explored the antiviral effect of CH25H and 25HC on porcine epidemic diarrhea virus (PEDV), which causes high mortality rates in newborn piglets with severe diarrhea, and considerable financial loss in the swine industry worldwide. Our results showed that PEDV infection downregulated the expression of CH25H in Vero cells. An overexpression and knockdown assay indicated that CH25H has significant antiviral action against PEDV, and a CH25H mutant (CH25H-M) that lacks hydroxylase activity also retains antiviral activity to a lesser extent. Furthermore, 25HC had a broad-spectrum antiviral effect against different PEDV strains by blocking viral entry. In addition, CH25H and 25HC inhibited the replication of porcine transmissible gastroenteritis virus (TGEV). Taken together, CH25H as a natural host restriction factor could inhibit PEDV and TGEV infection. Abstract We have examined six different JHMV variants, sp-4 (recloned wt JHMV), ~1-2, CNSV, DL, DS, and JHM-X, in terms of the sizes of the mRNA3 and E2 glycoprotein as well as their reactivity to a panel of monoclonal antibodies to the E2 glycoprotein.Two of these variants, sp-4 and JHM-X, were found to have smaller mRNA3 and E2 glycoprotein species compared with those of the other four variants. In addition, sp-4 and JHM-X were distinguished from the other four variants by their inability to bind to monoclonal antibodies recognizing two antigenic domains of the E2 molecule. Thus, six JHMV variants could clearly be divided into two groups with respect to the size and antigenicity of their E2 glycoproteins.Press, Inc. Abstract Dengue virus is the most common arboviral infection of humans in the tropical and subtropical regions of the world. This review briefly describes some of the challenges it presents. Dengue is an emerging disease; it is increasing in geographical distribution and severity, despite being significantly underreported. The World Health Organization case definition for the generally more severe manifestation of infection, dengue haemorrhagic fever (DHF), is controversial. The name DHF is something of a misnomer as the disease infrequently results in frank haemorrhage; the hallmark of DHF is actually plasma leakage. The existence of four closely related dengue virus serotypes contributes to difficulties in diagnosis and to original antigenic sin in the serological response to infection. The existence of multiple serotypes can result in more severe disease upon a second infection and complicates vaccine development. Nevertheless, a safe and effective vaccine is the greatest prospect for stemming the tide of dengue.Published by Elsevier B.V. on behalf of International Society of Chemotherapy. Abstract The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2 h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18 s/cycle; 40 cycles in less than 20 min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10 −1 plaque-forming unit/reaction for viruses in culture supernatants during 20 min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks. Abstract Two species of membrane-associated glycoproteins have been identified in the coronavirus virion. They are readily distinguished on the basis of size, radiolabeling characteristics, and location in relation to the lipid bilayer. The larger glycoprotein is highly labeled by both radiolabeled fucose and glucosamine. This species is found in two forms, GP180 and GP90, with apparent molecular weights of 180,000 and 90,000. GP180 can be converted to GP90 in vitro by treatment of virions with trypsin. Analysis of tryptic digests of GP90 and GPl80 give identical peptide patterns. Based on pronase and bromelain sensitiyities, GP180/90 is the only protein which is located entirely external to the viral envelope. It appears to comprise the characteristic long, petal-shaped peplomers of the virion. The smaller glycoprotein, GP23, has an apparent molecular weight of 23,000 and is labeled by radiolabeled glucosamine but not by fucose. The level of glucosamine-labeling of GP23 is about l/10 that of GP180/90. GP23 appears to possess two distinct domains: a smaller, carbohydrate containing region which is found outside the viral envelope, and a larger portion, highly labeled by methionine, which is integrally associated with the viral membrane. A new nomenclature is proposed for the three major coronavirus structural proteins. The two envelope glycoproteins, GP23 and GP180/90 are designated El and E2, respectively; the inner core protein, ~~50, is designated N. Abstract Airborne transmission is a main spread mode of respiratory infectious diseases, whose frequent epidemic has brought serious social burden. Identifying possible routes of the airborne transmission and predicting the potential infection risk are meaningful for infectious disease control. In the present study, an internal spread route between horizontal adjacent flats induced by air infiltration was investigated. Onsite measurements were conducted, and tracer gas technique was employed. Two measurement scenarios, closed window mode and open window mode, were compared. Using the calculated air change rate and mass fraction, the cross-infection risk was estimated using the WellseRiley model. It found that tracer gas concentrations in receptor rooms are one order lower than the source room, and the infection risks are also one order lower. Opening windows results in larger air change rate on the one hand, but higher mass fraction on the other hand. Higher mass fraction not necessarily results in higher infection risk as the pathogen concentration in the source room is reduced by the higher air change rate. In the present study, opening windows could significantly reduce the infection risk of the index room but slightly reduce the risks in receptor rooms. The mass fraction of air originated from the index room to the receptor units could be 0.28 and the relative cross-infection risk through the internal transmission route could be 9%, which are higher than the external spread through single-sided window flush. The study implicates that the horizontal transmission route induced by air infiltration should not be underestimated. Abstract A CFD-based numerical model was integrated with the WellseRiley equation to numerically assess the risk of airborne influenza infection in a popular means of public transportation, e.g. the bus microenvironment. Three mixing ventilation methods, which are widely used in current bus configurations, and an alternative displacement ventilation method were numerically assessed in terms of their ability to limit the risk of airborne influenza infection. Furthermore, both the non air-recirculation and airrecirculation with filtration ventilation modes were investigated in terms of the influenza infection probability. According to the simulation results, air-recirculation mode with high efficiency filtration was found to cause almost the same infection risk as non air-recirculation mode (100% outdoor air supply), which indicated a potential benefit of filtration in reducing the infection risk. Additionally, for the commonly used mixing ventilation methods, air distribution method, location of return/exhaust opening and seat arrangement affected the airborne transmission of influenza between passengers. The displacement ventilation method was found to be more effective in limiting the risk of airborne infection. Overall, the developed numerical model can provide insights into how the micro-environmental conditions affect airborne infection transmission in buses. This numerical model can assist in developing effective control strategies related to airborne transmitted diseases for other frequently used public transportation systems, such as trains and airplanes. Abstract Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus caffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo. Abstract Feline infectious peritonitis virus (FIPV) positive cells are present in pyogranulomas and exudates from cats with FIP. These cells belong mainly to the monocyte/macrophage lineage. How these cells survive in immune cats is not known. In this study, FIPV positive cells were isolated from pyogranulomas and exudates of 12 naturally FIPV-infected cats and the presence of two immunologic targets, viral antigens and MHC I, on their surface was determined. The majority of the infected cells were confirmed to be cells from the monocyte/macrophage lineage. No surface expression of viral antigens was detected on FIPV positive cells. MHC I molecules were present on all the FIPV positive cells. After cultivation of the isolated infected cells, 52 AE 10% of the infected cells re-expressed viral antigens on the plasma membrane.In conclusion, it can be stated that in FIP cats, FIPV replicates in cells of the monocyte/macrophage lineage without carrying viral antigens in their plasma membrane, which could allow them to escape from antibody-dependent cell lysis. # Abstract The constituents of Cimicifuga plants have been extensively investigated, and the principal metabolites are 9, 19-cyclolanostane triterpenoid glycosides, which often exhibit extensive pharmacological activities. 9, 19-Cyclolanostane triterpenoid glycosides are distributed widely in genus Cimicifuga rather than in other members of the Ranunculaceae family. So far, more than 140 cycloartane triterpene glycosides have been isolated from Cimicifuga spp.. The aim of this review was to summarize all 9, 19-cyclolanostane triterpenoid glycosides based on the available relevant scientific literatures from 2000 to 2014. Biological studies of cycloartane triterpene glycosides from Cimicifuga spp. are also discussed.[KEY WORDS] Cimicifuga spp.; 9, 19-Cyclolanostane glycosides; Chemical structure; Biological effects [CLC Number] R284, R965 [Document code] A [Article ID] 2095-6975(2016)10-0721-11 [Received on] Abstract Background: Advances have been made in selecting sensitive cell lines for isolation, in early detection of respiratory virus growth in cells by rapid culture assays, in production of monoclonal antibodies to improve many tests such as immunofluorescence detection of virus antigens in nasopharyngeal aspirates, in highly sensitive antigen detections by time-resolved fluoroimmunoassays (TR-FIAs) and biotin-enzyme immunoassays (BIOTH-E), and, finally, in the polymerase chain reaction (PCR) detection of respiratory virus DNA or RNA in clinical specimens. All of these advances have contributed to new or improved possibilities for the diagnosis of respiratory virus infections.Objectives and study design: This review summarizes our experiences during the last 15 years in the development of diagnostic tests for respiratory virus infections, and in use of these tests in daily diagnostic work and in epidemiological studies.Results: Immunofluorescence tests based on monoclonal antibodies, all-monoclonal TR-FIAs, and biotin-enzyme immunoassays (EIAs) have about the same sensitivities and specificities. They compare well with the sensitivity of virus culture. PCR followed by liquid-phase hybridization is a sensitive method for detecting adenovirus DNA and enterovirus and rhinovirus RNA in clinical specimens. IgG EIA on paired acute and convalescent phase sera is the most sensitive serological test for respiratory virus infections and is a valuable reference method when evaluating the sensitivity of new diagnostic tests. The IgG avidity test can distinguish primary infections from re-infections at least in respiratory syncytial virus (RSV) infections. IgM antibody assays, on the other hand, had low sensitivities in our studies.Conclusions: The choice of diagnostic methods for respiratory virus infections depends on the type and location of the laboratory, the number of specimens tested, and the previous experience of the laboratory. Virus culture, whenever possible, should be the basic diagnostic method; the results, including identification of the virus, should be available no more than 24 h later than the results of rapid diagnostic tests. In small laboratories, especially in hospitals where specimen transportation is well organized, immunofluorescence may be the best choice for antigen detection with the provision that an experienced microscopist and a good UV microscope are available. If the laboratory receives a large number of specimens and has previous experience with EIAs, then biotin-EIAs or TR-FIAs may be the most practical techniques. Their advantages include the stability of the antigens in clinical samples since intact, exfoliated epithelial cells are not required, treatment of specimens is practical, testing of large numbers of specimens is possible, and reading the printed test result is less subjective than reading fluorescence microscopy. The larger role of PCR in the diagnosis of respiratory virus infections depends on future developments such as practical methods to extract DNA or RNA and to purify the extracts from nonspecific inhibitors, plus further improvements to minimize cross-contamination. Abstract Porcine reproductive and respiratory syndrome virus (PRRSV)-specific cONA clones spanning the 3' terminal 5 kb of the genomic RNA were isolated, sequenced, and used as probes for identification of PRRSV-specific RNAs. The PRRSV genome is a positive-stranded polyadenylated RNA of about 15 kb. In infected cells, a 3' coterminal nested set of six major subgenomic mRNAs could be demonstrated. Within the 3' terminal 3.5 kb of the PRRSV genome, six overlapping reading frames (ORFs) were identified, each most likely expressed by one of the subgenomic mRNAs. Amino acid sequence comparisons revealed that the most 3' terminal ORF (ORF7) encodes the PRRSVnucleocapsid protein with a calculated molecular weight of 14 kna. It displays 44.8% amino acid identity with the capsid protein of lactate dehydroqenase-elevatinq virus (LOV) and 23.6% with that of equine arteritis virus (EAV). The product of ORF6, the second 3' terminal ORF, represents a putative membrane protein and exhibits 53.2 and 27.2% amino acid identity with the corresponding LOV and EAV polypeptides, respectively. Similar to EAV, ORFs 2 through 5 might encode glycosylated viral proteins. The polypeptide deduced from the most 5' ORF (ORF1b) contains two conserved domains common to EAV and coronavirus potymerases. Genome organization, strategy of gene expression, and the sequence of deduced proteins show that PRRSV belongs to the Arterivirus group of viruses. Abstract Zoonoses with a wildlife reservoir represent a major public health problem, affecting all continents. Hundreds of pathogens and many different transmission modes are involved, and many factors influence the epidemiology of the various zoonoses. The importance and recognition of wildlife as a reservoir of zoonoses are increasing. Costeffective prevention and control of these zoonoses necessitate an interdisciplinary and holistic approach and international cooperation. Surveillance, laboratory capability, research, training and education, and communication are key elements. Abstract We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gammainterferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta (αβ) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta (γδ) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chain and a minority of vaccinated immunoglobulin heavy chain-deficient (μMT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3 + T cells are required for protection. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. practical requirement in the development of any EIAV vaccine is compatibility with established regulatory policies and diagnostic assays. The ability of EIAV-infected horses to routinely establish immunologic control over virus replication and disease suggests that an effective vaccine can indeed be developed, if the critical natural immune correlates of protection can be elicited by a candidate vaccine. An attenuated live EIAV vaccine with a reported protection efficacy of about 70% has been used in China since the early 1980s, but the effectiveness of this vaccine remains to be confirmed outside of that country. Evaluation of other candidate EIAV vaccines (live-attenuated, inactivated whole virus, subunit vaccines, synthetic peptides, etc.) under experimental conditions has revealed a spectrum of vaccine efficacy that ranges from 'sterile protection' (prevention of infection upon inoculation with EIAV) to severe elevation of EIAVreplication, and exacerbation of disease. These results indicate that immune responses to EIAVare a double-edged sword that can either mediate protection or yield vaccine enhancement. Vaccine enhancement has previously been reported for other viral infections (dengue virus, respiratory syncitial virus, feline infectious peritonitis virus) and is of special concern with macrophage-tropic viruses. Similar examples of vaccine protection and enhancement have been reported in studies of experimental vaccines for other lentiviruses, including feline immunodeficiency virus, caprine arthritis-encephalitis virus, and visna-maedi virus. These observations in several diverse animal lentivirus systems suggest that the potential for immune enhancement may be a general property of lentiviruses, including HIV-1. Current efforts in the production of a commercial EIAV vaccine are focused on the development of a vaccine that can achieve sufficient maturation of immune responses to provide protection from virus infection, but allow the serological differentiation between vaccinated and infected horses. In this regard, DNA vaccine strategies appear to be well suited to accomplish these criteria for a commercial EIAV vaccine.EIAV provides a dynamic system for examining the interaction between virus populations and host immune responses that are evolving in response to each other. In addition, EIAV offers a remarkable model for studying the delicate balance between immune responses to a persistent virus infection that result in disease and those that have beneficial results. A characterization of the nature of protective and enhancing immune responses can provide important information about the mechanisms of lentivirus disease and the type of immune responses to be elicited or avoided by a vaccine. The results of these studies in the EIAV system should be applicable to other lentiviruses, including HIV-1. Abstract Background: Rapid diagnosis of influenza is important for controlling outbreaks and starting antiviral therapy. Direct antigen detection (DAD) is rapid, but lacks sensitivity, whereas nucleic acid amplification testing (NAT) is more sensitive, but also more time-consuming. Objectives: To evaluate the performance of a rapid isothermal NAT and two DADs. Study design: During February-May 2014, we tested 211 consecutive patients with influenza-like illness using a commercial isothermal NAT (Alere TM Influenza A&B) as well as the DAD Sofia ® Influenza A + B and BinaxNOW ® Influenza A&B for detection of influenza-A and -B virus. RespiFinder-22 ® a commercial multiplex NAT served as reference test. Serial 10-fold dilutions of influenza-A and -B cell culture supernatants were examined. Another 225 patient samples were tested during December 2014-February 2015.Results: Compared to RespiFinder-22 ® , the isothermal NAT Alere TM Influenza A&B, and the DAD Sofia ® Influenza A + B and BinaxNOW ® Influenza A&B had sensitivities of 77.8%, 59.3% and 29.6%, and specificities of 99.5%, 98.9% and 100%, respectively, for the first 211 patient samples. Alere TM Influenza A&B showed 85.7% sensitivity and 100% specificity in the second cohort. Isothermal NAT was 10-100-fold more sensitive compared to DAD for influenza virus culture supernatants with a lower limit of detection of 5000-50,000 copies/mL. The average turn-around time (TAT) of isothermal NAT and DADs was 15 min, but increased to 110 min for Alere TM Influenza A&B, 30 min for BinaxNOW ® Influenza A&B, and 45 min for Sofia ® Influenza A + B, when analyzing batches of 6 samples. Conclusion: Simple sample processing and a TAT of 15 min render isothermal NAT Alere TM Influenza A&B suitable for sequential near-patient testing, but the TAT advantage is lost when testing of larger series. Abstract Rotavirus is the main cause of severe diarrhea in children aged less than 5 years, leading to elevated morbidity and mortality worldwide. The aim of the present study was to determine the etiology of enteric viruses reported to the Catalan Microbiology Notification System (CMNS) (rotavirus and adenovirus 40-41) and to evaluate the importance of these viruses in enteric disease. Material and methods. We retrospectively reviewed the notifications made from 1995-2002 by the laboratories of hospitals participating in the CMNS. Cases of acute gastroenteritis (AGE) were diagnosed by antigen detection in stools. The variables analyzed were: age, sex, month, and year of isolation. Statistical analysis was performed using the χ 2 test Abstract Feline infectious peritonitis (FIP) virus antigen was demonstrated after methanol, ethanol or formalin fixation in paraffin-embedded tissues by means of monoclonal and polyclonal antibodies. The monoclonal antibody was induced by immunization with transmissible gastroenteritis virus. Polyclonal antibodies were obtained by purification on protein A-Sepharose of ascites fluid from a cat with FIP. Almost all cats diagnosed as suffering from FIP by postmortem and histological examination exhibited FIP virus (FIPV) antigen in macrophages in granulomas whereas FIPV antigen was only once demonstrable in another location. Abstract Ribavirin, a triazole derivative has a wide application in the medical field as an antiviral drug. In the present work, a quantum chemical approach was followed to study the vibrational modes and the reactivity. Experimental techniques of FT-IR, FT-Raman were used to study the vibrational spectrum. A complete vibrational analysis was carried out and assignments of the fundamental modes were proposed. Molecular electrostatic potential, frontier molecular orbitals, electronic localization function and fukui functions were analyzed by using wavefunction analyser, Multiwfn 3.4.1 to study the chemical reactivity. Band gap energy of the title molecule is found to be 6.01 eV, as calculated from the HOMO-LUMO energies. The intermolecular charge transfer within the molecule was confirmed from the charge transfer interactions. Molecular docking studies were carried out to study the biological activity of the compound. Viral target proteins such as Dengue and Hepatitis C were chosen and the respective docking parameters were calculated. Abstract We determined the molecular sequence of monoclonal antibodies (mAbs) to serogroups B and C capsular polysaccharides (PS) of Neisseria meningitidis. N. meningitidis infections are a leading cause of bacterial septicemia and meningitis in humans. Antibodies to PS are fundamental to host defense and diagnostics. The polysaccharide capsule of group B N. meningitidis is poorly immunogenic and thus is an important model for studying pathogen-host co-evolution through understanding the molecular basis of the host immune response. We used a modified reversetranscriptase PCR to amplify and sequence the V-genes of murine hybridomas produced against types B and C capsular PS. Databank analysis of the sequences encoding the V-genes of type C capsular PS mAb, 4-2-C, reveal that heavy chain alleles are recurrently used to encode this specificity in mice. Interestingly, a V-gene from the same germline family also encodes the V-domain of mAbs 2-2-B, which targets the antigenically distinct serogroup B capsular PS. Somatic mutation, junctional diversity and alternative light chains collectively impart the specificity for these serologically distinct epitopes. Knowledge of the specific immunoglobulin genes used to target common bacterial virulence factors may lead to insights on pathogen-host co-evolution, and the potential use of this information in pre-symptomatic diagnosis is discussed. Abstract Biotechnological innovation is gaining increased recognition as an important tool for improving global health. The challenge, however, lies in defining the role of technology transfer to develop therapies for diseases prevalent in developing countries. During the past decade, a large disparity emerged between the developed and developing world in accessing affordable medicines because of the pharmaceutical industry's focus on health areas bearing greatest profits. Discussed herein are several mechanisms that provide partial solutions to this challenge.The Office of Technology Transfer of the US National Institutes of Health has increased its technology licensing pertaining to neglected diseases to partners in developing regions. Establishing partnerships through the transfer of technologies and assisting indigenous institutions build R and D capacity may positively impact policies on protection of intellectual property rights and increase multinational company investments in lesser-developed countries. This will most probably result in the development of more accessible therapies for those in need. Published by Elsevier Inc. Abstract Nature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list items might not apply to an individual manuscript, but all fields must be completed for clarity.Describe how sample size was determined.With baseline carriage rates of 50% (expected based on prior data from our model), 73 participants in each arm were required for 80% power to detect a 50% relative increase in pneumococcal acquisition at any time point, after 10% drop-out.Describe any data exclusions.Of 130 vaccinated volunteers, five were natural pneumococcal carriers (two in LAIV arm and three in control arm) and were excluded from further analysis. Another 8 subjects in the LAIV arm were excluded following a systematic LAIV dispensing error by a single practitioner, as recommended by the trial steering group. This resulted in a final 55 subjects analysed in the LAIV arm and 62 subjects in the control arm.Describe the measures taken to verify the reproducibility of the experimental findings.Key findings, including monocyte recruitment to the nasopharynx were validated in an independent patient cohort. All attempts are replication findings were successful.Describe how samples/organisms/participants were allocated into experimental groups.Using a permuted-block algorithm (1:1, blocks of 10) held in sealed envelopes, participants were randomised to receive either nasal LAIV (Fluenz Tetra or FluMist Tetra, AstraZeneca, UK, used interchangeably due to procurement shortages) paired with intramuscular placebo (0.5ml normal saline), or nasal placebo [control] (0.2ml normal saline) paired with intramuscular Quadrivalent Inactivated Influenza Vaccination (Fluarix Tetra, GlaxoSmithKline, UK) (see supplemental methods for flu strains).Describe whether the investigators were blinded to group allocation during data collection and/or analysis.This was a double-blinded, randomized trial where investigators were blinded during data collection and data analysis Note: all in vivo studies must report how sample size was determined and whether blinding and randomization were used. Abstract We investigate the effects of delaying the time to recovery (delayed recovery) and of nonuniform transmission on the propagation of diseases on structured populations. Through a mean-field approximation and large-scale numerical simulations, we find that postponing the transition from the infectious to the recovered states can largely reduce the epidemic threshold, therefore promoting the outbreak of epidemics. On the other hand, if we consider nonuniform transmission among individuals, the epidemic threshold increases, thus inhibiting the spreading process. When both mechanisms are at work, the latter might prevail, hence resulting in an increase of the epidemic threshold with respect to the standard case, in which both ingredients are absent. Our findings are of interest for a better understanding of how diseases propagate on structured populations and to a further design of efficient immunization strategies. Abstract Extraordinary incidents resulting in airborne infectious disease outbreaks could produce patient isolation requirements that exceed most hospitals' capacity. This article investigates expedient methods to establish airborne infection isolation areas using a commercially available portable filtration unit and common hardware supplies. The study was conducted within a conventional, nonisolation hospital room, and researchers evaluated several airborne isolation configurations that did not require building ventilation or structural modifications. A portable high-efficiency particulate air filtration unit and full-length plastic curtains established a ''zone-within-zone'' protective environment using local capture and directional airflows. The cost of constructing the expedient configurations was less than US$2,300 and required fewer than 3 person-hours to construct. A medical nebulizer aerosolized polystyrene latex microspheres to generate respirable condensation nuclei. Aerosol spectrometers sized and counted respirable particles at the source patient and health care worker positions and in areas outside the inner zone. The best-performing designs showed no measurable source migration out of the inner isolation zone and mean respirable particle counts up to 87% lower at the health care worker position(s) than those observed directly near the source patient location. Investigators conclude that with careful implementation under emergency circumstances in which engineered isolation rooms are unavailable, expedient methods can provide affordable and effective patient isolation while reducing exposure risks and potential disease transmission to health care workers, other patients, and visitors. Abstract Isatin and its metabolites are constituents of many natural substances. They are also components of many synthetic compounds exhibiting a wide range of effects, including antiviral activity, antitumor and antiangiogenic activity, antibacterial, antitubercular, antifungal, antiaptotic, anticonvulsant and anxyolytic activities. Isatin itself is an endogenous oxidized indole with a wide spectrum of behavioral and metabolic effects. It has a distinct and discontinuous distribution in the brain, peripheral tissues and body fl uids and isatin binding sites are widely distributed also. Its output is increased during stress. Its most potent known in vitro actions are as an antagonist of atrial natriuretic peptide (ANP) function and NO signaling. As we understand more about its function and sites of action we may be able to develop new pharmacological agents to mimic or counteract its activity. We consider here the most promising biological targets for various isatin analogues and/or metabolites, which are employed for the development of various groups of therapeutics. It is also possible that the level of endogenous isatin may infl uence the in vivo pharmacological activity of compounds possessing the isatin moiety. Abstract Background: Since August 2014, an increase in infections caused by enterovirus D68 (EV-D68) was reported in the USA and Canada, for the most part in children presenting with severe respiratory symptoms. Objectives: To determine whether an increase in severe EV-D68 respiratory infections was observed in our region. Study design: Samples from patients with respiratory symptoms were screened for viral pathogens, including rhinovirus and enterovirus. Subsequently, samples positive for rhinovirus and enterovirus were routinely sequenced for phylogenetic analysis. Furthermore, an additional method was used to detect EV-D68 specifically. Results: During the first three quarters of the year 2014, 1896 respiratory samples were analyzed; 39 (2%) of them tested positive for enterovirus. Eighteen samples tested positive for EV-D68, obtained from 16 different patients admitted to our hospital. Eleven were children below the age of 18, of whom five children needed intensive care treatment. The remaining five samples were from adults, who all had an underlying disease; three were transplant patients (heart, lung and renal transplantation), the other two had an underlying lung condition (COPD, asthma). Phylogenetic analysis showed a close relationship with the strains circulating currently in the USA, all belonging to the known EV-D68 genetic subtypes. Conclusions: We observed an increase of EV-D68 infections in our population, both in children as well as in adult. In 2014 there have been 16 cases so far, compared to none in 2011 and 2013 and a single case in 2012. Phylogenetic analysis identified two similar clusters as shown in the USA and Canada. Abstract The usefulness of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid preclinical detection of classical swine fever virus (CSFV) infection was evaluated. The RT-LAMP reaction could be finished in 60 min under isothermal condition at 65 C by employing a set of four primers targeting the 5 0 untranslated region of CSFV. The RT-LAMP assay of CSFV showed higher sensitivities than that of RT-PCR, with a detection limit of 5 copies per reaction. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 2, porcine parvovirus, porcine pseudorabies virus, Japanese encephalitis virus, and porcine reproductive and respiratory syndrome virus. The detection rates of CSFV RT-LAMP, RT-PCR and virus isolation for samples including blood, tonsil, nasal and rectal swabs from uninoculated pigs without any clear clinical symptom were 89%, 78% and 71%, respectively. Furthermore, all of the assays showed higher sensitivity for blood and tonsil swabs samples than nasal and rectal swabs. These results indicate that the CSFV RT-LAMP assay is a valuable tool for its rapid, cost-effective detection and has potential usefulness for rapid pre-clinical detection and surveillance of classical swine fever in developing countries. Abstract We describe the use of random peptide sequences for the mapping of antigenic determinants. An oligonueleotide with a completely degenerate sequence of 17 or 23 nucleotides was inserted into a bacterial expression vector. This resulted in an expression library producing random hexa-or octapeptides attached to a fl-galactosidase hybrid protein. Mimotopes, or antigenic sequences that mimic an epitope, were selected by immur.oscteening of colonies with monoclonal antibodies, which were specific for antigenic sites on the spike protein of the coronavirus transmissible gastroenteritis virus. We report one mimotope for antigenic site II, eight for site I!I and one for site IV. The site I!1 and site IV mimotopes were closely similar to the corresponding linear epitopes, localized previously in the amino acid sequence of the S protein. An alignment of the site II mimotope and the sequence of the S protein around Trp97, which is substituted in escape mutants, suggests that this mimotope mimics a conformational epitope located around residues 97-103. Applications of mimotopes to epitope mapping, serodiagnosis and vaccine development are discussed. Abstract Genetic sequences highly related to Bovine coronavirus (BCoV) were detected in fecal samples from Peruvian 1-3 week old alpaca crias located on six farms in Puno department, some of which shared pastures with cattle. A total of 60 samples were screened for coronavirus using a nested PCR amplification of a fragment of the RNA-dependent RNA polymerase (RdRp) gene. Sequences from 11 positive samples were highly similar to the Kakegawa, Quebec and Mebus BCoV strains (99.5-100.0%) and 99.2% identical to an alpaca Coronavirus (CoV) previously detected in the USA. The detection of genetic sequences related to BCoV from Peruvian alpaca crias suggests possible role of this virus on enteric disorders etiology in the High Andes. Abstract Recent progress in lung biology includes the description of a series of pulmonary stem and progenitor cells involved in homeostasis and regeneration of the respiratory system. Moreover, the contribution of extrapulmonary stem cells to healthy and pathological lung tissue has been observed and the developmental biology of such processes should provide important hints for understanding maintenance and repair of adult lung structure and function. Despite such remarkable advances, the phenotypic and especially the functional characterization of these stem and progenitor cells, and their derivatives, along with an understanding of the molecular cues and pathways underlying differentiation into specific respiratory lineages is still in its infancy. Accordingly, the role of endogenous and extrapulmonary stem cells in normal tissue repair and pathogenesis is still largely mysterious and added basic knowledge is required in order to explore their potential for novel regenerative therapies. This review provides an overview of the current state of the art in adult lung stem cell biology including technical aspects of isolation, characterization and differentiation, and a discussion of perspectives for future regenerative therapies. Abstract Particulate air pollution is a serious problem in Beijing. The annual concentration of particulate matter with aerodynamic diameter less than 10 μm (PM 10 ), ranging from 141 to 166 μg m − 3 in 2000-2004, could be very harmful to human health. In this paper, we presented the mortality and morbidity effects of PM 10 pollution based on statistical data and the epidemiological exposure-response function. The economic costs to health during the 5 years were estimated to lie between US$1670 and $3655 million annually, accounting for about 6.55% of Beijing's gross domestic product each year. The total costs were apportioned into two parts caused by: the local emissions and long-range transported pollution. The contribution from local emissions dominated the total costs, accounting on average for 3.60% of GDP. However, the contributions from transported pollution cannot be neglected, and the relative percentage to the total costs from the other regions could account for about 45%. An energy policy and effective measures should be proposed to reduce particulate matter, especially PM 2.5 pollution in Beijing to protect public health. The Beijing government also needs to cooperate with the other local governments to reduce high background level of particulate air pollution. Abstract Emerging infectious diseases (EIDs) have always been one of the major threats to public health. Although the implementation of mandatory testing for 4 classical transfusion-transmitted infectious-human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and syphilis-has reduced the transfusion risk of these pathogens, the potential threat of various EID agents and their constantly evolving variants to blood safety in China is not fully understood. This review presents 9 representative EID agents that are autochthonous and epidemic nationally or regionally in China. The epidemiologic status and distribution of these EID agents among donors and/or healthy populations are summarized. The potential risks of these EID agents to blood safety are discussed. The review also explores strategies to strengthen hemovigilance systems and studies to further evaluate the impact of EID agents on blood safety. Abstract Porcine epidemic diarrhea virus (PEDV) has caused severe economic losses both recently in the United States (US) and historically throughout Europe and Asia. Traditionally, analysis of the spike gene has been used to determine phylogenetic relationships between PEDV strains. We determined the complete genomes of 93 PEDV field samples from US swine and analyzed the data in conjunction with complete genome sequences available from GenBank (n = 126) to determine the most variable genomic areas. Our results indicate high levels of variation within the ORF1 and spike regions while the C-terminal domains of structural genes were highly conserved. Analysis of the Receptor Binding Domains in the spike gene revealed a limited number of amino acid substitutions in US strains compared to Asian strains. Phylogenetic analysis of the complete genome sequence data revealed high rates of recombination, resulting in differing evolutionary patterns in phylogenies inferred for the spike region versus whole genomes. These finding suggest that significant genetic events outside of the spike region have contributed to the evolution of PEDV. Abstract During the 2013-2016 Ebola outbreak in West Africa an expert panel was established on the instructions of the UK Prime Minister to identify priority pathogens for outbreak diseases that had the potential to cause future epidemics. A total of 13 priority pathogens were identified, which led to the prioritisation of spending in emerging diseases vaccine research and development from the UK. This meeting report summarises the process used to develop the UK pathogen priority list, compares it to lists generated by other organisations (World Health Organisation, National Institutes of Allergy and Infectious Diseases) and summarises clinical progress towards the development of vaccines against priority diseases. There is clear technical progress towards the development of vaccines. However, the availability of these vaccines will be dependent on sustained funding for clinical trials and the preparation of clinically acceptable manufactured material during inter-epidemic periods. Abstract In recent years, the outbreaks of porcine epidemic diarrhea (PED) caused by the highly virulent porcine epidemic diarrhea virus (PEDV) variants occurred frequently in China, resulting in severe economic impacts to the pork industry. In this study, we selected and analyzed the genetic evolution of 15 PEDV representative strains that were identified in fecal samples of diarrheic piglets in 10 provinces and cities during 2011-2017. The phylogenetic analysis indicated that all the 15 PEDV isolates clustered into G2 genotype associated with the current circulating strains. Compared with the genome of the prototype strain CV777, these strains had 103-120 amino acid mutations in their S proteins, most of which were in the N terminal domain of S1 (S1-NTD). We also found 37 common mutations in all these 15 strains, although these strains shared 96.9-99.7% nucleotide homology and 96.3-99.8% amino acid homology in the S protein compared with the other original pandemic strains. Computational analysis showed that these mutations may lead to remarkable changes in the conformational structure and asparagine (N)-linked glycosylation sites of S1-NTD, which may be associated with the altered pathogenicity of these variant PEDV strains. We evaluated the pathogenicity of the PEDV strain FJzz1 in piglets through oral and intramuscular infection routes. Compared with oral infection, intramuscular infection could also cause typical clinical signs but with a slightly delayed onset, confirming that the variant PEDV isolate FJzz1 was highly pathogenic to suckling piglets. In conclusion, we analyzed the genetic variation and pathogenicity of the emerging PEDV isolates of China, indicating that G2 variant PEDV strains as the main prevalent strains that may mutate continually. This study shows the necessity of monitoring the molecular epidemiology and the etiological characteristics of the epidemic PEDV isolates, which may help better control the PED outbreaks. Stevenson et al., 2013; Vlasova et al., 2014) . Subsequently, the outbreak of PED occurred in Okinawa, Japan in October 2013, followed by https://doi. Abstract In South Africa, until recently, veterinary waste has not been included in definitions of health care waste, and so has been neglected as a contributor to the hazardous waste stream. Despite the application of, for example, the ''Polluter Pays'' principle in South African environmental legislation, to generators of waste, which would include veterinarians, there appears to be little awareness of and even less enforcement of the legislation in this regard. This paper reports on a 2001-2003 survey of management practices of the five waste contractors servicing just over half of the veterinarians in Durban, South Africa's second largest city. Some of their activities, when evaluated in terms of the legislation, guidelines and policies relating to waste handling and disposal, were found to be non-compliant. Since any discussion on waste management should take cognisance of waste from generation to final disposal, the responsibility of veterinarians as waste generators is also discussed in the light of the recent developments in health care waste management in South Africa. This study presents a review of past and current policies, legislation and guidelines that have application to veterinary waste. This is the first study to address veterinary waste disposal in any South African city. Abstract The definition of mitigation includes a wide variety of measures taken before an event occurs that will prevent illness, injury, and death and limit the loss of property. Taking steps to mitigate potential hazards has taken on increasing favor in disaster preparedness circles, particularly in the international arena, where the pursuit of disaster risk reduction (DRR) and disaster risk management (DRM) is emphasized above efforts focused simply on disaster event response. The absolutely stunning loss of life, illnesses, injury, psychological impact, displacement from home and community, and social and financial consequences of a disaster, coupled with its disproportionate impact on the already disadvantaged, makes it imperative to fully implement the best principles and practices of disaster mitigation. 1 These principles and practices fall into two types: 1. Disaster Risk Reduction (DRR) aims to reduce the damage caused by natural hazards like earthquakes, floods, droughts, and cyclones, through the ethic of prevention. 2 2. Disaster Risk Management (DRM) includes management activities that address and seek to correct or reduce disaster risks that are already present. 3The Hyogo Framework for Action 4 offers guiding principles, priorities for action, and practical means to achieve disaster resilience for vulnerable communities. Priorities for action include the following: 1. Ensure that DRR is a national and local priority with a strong institutional basis for implementation 2. Identify, assess, and monitor disaster risks and enhance early warning 3. Use knowledge, innovation, and education to build a culture of safety and resilience at all levels 4. Reduce the underlying risk factors 5. Strengthen disaster preparedness for effective response at all levels Although the primary emphasis on the Hyogo Framework is natural disasters, the processes discussed and framework for community resiliency and partnerships have application to all types of hazard responses.The Federal Emergency Management Agency (FEMA) reinforces the importance of engaging "not only FEMA and its federal partners, but also local, tribal, state and territorial partners; non-governmental faith-based and nonprofit organizations, and private sector industry; to individuals, families and communities, who continue to be the nation's most important assets as first responders during a disaster." Engaging local communities and a diverse set of partners ensures that the "unique and diverse needs of a population" are met and helps communities become more resilient after a disaster. 5 Abstract The problem of the asymptotic dynamics of a quarantine/isolation model with time delay is considered, subject to two incidence functions, namely standard incidence and the Holling type II (saturated) incidence function. Rigorous qualitative analysis of the model shows that it exhibits essentially the same (equilibrium) dynamics regardless of which of the two incidence functions is used. In particular, for each of the two incidence functions, the model has a globally asymptotically stable disease-free equilibrium whenever the associated reproduction threshold quantity is less than unity. Further, it has a unique endemic equilibrium when the threshold quantity exceeds unity. For the case with the Holling type II incidence function, it is shown that the unique endemic equilibrium of the model is globally asymptotically stable for a special case. The permanence of the disease is also established for the model with the Holling type II incidence function. Furthermore, it is shown that adding time delay to and/or replacing the standard incidence function with the Holling type II incidence function in the corresponding autonomous quarantine/isolation model with standard incidence (considered in Safi and Gumel (2010) [10]) does not alter the qualitative dynamics of the autonomous system (with respect to the elimination or persistence of the disease). Finally, numerical simulations of the model with standard incidence show that the disease burden decreases with increasing time delay (incubation period). Furthermore, models with time delay seem to be more suitable for modeling the 2003 SARS outbreaks than those without time delay. Abstract The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. Using mice with granulocyte-and macrophage-specific deletion of the essential autophagy protein Atg5, we show that Atg5 is required for in vivo resistance to the intracellular pathogens Listeria monocytogenes and Toxoplasma gondii. In primary macrophages, Atg5 was required for interferong (IFN-g)/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect classical hallmarks of autophagy, such as autophagosomes enveloping T. gondii, Atg5 was required for recruitment of IFN-g-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane, an event that mediates IFN-g-mediated clearance of T. gondii. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo, and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking. Abstract As infectious diseases circle the globe, medical costs skyrocket and the waste stream continues to grow, it is imperative to look for medical textiles with improved protective performance, low costs, and minimized environmental impacts. Medical textiles include surgical gowns, gloves, drapes, facemasks, dresses, and linens, which could be disposable or reusable based on uses. The selection of reusable and disposable textiles is determined by many factors, such as cost, protective and comfort properties of the textiles, government regulations, and possibly social and psychological perceptions of both types of textile. This chapter intends to provide a broad view on both disposable and reusable textiles, as well as suggestions on improved protection against transmission of infectious diseases by textile materials. Abstract Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenzavaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPY-MEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains. Abstract Fordism A system of mass production and consumption that originated with Henry Ford, and led to stabilization of wage relations, guarding against overproduction or underconsumption through state entitlements, regulation of trade agreements, and a culture of mass consumption. Keynesianism A system of social entitlements that included unemployment insurance, health care, mother's allowance, and welfare, originating with John Maynard Keynes and instituted after the Great Depression of the 1930s in many industrialized nations. Neoliberalism A strategy of governance that attempts as much as possible to reduce a state's responsibility for social entitlements and to download responsibilities for care onto cities, communities, families, and individuals. Post-Fordism A globalized system of production and consumption characterized by the increasing use of robotics in production systems, externalizing uncertainties of the market to small-time suppliers who produce on demand (just in time), and the marketing of specialized goods to globalized "niches" of consumers. Post-Structuralism A movement in French continental philosophy centered on the destabilization of claims to truth and variously connected to the antihumanist rejection of a unified rational subject; a rejection of binary oppositions; and a view which sees knowledge as fluid, unstable, and discursively produced rather than ideological.When figures as divergent as conservative British Prime Minister, Margaret Thatcher, and left leaning political philosopher, Ernesto Laclau, declare society is an "impossible object" we might askdwhy investigate the relationship between society and space? The topic appears so vast that when properly charteddlike Borges' famous map drawn at a 1:1 scaledso much ground is covered as to become practically and analytically useless. Moreover, the relationship would seem a truism: societies create spaces that best express their needs; spaces in turn constrain or enable societal developments. But how we conceive of this relationship subtends any attempt to understand, critique, or transform, the social world. Our understanding of society-space, as a totality, determines the vantage point from which we understand the social world. It locates these delimited processes, both literally and figuratively, in relation to a social whole.The first approach views society-space in terms of exploitative relations organized in a "structured coherence"da social formation, whose territorial boundaries loosely approximate politicojuridical boundaries. Rooted in a Marxist vision of social inequity, whereby societal relations are defined by exploitationdalienated labordits debates have focused on the logical primacy of particular unequal relations (classed, gendered, and racialized) and their attendant spatialities (workplace, home, underdeveloped neighborhoods, regions, and nations). The second approach sees society-space as a "strategic field"dconstituted through productive relations of governancedan occupied zone in which struggles resemble the engagements of a war. Underpinned by philosophical approaches rooted in the work of Foucault, Nietzsche, Heidegger, and Clausewitz, in this view, societies are organized through a constellation of discursive logics, enacted in everyday spaces, and through these practices the abnormal are contained, controlled, excluded, or policed. The third sees society-space as "performative field", beginning with the premise that society is fundamentally unstable, its subjects perpetually hybridizing, caught in structured instabilitiesdin a social field that is conflictual and paradoxical. In the fourth approach, society-space is an "immanent field", an expression of continuous differentiation, its subjects are unstable because they continuously transform themselves, engaging in acts of conjunction, connection, or collaboration with their milieu.These four approaches share a recognition of persistent structural social inequities, a conviction that space is complicit in the production and possible transformation of these relationships, and the belief that one should not merely analyze societies but attempt to change themdto create a more equitable world.The contemporary view is that the relationship between the spatial and social is an iterative one. This is by now so commonplace as to seem self-evident. However, this insight has only recently returned to geography after a long conceptual slumber in the seductive embrace of mathematical description. Mathematical approaches enabled a complex description of either social or spatial processes, but little theorization, and as David Harvey noted, no strategy at the interface between the spatial and the social. Geography's Abstract Little is known about the long-term consequence of severe acute respiratory syndrome (SARS). We carried out an assessment on SARS patients after their recovery from their acute illness. Method: Postal survey comprising Health-Related Quality of Life (HRQoL) questionnaires and anxiety and depression measures was sent to them at 3 months' postdischarge. Results: There was a significant impairment in both the HRQoL and mental functioning. Forty-one percent had scores indicative of a posttraumatic stress disorder (PTSD); about 30% had likely anxiety and depression. Conclusion: SARS has significant impact on HRQoL and psychological status at 3 months. D Abstract Global warming is believed to induce a gradual climate change. Hence, it was predicted that tropical insects might expand their habitats thereby transmitting pathogens to humans. Although this concept is a conclusive presumption, clear evidence is still lacking-at least for viral diseases. Epidemiological data indicate that seasonality of many diseases is further influenced by strong single weather events, interannual climate phenomena, and anthropogenic factors. So far, emergence of new diseases was unlinked to global warming. Re-emergence and dispersion of diseases was correlated with translocation of pathogen-infected vectors or hosts. Coupled ocean/ atmosphere circulations and 'global change' that also includes shifting of demographic, social, and economical conditions are important drivers of viral disease variability whereas global warming at best contributes. Abstract Background: Viral infections contribute to morbidity in cystic fibrosis (CF), but the impact of respiratory viruses on the development of airway disease is poorly understood. Methods: Infants with CF identified by newborn screening were enrolled prior to 4 months of age to participate in a prospective observational study at 4 centers. Clinical data were collected at clinic visits and weekly phone calls. Multiplex PCR assays were performed on nasopharyngeal swabs to detect respiratory viruses during routine visits and when symptomatic. Participants underwent bronchoscopy with bronchoalveolar lavage (BAL) and a subset underwent pulmonary function testing. We present findings through 8.5 months of life. Results: Seventy infants were enrolled, mean age 3.1 ± 0.8 months. Rhinovirus was the most prevalent virus (66%), followed by parainfluenza (19%), and coronavirus (16%). Participants had a median of 1.5 viral positive swabs (range 0-10). Past viral infection was associated with elevated neutrophil concentrations and bacterial isolates in BAL fluid, including recovery of classic CF bacterial pathogens. When antibiotics were prescribed for respiratory-related indications, viruses were identified in 52% of those instances. Conclusions: Early viral infections were associated with greater neutrophilic inflammation and bacterial pathogens. Early viral infections appear to contribute to initiation of lower airway inflammation in infants with CF. Antibiotics were commonly prescribed in the setting of a viral infection. Future investigations examining longitudinal relationships between viral infections, airway microbiome, and antibiotic use will allow us to elucidate the interplay between these factors in young children with CF. Abstract Clinical Signs As with other animals, infection with Giardia sp. is most often asymptomatic and self-limitingThe Common Marmoset in Captivity and Biomedical Research Abstract Background: Acute respiratory tract infections (RTI) cause substantial morbidity during childhood, and are responsible for the majority of pediatric infectious diseases. Although most acute RTI are thought to be of viral origin, viral etiology is still unknown in a significant number of cases. Objectives: Multiplexed whole genome sequencing (WGS) was used for virome determination directly on clinical samples as proof of principle for the use of deep sequencing techniques in clinical diagnosis of viral infections. Study design: WGS was performed with nucleic acids from sputum and nasopharyngeal aspirates from four pediatric patients with known respiratory tract infections (two patients with human rhinovirus, one patient with human metapneumovirus and one patient with respiratory syncytial virus), and from four pediatric patients with PCR-negative RTI, and two control samples. Results: Viral infections detected by routine molecular diagnostic methods were confirmed by WGS; in addition, typing information of the different viruses was generated. In three out of four samples from pediatric patients with PCR-negative respiratory tract infections and the two control samples, no causative viral pathogens could be detected. In one sample from a patient with PCR-negative RTI, rhinovirus type-C was detected. Almost complete viral genomes could be assembled and in all cases virus species could be determined. Conclusions: Our study shows that, in a single run, viral pathogens can be detected and characterized, providing information for clinical assessment and epidemiological studies. We conclude that WGS is a powerful tool in clinical virology that delivers comprehensive information on the viral content of clinical samples. Abstract goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEVinfected gosling cells displayed characteristic 180∼200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease. Abstract Abstraet--A battery of in vitro and in vivo tests were conducted on HCFC-141b as a vapour. Bacterial gene mutation assays with Escherichia coli and Salmonella typhimurium were negative in all tester strains. In vitro chromosomal aberration assays were positive on CHO cells but negative on human lymphocytes. Moreover, HCFC-141b was negative in vivo in a mouse micronucleus inhalation assay. On the basis of these data and previously reported genotoxicity testing, HCFC-141b is considered non-genotoxic. Groups of 80 male and 80 female Sprague-Dawley rats were exposed, by inhalation (6 hr/day, 5 days/wk) to vapours of HCFC-141b for 104 wk at target concentrations of 0 (control), 1500, 5000 and 20,000 ppm (increased from 15,000ppm after 17wk of exposure). No exposure-related effects of toxicological significance were noted with respect to survival, clinical signs, ophthalmoscopy, haematology, clinical chemistry, urinalysis or organ weight analysis. Reduced food intake and body weight gain were noted in both sexes of the 15,000 ppm group during the first 16 wk; thereafter, body weight gains in all groups were similar although the intergroup differences in body weight remained evident. Reduced food intake persisted in both sexes through wk 52 and in females during the second year of exposure. Treatmentrelated effects on macroscopic pathology were confined to increased incidences of testicular masses and altered appearance. Microscopic pathology examinations confirmed the testes as the target organ with findings of increased incidences of benign interstitial cell tumours and hyperplasia at 5000 and 20,000 ppm. The no-observable-adverse-effect level (NOAEL) was 1500 ppm. The testicular changes at high exposure levels were considered to be due to a change of the senile hormonal imbalance in geriatric rats and of little significance for the assessment of human health effects. Abstract Background and Objective: Viruses are infectious agents that replicate inside organisms and reveal a plethora of distinct characteristics. Viral infections spread in many ways, but often have devastating consequences and represent a huge danger for public health. It is important to design statistical and computational techniques capable of handling the available data and highlighting the most important features.Methods: This paper reviews the quantitative and qualitative behaviour of 22 infectious diseases caused by viruses. The information is compared and visualized by means of the multidimensional scaling technique.The results are robust to uncertainties in the data and revealed to be consistent with clinical practice.The paper shows that the proposed methodology may represent a solid mathematical tool to tackle a larger number of virus and additional information about these infectious agents. c o m p u t e r m e t h o d s a n d p r o g r a m s i n b i o m e d i c i n e 1 3 1 ( 2 0 1 6 ) 9 7 -1 1 0 j o u r n a l h o m e p a g e : w w w. i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / c m p b Abstract Mannheimia varigena was identified as the etiologic agent of meningitis in a young Belgian White Blue heifer calf. Species identification of the bacterium was done by phenotyping and molecularly confirmed by tDNA-PCR. Standard bacteriological examination might fail to differentiate species belonging to the genus Mannheimia. Abstract Thread-based microfluidics Point-of-care Cotton Colorimetric Electrochemical A B S T R A C T Over the past decades, researchers have been seeking attractive substrate materials to keep microfluidics improving to outbalance the drawbacks and issues. Cellulose substrates, including thread, paper and hydrogels are alternatives due to their distinct structural and mechanical properties for a number of applications. Thread have gained considerable attention and become promising powerful tool due to its advantages over paper-based systems thus finds numerous applications in the development of diagnostic systems, smart bandages and tissue engineering. To the best of our knowledge, no comprehensive review articles on the topic of thread-based microfluidics have been published and it is of significance for many scientific communities working on Microfluidics, Biosensors and Lab-on-Chip. This review gives an overview of the advances of thread-based microfluidic diagnostic devices in a variety of applications. It begins with an overall introduction of the fabrication followed by an in-depth review on the detection techniques in such devices and various applications with respect to effort and performance to date. A few perspective directions of thread-based microfluidics in its development are also discussed. Thread-based microfluidics are still at an early development stage and further improvements in terms of fabrication, analytical strategies, and function to become low-cost, low-volume and easy-to-use pointof-care (POC) diagnostic devices that can be adapted or commercialized for real world applications. Abstract The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo-and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform 13 C and 15 N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the β-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution. Abstract Background: Hospital emergency management plans are essential and must include input from an infection preventionist (IP). Multiple hospital planning documents exist, but many do not address infection prevention issues, combine them with noninfection prevention issues, or are disease/event specific. An all-encompassing emergency management planning guide for IPs is needed. Methods: A literature review and Internet search were conducted in December 2008. Data from relevant sources were extracted. A spreadsheet was created that delineated hospital emergency management plan components of interest to IPs. Results: Of the sources screened, 49 were deemed relevant. Eleven domains were identified: (1) having a plan; (2) assessing hospital readiness; (3) having infection prevention policies and procedures; (4) having occupational health policies and procedures; (5) conducting surveillance and triage; (6) reporting incidents, having a communication plan, and managing information; (7) having laboratory support; (8) addressing surge capacity issues; (9) having anti-infective therapy and/or vaccines; (10) providing infection prevention education; and (11) managing physical plant issues. Conclusion: Infection preventionists should use this article as an assessment tool for evaluating their hospital emergency management plan and for developing policies and procedures that will decrease the risk of infection transmission during a mass casualty event.Rebmann 713 Continued www.ajicjournal.org Vol. 37 No. 9 Rebmann 714.e1 d Identifies a process for monitoring individuals who are quarantined on-site for signs/symptoms of contagious diseases 4,9 d Includes plans for a designated location that is physically separated from other triage/evaluation areas for triaging patients with possible communicable diseases during a biologic event 4,5,7,26,27 d Has a protocol for creating or has triage signage that addresses language barriers, individuals with disabilities (visual, hearing, or others), and varying reading levels 4,24,26 Reporting, communication plan, and information management d Includes a protocol for 24/7 notification of infection prevention or hospital epidemiology of a known or suspected biologic event, including bioterrorism, outbreaks of emerging infectious diseases, and pandemics [2] [3] [4] [5] 7, 9, 19, [25] [26] [27] [28] ,43 d Includes a protocol for notifying local health officials and local law enforcement of a known or suspected biologic event, including bioterrorism, outbreaks of emerging infectious diseases, and pandemics [2] [3] [4] [5] 7, 9, 14, 19, [24] [25] [26] [27] [28] [29] 43 ,47 d Identifies the name, title, and contact information of a primary and back-up person assigned to communicate with hospital staff and volunteers regarding activation of the Plan and the status and impact of the biologic event during the incident 4,9,16 d Identifies the name, title, and contact information of a primary and back-up person assigned to communicate with public health authorities during a biologic event 4,11 d Identifies a person assigned to monitor state and federal public health advisories and update the pandemic response coordinator and members of the hospital emergency management committee when pandemic flu has been reported in the United States and is nearing the geographic area 4,5,9,10,24,42 d Includes a list and contact information of other health care entities and key community response entities (fire safety, law enforcement, EMS, public health, and governmental agencies) within the region with which it will be necessary to maintain real-time communication during a biologic event 4 d Describes the process with which health alert messages, information about contagious diseases, hospital rules for visitors, and need for and use of infection control measures (social distancing practices, isolation, PPE, and others) will be posted and/or communicated within the facility during an MCE (such as through an intranet, hotline, closed-circuit TV, and others) 2-5, Abstract Administration of recombinant feline interferon-v (rFeIFN) has been proposed for the prophylaxis of canine and feline parvovirosis. In the present study, the influence of the administration of rFeIFN on blood markers of inflammation (a-globulins, a 1acid glycoprotein) and immune system activation (g-globulins, IgG, IgM, specific anti-feline parvovirus IgG or IgM) was evaluated in a cattery developing an outbreak of feline panleukopenia due to feline parvovirus (FPV) infection few days after initial administration of rFeIFN. Kittens (n = 23) were injected with rFeIFN (1 MU/kg subcutaneoulsy, once a day for 3 days) and their blood parameters were compared with those of 17 untreated cats. Cats that survived the outbreak were vaccinated and re-sampled 1 month after the last rFeIFN administration. Time of emergence of clinical signs and survival rate were not significantly different between the two groups. Controls and treated cats surviving the infection had high levels of g-globulins, total-and anti-FPV specific IgGs, likely due to passive transfer of maternal immunity. Compared to controls, treated kittens had lower levels of a 1 -globulins and higher mean values of g-globulins and immunoglobulins. Data from samples collected after vaccination revealed a higher level of gglobulins, total-and anti-FPV specific IgGs in treated kittens, compared with controls, suggesting that rFeIFN stimulates antibody production. Based on this results, rFeIFN should be administered to the queen, to increase passive maternal immunity, or to kittens before introduction in a potentially contaminated environment. # * P < 0.05 vs. CI. y P < 0.05 vs. CII and vs. TI. Abstract Influenza was detected in 308 of 1027 (30%) samples tested; influenza A in 157 (15.3%), influenza B in 149 (14.5%) and RSV in 28 (2.7%). When compared against Fast Track Diagnostics Respiratory Pathogens 21 multiplex polymerase chain reaction and Cepheid Xpert Xpress Flu/RSV assay, Liat performance for the detection of influenza A or B was: sensitivity 85% [95% confidence interval (CI) 76e92)], specificity 98% (95% CI 97e99), negative predictive value 94% (95% CI 92e96) and positive predictive value 95% (95% CI 91e97). ª Abstract A B S T R A C T Abortion and neonatal mortality are events that can occur in breeding bitches and queens. It has been reported that up to 55% and 33% of these cases remain without a known cause, respectively, in canine and feline pregnancies. Unusual abortigenic and potentially zoonotic agents, including Coxiella burnetii and Leptospira spp., may be involved in these cases. C. burnetii is able to cause reproductive disorders in cattle, sheep and goats, and cases of abortion have been observed in dogs and cats. Moreover, several outbreaks of C. burnetii infection in humans have been caused by delivering bitches and queens, and some of these animals experienced abortion. Leptospira interrogans sensu lato is able to cause abortion or stillbirth in several animal species and its abortigenic role has occasionally been described in bitches and queens. The aim of this study was to search for C. burnetii and Leptospira spp. DNA in a retrospective series of 103 cases of canine and feline abortion, stillbirth, and neonatal mortality submitted for the identification of possible infectious agents. One hundred and fifty-one specimens were tested using PCR assays and found negative for C. burnetii and Leptospira DNA. However, in 49 samples (47.6%) other infectious causes of abortion, stillbirth, and neonatal mortality were identified. These results showed that C. burnetii and Leptospira spp. are probably not common abortigenic agents or causes of neonatal deaths in dogs. However, given the potential abortigentic and zoonotic role of these agents, surveillance of canine and feline abortion, stillbirth, and neonatal mortality could be advisable for a systematic investigation of these events. Abstract Respiratory infections and subsequent complications are one of the leading causes of high mortality in immunocompromised patients. Although chest radiograph and computed tomography are the commonly used diagnostic tools for the early diagnosis of lung manifestations of infections, they lack the specificity for the wide range of chest infections which can occur in immunocompromised patients. Systematic analysis of the imaging findings in correlation with the clinical settings along with comparison with the old images can expedite early and accurate diagnosis for subsequent appropriate management. Computer tomography findings in immunocompromised patients with respiratory infections, with regards to various clinical settings, will be discussed here. Abstract A B S T R A C T Astroviruses (AstV) are single-stranded, positive-sense RNA viruses and one of the major causes of infant diarrhoea worldwide. Diarrhoea is a common and important cause of morbidity and mortality in calves; therefore, we investigated whether the presence of AstV is associated with calf diarrhoea. We identified diverse AstV lineages from faecal samples of both healthy and diarrhoeic calves and healthy adult cattle in South West Scotland. AstV was common in calves (present in 74% (85/115) of samples) but uncommon in adult cattle (present in 15% (3/20) of samples). No association was found between the presence of AstV and calf diarrhoea or the presence of a specific AstV lineage and calf diarrhoea. AstV was strongly associated with the presence of rotavirus Group A (RVA), and a protective effect of age was evident for both AstV and RVA. Co-infections with multiple AstV lineages were detected in several calves and serial infection with different viruses could also be seen by longitudinal sampling of individuals. In summary, our study found genotypically diverse AstV in the faeces of calves in South West Scotland. However, no association was identified between AstV and calf diarrhoea, which suggests the virus does not play a primary role in the aetiology of calf diarrhoea in the group studied. Abstract Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a severe, clinically acute, vesicular disease of cloven-hoofed animals. RNA interference (RNAi) is a mechanism for silencing gene expression post-transcriptionally that is being exploited as a rapid antiviral strategy. To identify efficacious small interfering RNAs (siRNAs) to inhibit the replication of FMDV, candidate siRNAs corresponding to FMDV VP1 gene were designed and synthesized in vitro using T7 RNA polymerase. In reporter assays, five siRNAs showed significant sequence-specific silencing effects on the expression of VP1-EGFP fusion protein from plasmid pVP1-EGFP-N1, which was cotransfected with siRNA into 293T cells. Furthermore, using RT-qPCR, viral titration and viability assay, we identified VP1-siRNA517, VP1-siRNA113 and VP1-siRNA519 that transiently acted as potent inhibitors of FMDV replication when BHK-21 cells were infected with FMDV. In addition, variations within multiple regions of the quasispecies of FMDV were retrospectively revealed by sequencing of FMDV genes, and a single nucleotide substitution was identified as the main factor in resistance to RNAi. Our data demonstrated that the three siRNA molecules synthesized with T7 RNA polymerase could have transient inhibitory effects on the replication of FMDV. Abstract Vaccines have features that require special consideration when assessing their cost-eff ectiveness. These features are related to herd immunity, quality-of-life losses in young children, parental care and work loss, time preference, uncertainty, eradication, macroeconomics, and tiered pricing. Advisory committees on public funding for vaccines, or for pharmaceuticals in general, should be knowledgable about these special features. We discuss key issues and diffi culties in decision making for vaccines against rotavirus, human papillomavirus, varicella-zoster virus, infl uenza virus, and Streptococcus pneumoniae. We argue that guidelines for economic evaluation should be reconsidered generally to recommend (1) modelling options for the assessment of interventions against infectious diseases; (2) a wider perspective to account for impacts on third parties, if relevant; (3) a wider scope of costs than health-care system costs alone, if appropriate; and (4) alternative discounting techniques to explore social time preference over long periods.Lancet Infect Dis 2008; Abstract The mucosal surfaces of the lungs are a major portal of entry for virus infections and there are urgent needs for new vaccines that promote effective pulmonary immunity. However, we have only a rudimentary understanding of the requirements for effective cellular immunity in the respiratory tract. Recent studies have revealed that specialized cellular immune responses and lymphoid tissues are involved in the protection of distinct anatomical microenvironments of the respiratory tract, such as the large airways of the nose and the alveolar airspaces. This review discusses some of the anatomical features of anti-viral immunity in the respiratory tract including the role of local lymphoid tissues and the relationship between effector and memory T cells in the airways, the lung parenchyma, and lymphoid organs. Abstract Examples of vaccine-induced enhancement of susceptibility to virus infection or of aberrant viral pathogenesis have been documented for infections by members of different virus families. Several mechanisms, many of which still are poorly understood, are at the basis of this phenomenon. Vaccine development for lentivirus infections in general, and for HIV/AIDS in particular, has been little successful. Certain experimental lentiviral vaccines even proved to be counterproductive: they rendered vaccinated subjects more susceptible to infection rather than protecting them. For vaccine-induced enhanced susceptibility to infection with certain viruses like feline coronavirus, Dengue virus, and feline immunodeficiency virus, it has been shown that antibody-dependent enhancement (ADE) plays an important role. Other mechanisms may, either in the absence of or in combination with ADE, be involved. Consequently, vaccine-induced enhancement has been a major stumble block in the development of certain flavi-, corona-, paramyxo-, and lentivirus vaccines. Also recent failures in the development of a vaccine against HIV may at least in part be attributed to induction of enhanced susceptibility to infection. There may well be a delicate balance between the induction of protective immunity on the one hand and the induction of enhanced susceptibility on the other. The present paper reviews the currently known mechanisms of vaccine-induced enhancement of susceptibility to virus infection or of aberrant viral pathogenesis. Abstract Objectives: The aims of this article were: to summarize the pharmacology, pharmacokinetics, and efficacy of daptomycin; to explore its safety profile', and to discuss its current and potential roles as an antimicrobial therapy Results: Phase III study results suggest no difference in efficacy or tolerability between daptomycin 4 mg/kg IV QD and vancomycin or semisynthetic penicillins for complicated skin and skin-structure infections. Animal studies suggest daptomycin may be useful for the treatment of endocarditis. Daptomycin is not indicated for pneunionia, with poorer outcomes than conventional treatnient. It is available as an IV niedication and exhibits 92% plasnia protein binding in vitro. In healthy adult humans, daptoniycin has a volume of distribution of 0.1 Ukg and a plasma elimination half-life of -9 hours, and is eliminated primarily by renal excretion (-54%). In patients with reduced renal function, including those receiving hemodialysis and peritoneal dialysis, the dose interval should be 48 hours. No dosage adjustnient appears to be necessary for mild to nioderate hepatic impairnient. The use of daptomycin in patients with severe hepatic irnpairnient has not been assessed. The niost comnionly reported adverse events include constipation, nausea, injection-site reactions, headache, and diarrhea. Patients should also be nionitored regularly for skeletal niuscle toxicity.Conclusions: Daptoniycin may be useful for complicated skin and skin-structure infections and gram-positive pathogens resistant to conventional antimicrobials. However, limited data are currently available for duration of treatment beyond 14 days and at doses >4 mg/kg QD. (Clin Ther. Abstract The Middle East respiratory syndrome coronavirus (MERS-CoV) was first discovered in late 2012 and has gone on to cause over 1800 infections and 650 deaths. There are currently no approved therapeutics or vaccinations for MERS-CoV. The MERS-CoV spike (S) protein is responsible for receptor binding and virion entry to cells, is immunodominant and induces neutralizing antibodies in vivo, all of which, make the S protein an ideal target for anti-MERS-CoV vaccines. In this study, we demonstrate protection induced by vaccination with a recombinant MERS-CoV S nanoparticle vaccine and Matrix-M1 adjuvant combination in mice. The MERS-CoV S nanoparticle vaccine produced high titer anti-S neutralizing antibody and protected mice from MERS-CoV infection in vivo. Abstract This article quantifies the impact of H7N9 bird flu on chicken demand and consumer willingness to pay (WTP) in China. We measure risk perception, fear and trust against actual reduction in consumption and stated change in WTP for safe chicken between 2012 and 2013. Through a survey conducted in each year on the same Chinese urban consumers, we found that the consumption of chicken never increased after the emergence of H7N9 in 2013, and WTP for safe chicken did not necessarily increase relative to generic risks associated with consuming chicken in 2012. Factors such as the fear of H7N9's spreading, the impact of distrust (especially the distrust in government) enhanced the deviation of consumption and WTP; and the sheer mentioning of H7N9 is more important and negative than whether it was associated with a risk-perception reducing or risk-perception elevating message given to consumers. Abstract A pro-active emerging risk identification system starts with the selection of critical factors related to the occurrence of emerging hazards. This paper describes a method to derive the most important factors in dynamic production chains starting from a gross list of critical factors. The method comprised the semiquantitative evaluation of the critical factors for a relatively novel product on the Dutch market and a related traditional product. This method was tested in an expert study with three case studies. The use of group discussion followed by individual ranking in an expert study proved to be a powerful tool in identifying the most important factors for each case. Human behaviour (either producers' behaviour or human knowledge) was the most important factor for all three cases. The expert study showed that further generalization of critical factors based on product characteristics may be possible. Abstract Giardiosis and cryptosporidiosis are frequently diagnosed in calves at the large animal clinic of the veterinary school. Few studies have been reported in the literature regarding pathogenesis of these two intestinal protozoa. The aims of this study were to follow the histological changes in the villi and crypts and the changes in the number of intraepithelial lymphocytes in the jejunum of naturally infected calves during the acute phase of infection. For this purpose, 29 calves aged between 7 and 10 days were bought at a local auction. The animals were housed in individual pens to avoid cross-contamination. Fecal samples were examined microscopically for the presence of Giardia cysts and Cryptosporidium oocysts, three times per week for a period of 45 days. Six calves did not pass any cysts or oocysts and were used as controls. Fifteen calves passed Giardia cysts only, five passed both cysts and oocysts, and three passed oocysts only. The villus to crypt ratio index was 1.76 in the control group and 1.08 in the Giardia-infected group. In the Cryptosporidium-infected calves, the ratio was 1.18 and calves infected with both parasites had an index of 1.37. The number of intraepithelial lymphocytes per millimeter of jejunum tissue was 21 in the control group. This number was doubled in the calves infected with Giardia, but was slightly lower in the other infected groups. All of the infected calves had intermittent diarrhea and mucus was seen in many fecal samples. © 1997 Elsevier Science B.V. Abstract A major polyadenylated viral RNA of approximately 0.8 X lo6 daltons was isolated from murine hepatitis virus (A59)-infected cells by preparative polyacrylamide gel electrophoresis in formamide. This RNA was shown to encode the viral nucleocapsid protein by direct in vitro translation in a cell-free, reticulocyte-derived system. Single stranded q-labeled complementary DNA was prepared from this RNA and was demonstrated to be virus specific. Using this complementary DNA in a Northern blotting procedure, we were able to identify six major virus-specific intracellular RNA species with estimated molecular weights of 0.8, 1.1, 1.4, 1.6, 3, and 4 X l@ daltons. All of these RNA species were polyadenylated. Our results support the idea that coronavirus-infected cells contain multiple intracellular polyadenylated RNAs which share common sequences. et al., 1959) of mouse fibroblasts. Abstract Using the pH gradient electrophoretic technique of Vrijsen and Boeye, the coat protein of poliovirus types 1 and 2 was resolved into six components, Cl to C6, instead of the classical VPl-2-3 (the much smaller polypeptide VP4 was excluded from this study). The multiple components ran true upon reelectrophoresis, and there were no technical artifacts. Their resolution did not depend on a particular method for the preparation or disruption of the virion. The Cl-C6 components of pH gradient electrophoresis and the classical VPl-VP3 polypeptides were examined with regard to (i) their migration in normal and pH gradient reelectrophoresis and (ii) their kinetics of leucine incorporation in emetine-stopped pulses. It is concluded that Cl and C2 were both derived from VPI, C3 and C4 from VP2, and C5 and C6 from VP3. Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) Genotype 2 Nonstructural protein 9 (Nsp9) Retinoblastoma protein (pRb) Interaction Replication a b s t r a c t The nonstructural protein 9 (Nsp9) of porcine reproductive and respiratory syndrome virus (PRRSV) is a RNA-dependent RNA polymerase (RdRp) that plays a vital role in viral replication. This study first demonstrated that the Nsp9 of genotype 2 PRRSV interacted with cellular retinoblastoma protein (pRb), and Nsp9 co-localized with pRb in the cytoplasm of PRRSV-infected MARC-145 cells and pulmonary alveolar macrophages (PAMs). Next, the overexpression of truncated pRb was shown to inhibit the PRRSV replication and silencing the pRb gene could facilitate the PRRSV replication in MARC-145 cells. Finally, the pRb level was confirmed to be down-regulated in PRRSV-infected MARC-145 cells, and Nsp9 was shown to promote the pRb degradation by proteasome pathway. These findings indicate that the interaction of Nsp 9 with pRb benefits the replication of genotype 2 PRRSV in vitro, helping to understand the roles of Nsp9 in the replication and pathogenesis of PRRSV. Abstract Toll-like receptors (TLRs) are highly conserved type 1 membrane proteins that initiate a multiplicity of transient gene transcriptions, resulting in innate and adaptive immune responses. These essential immune responses are triggered by common TLR pattern recognition receptors of microbial products expressed through the cytoplasmic carboxy-terminal Toll/IL-1 domain. Toll/IL-1 adapter protein cascades are induced by an activated Toll/IL-1 to induce transient transcription responses. All TLRs, with the exception of TLR3, use an MyD88 adapter to Toll/IL-1 to initiate a proinflammatory cascade. TLR3 uses the toll receptor 3/4 induction factor adapter to initiate a different cytosolic adapter cascade with double-stranded RNA agonists. This non-MyD88 pathway induces both NF-kB and type 1 interferon responses. By using a TLR3-restricted double-stranded RNA agonist, rintatolimod, we demonstrate significant unexpected differences in toxic responses between rats and primates. The mechanism of this differential response is consistent with a relative down-regulation of the NF-kB inflammatory cytokine induction pathway in the cynomolgus monkey and humans, but not observed systemically in rat. Our findings suggest evaluation of TLR3 agonists in drug therapy. (Am J Pathol 2014, 184: 1062e1072; http://dx. Abstract Like asthma itself, the association between respiratory infections and the inflammatory immune response is complex and incompletely understood. One can clearly see the effects of infections on wheezing, asthma inception, and exacerbations, but dissecting out the mechanistic details is the challenge confronting research in this decade. As always, it is hoped that a more complete understanding of the cells, cytokines, and signaling pathways involved, along with knowledge of the temporal progression of events and the role of regulatory factors such as microRNAs, will lead to more effective treatments, individualized to the patient.Asthma afflicts more than 300 million individuals of all ages and ethnic groups, and results in approximately 250,000 deaths worldwide. More than 7 million children younger than 18 years have been diagnosed with asthma in the United States alone. 1 In 2003, asthma-related hospitalizations neared 500,000 in the United States and the annual direct medical expenses associated with asthma care were $12.7 to $15.6 billion. 2,3 According to a cross-sectional international study conducted from 2002 to Abstract Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury. It is a response to various diseases of variable etiology, including SARS-CoV infection. To date, a comprehensive study of the genomic physiopathology of ARDS (and SARS) is lacking, primarily due to the difficulty of finding suitable materials to study the disease process at a tissue level (instead of blood, sputa or swaps). Hereby we attempt to provide such study by analyzing autopsy lung samples from patient who died of SARS and showed different degrees of severity of the pulmonary involvement. We performed real-time quantitative PCR analysis of 107 genes with functional roles in inflammation, coagulation, fibrosis and apoptosis; some key genes were confirmed at a protein expression level by immunohistochemistry and correlated to the degree of morphological severity present in the individual samples analyzed. Significant expression levels were identified for ANPEP (a receptor for CoV), as well as inhibition of the STAT1 pathway, IFNs production and CXCL10 (a T-cell recruiter). Other genes unassociated to date with ARDS/SARS include C1Qb, C5R1, CASP3, CASP9, CD14, CD68, FGF7, HLA-DRA, IGF1, IRF3, MALAT-1, MSR1, NFIL3, SLPI, USP33, CLC, GBP1 and TAC1. As a result, we proposed to therapeutically target some of these genes with compounds such as ANPEP inhibitors, SLPI and dexamethasone. Ultimately, this study may serve as a model for future, tissue-based analyses of fibroinflammatory conditions affecting the lung. Abstract Temperature-sensitive mutants of the munne coronavirus JHM induced a sub~ acute demyelinatin 8 encephalomyelitis (SD~E) in youn~ rats. Neurological symptol~,; were associated with marked lesion3 of primary d~my¢;lination ".m the white matter of the central nervous system (CNS), and developing after an incubation time of several weeks to months. Many rats survived this infection and rco3vered completely from this CNS disease. Amon 8 43 survivors of SDE, 9 ra'~s developed a ~'elapse 27-153 days after onset of the first attack. Neuropathologica.l ex~n~ation ol~ these animals revealed areas of fresh| ¢~emyelination together with old remyelinated lesions. V'md antisens were detectable in the neighbourhood of fresh les:ions and in some ca~ infectious virus was re-i~lated from rats revealing low antibl~y titers to JHM virus. These results demonstrate that mutants of JHM virus can induca~ a relapsing demyelinating disease process, associated with a persistent in fection, which some similarities to chronic experimental allergic enceph~dor~ayel/tis. Abstract The launch of the new journal, Biosafety and Health, presents me with a unique opportunity to recount the progress of laboratory biosafety (LB) in China and my contribution to this area over the past 30 years. Since the severe acute respiratory syndrome epidemic in 2003, China has constructed a primary network of high-level biosafety laboratories at different levels and established an expert team on LB. Furthermore, a series of LB management documents, including laws, regulations, standards, and guidelines, have been developed and published. This gradually maturing LB system has played a pivotal role in emerging infectious disease control and prevention, as well as in research, which in turn contributes to public health. In recent years, international collaboration between China and other countries has also been accelerated. Despite these achievements, we are still facing many challenges and opportunities in the field of LB. Sustainable LB development requires the joint efforts of the entire society and continuous international cooperation to safeguard global public health. Abstract Since the 1950s, medical communities have been facing with emerging and reemerging infectious diseases, and emerging pathogens are now considered to be a major microbiologic public health threat. In this review, we focus on bacterial emerging diseases and explore factors involved in their emergence as well as future challenges. We identified 26 major emerging and reemerging infectious diseases of bacterial origin; most of them originated either from an animal and are considered to be zoonoses or from water sources. Major contributing factors in the emergence of these bacterial infections are: (1) development of new diagnostic tools, such as improvements in culture methods, development of molecular techniques and implementation of mass spectrometry in microbiology; (2) increase in human exposure to bacterial pathogens as a result of sociodemographic and environmental changes; and (3) emergence of more virulent bacterial strains and opportunistic infections, especially affecting immunocompromised populations. A precise definition of their implications in human disease is challenging and requires the comprehensive integration of microbiological, clinical and epidemiologicaspects as well as the use of experimental models. It is now urgent to allocate financial resources to gather international data to provide a better understanding of the clinical relevance of these waterborne and zoonotic emerging diseases.Clinical Microbiology and Infection Abstract In China, the current situation is that people under indirect threat from unprotected lead-zinc mining tends to oppose it, whereas people under direct threat are likely to 'sail close to the wind'. To understand this puzzle-like phenomenon, we surveyed 220 residents in a lead-zinc mining area located in Fenghuang County of China. We found that: 1) The degree of risk perception of villagers living around the mining site correlated inversely with their degree of involvement in mining risk. We refer to this as the ''involvement'' version of the psychological typhoon eye effect. 2) Perceived benefit and perceived harm provided a satisfactory explanation for this ''involvement'' version of the psychological typhoon eye effect. 3) Risk perception was negatively related to support for the relevant policy which we viewed as constituting a sort of voting behavior. The results may have implications for better understanding how benefited individuals respond to environmental health risks. Abstract Influenza A and B, and many unrelated viruses including rhinovirus, RSV, adenovirus, metapneumovirus and coronavirus share the same seasonality, since these viral acute respiratory tract infections (vARIs) are much more common in winter than summer. Unfortunately, early investigations that used recycled ''pedigree" virus strains seem to have led microbiologists to dismiss the common folk belief that vARIs often follow chilling. Today, incontrovertible evidence shows that ambient temperature dips and host chilling increase the incidence and severity of vARIs. This review considers four possible mechanisms, M1 -4, that can explain this link: (M1) increased crowding in winter may enhance viral transmission; (M2) lower temperatures may increase the stability of virions outside the body; (M3) chilling may increase host susceptibility; (M4) lower temperatures or host chilling may activate dormant virions. There is little evidence for M1 or M2, which are incompatible with tropical observations. Epidemiological anomalies such as the repeated simultaneous arrival of vARIs over wide geographical areas, the rapid cessation of influenza epidemics, and the low attack rate of influenza within families are compatible with M4, but not M3 (in its simple form). M4 seems to be the main driver of seasonality, but M3 may also play an important role. Abstract Total internal reflection microscopy combined with microfluidics and supported bilayers is a powerful, single particle tracking (SPT) platform for host-pathogen membrane fusion studies. But one major inadequacy of this platform has been capturing the complexity of the cell membrane, including membrane proteins. Because of this, viruses requiring proteinaceous receptors, or other unknown cellular co-factors, have been precluded from study. Here we describe a general method to integrate proteinaceous receptors and cellular components into supported bilayers for SPT fusion studies. This method is general to any enveloped virus-host cell pair, but demonstrated here for feline coronavirus (FCoV). Supported bilayers are formed from mammalian cell membrane vesicles that express feline aminopeptidase N (the viral receptor) using a cell blebbing technique. SPT is then used to identify fusion intermediates and measure membrane fusion kinetics for FCoV. Overall, the fusion results recapitulate what is observed in vivo, that coronavirus entry requires binding to specific receptors, a low-pH environment, and that membrane fusion is receptorand protease-dependent. But this method also provides quantitative kinetic rate parameters for intermediate steps in the coronavirus fusion pathway, which to our knowledge have not been obtained before. Moreover, the platform offers versatile, precise control over the sequence of triggers for fusion; these triggers may define the fusion pathway, tissue tropism, and pathogenicity of coronaviruses. Systematically varying these triggers in this platform provides a new route to study how viruses rapidly adapt to other hosts, and to identify factors that led to the emergence of zoonotic viruses, such as human SARS-CoV and the newly emerging human MERS-CoV. Abstract practices and implementation varied. Essential aspects such as compliance measurement and audits to assess guideline effectiveness were neglected in most documents. In conclusion, there is a need for a more consistent approach leading to recommendations based on a thorough evaluation of evidence and applicable worldwide. Aspects related to implementation and impact monitoring deserve greater attention. Abstract RNA interference (RNAi), initially recognized as a natural antiviral mechanism in plants, has rapidly emerged as an invaluable tool to suppress gene expression in a sequence-specific manner in all organisms, including mammals. Its potential to inhibit the replication of a variety of viruses has been demonstrated in vitro and in vivo in mouse and monkey models. These results have generated profound interest in the use of this technology as a potential treatment strategy for viral infections for which vaccines and drugs are unavailable or inadequate. In this review, we discuss the progress made within the past 2-3 years towards harnessing the potential of RNAi for clinical application in viral infections and the hurdles that have yet to be overcome. Abstract The emergence of new infectious bronchitis virus (IBV) variants is often disastrous in the poultry industry. In this study, an IBV, CK/CH/2010/JT-1, was isolated from an H120-and 4/91-IBV-vaccinated flock in China. Antisera against vaccine strains H120 and 4/91 could not provide effective protection against CK/CH/2010/JT-1 in virus neutralization assays. CK/CH/2010/JT-1 could cause 43.75% mortality with respiratory and severe renal lesions in inoculated chickens. Phylogenetic analysis of the S1 gene showed that CK/CH/2010/JT-1 and 31 other isolates could be grouped as a new genotypic cluster. Recombination analysis revealed that three recombination events could be found in the genome of CK/ CH/2010/JT-1 at positions 24709-365, 17160-19811 and 21136-21770. Whole-genome sequence analysis showed that CK/CH/2010/JT-1 originated from multiple template switches among QX-like, CK/CH/LSC/ 99I-, tl/CH/LDT3/03-and 4/91-type IBVs. All of these data demonstrated that CK/CH/2010/JT-1 is a new recombinant genotype IBV with high virulence. Our findings suggest that the surveillance of new genotype strains of IBV is very important for developing more effective anti-IBV strategies. Abstract Infectious bronchitis virus (IBV) is a Gammacoronavirus that causes a highly contagious respiratory disease in chickens. A QX-like strain was analysed by high-throughput Illumina sequencing and genetic variation across the entire viral genome was explored at the sub-consensus level by single nucleotide polymorphism (SNP) analysis. Thirteen open reading frames (ORFs) in the order 5 0 -UTR-1a-1ab-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3 0 UTR were predicted. The relative frequencies of missense: silent SNPs were calculated to obtain a comparative measure of variability in specific genes. The most variable ORFs in descending order were E, 3b, 5 0 UTR, N, 1a, S, 1ab, M, 4c, 5a, 6b. The E and 3b protein products play key roles in coronavirus virulence, and RNA folding demonstrated that the mutations in the 5 0 UTR did not alter the predicted secondary structure. The frequency of SNPs in the Spike (S) protein ORF of 0.67% was below the genomic average of 0.76%. Only three SNPS were identified in the S1 subunit, none of which were located in hypervariable region (HVR) 1 or HVR2. The S2 subunit was considerably more variable containing 87% of the polymorphisms detected across the entire S protein. The S2 subunit also contained a previously unreported multi-A insertion site and a stretch of four consecutive mutated amino acids, which mapped to the stalk region of the spike protein. Template-based protein structure modelling produced the first theoretical model of the IBV spike monomer. Given the lack of diversity observed at the sub-consensus level, the tenet that the HVRs in the S1 subunit are very tolerant of amino acid changes produced by genetic drift is questioned. Abstract Health policyPandemic response s u m m a r y Objectives: University students, both travelling abroad on holiday or exchange students entering a country, can serve as mobile carriers of infectious diseases during a pandemic, and thus require special attention when considering preventive measures. The objectives of this study were to evaluate student compliance and opinions on preventive measures of a university before and during an H1N1 influenza pandemic, and to explore environmental and behavioural factors that might contribute towards compliance.Study design: Cross-sectional, self-administered questionnaire.Methods: Local and foreign students attending an international summer school programme were invited to participate in a self-administered survey.Results: Respondents complied with most of the preventive measures, excluding website viewing and mask wearing. Significant differences in compliance and perceived necessity were found amongst students from Singapore, Hong Kong and the USA. Singaporean students were significantly more likely to comply with all measures and consume antiviral medication in response to the pandemic than students studying in the US.Conclusions: Students' responses towards university pandemic measures were largely positive, but sensitivity towards these measures varied between groups by country of study. This should be considered in further comparative studies. Abstract The Severe Acute Respiratory Syndrome (SARS) is a serious respiratory illness that has recently been reported in parts of Asia and Canada. In this study, we use molecular dynamics (MD) simulations and docking techniques to screen 29 approved and experimental drugs against the theoretical model of the SARS CoV proteinase as well as the experimental structure of the transmissible gastroenteritis virus (TGEV) proteinase. Our predictions indicate that existing HIV-1 protease inhibitors, l-700,417 for instance, have high binding affinities and may provide good starting points for designing SARS CoV proteinase inhibitors. # Abstract Various methods for the control of PRRS virus have been published. The technology of nursery depopulation (ND) appears to effectively control the spread of virus between members of endemically infected populations. ND consists of a strategic adjustment in pigflow based on the presence of specific serologic patterns as detected by the indirect fluorescent antibody test. This pattern indicates a low seroprevalence of antibodies detected in the breeding herd and recently weaned piglets ( I 10%). in contrast to a high ( > 50%) seroprevalence in 8 to 10 week old piglets. ND has been carried out on swine farms in the US and results indicate improvements in nursery piglet growth rate and mortality levels. Three examples are provided in the following text. Recently a modified live virus vaccine (RespPRRS, NOBL Laboratories/Boerhinger Ingleheim) has become commercially available. It is currently approved for use in piglets from 3 to 18 weeks of age: however. potential for the use in adult animals is currently under investigation. 0 1997 Elsevier Science B.V. Abstract Porcine epidemic diarrhea virus (PEDV), a member of the genera Alphacoronavirus in the family Coronaviridae, causes acute diarrhea/vomiting, dehydration and high mortality in seronegative neonatal piglets. For the last three decades, PEDV infection has resulted in significant economic losses in the European and Asian pig industries, but in 2013-2014 the disease was also reported in the US, Canada and Mexico. The PED epidemic in the US, from April 2013 to the present, has led to the loss of more than 10% of the US pig population.The disappearance and re-emergence of epidemic PED indicates that the virus is able to escape from current vaccination protocols, biosecurity and control systems. Endemic PED is a significant problem, which is exacerbated by the emergence (or potential importation) of multiple PEDV variants. Epidemic PEDV strains spread rapidly and cause a high number of pig deaths. These strains are highly enteropathogenic and acutely infect villous epithelial cells of the entire small and large intestines although the jejunum and ileum are the primary sites. PEDV infections cause acute, severe atrophic enteritis accompanied by viremia that leads to profound diarrhea and vomiting, followed by extensive dehydration, which is the major cause of death in nursing piglets. A comprehensive understanding of the pathogenic characteristics of epidemic or endemic PEDV strains is needed to prevent and control the disease in affected regions and to develop an effective vaccine. This review focuses on the etiology, epidemiology, disease mechanisms and pathogenesis as well as immunoprophylaxis against PEDV infection. Abstract Long non-coding RNAs (lncRNAs) are found not only in mammals but also in other organisms, including viruses. Recent findings suggest that lncRNAs play various regulatory roles in multiple major biological and pathological processes. During viral life cycles, lncRNAs are involved in a series of steps, including enhancing viral gene expression, promoting viral replication and genome packaging, boosting virion release, maintaining viral latency and assisting viral transformation; additionally, lncRNAs antagonize host antiviral innate immune responses. In contrast to proteins that function in viral infection, lncRNAs are expected to be novel targets for the modulation of all types of biochemical processes due to their broad characteristics and profound influence. This review highlights our current understanding of the regulatory roles of lncRNAs during viral infection processes with an emphasis on the potential usefulness of lncRNAs as a target for viral intervention strategies, which could have therapeutic implications for the application of a clinical approach for the treatment of viral diseases. Abstract on behalf of the IRC002 Study Team Summary Background-Influenza causes significant morbidity and mortality despite currently available treatments. Anecdotal reports suggest plasma with high antibody titers towards influenza may be of benefit in the treatment of severe influenza.Methods-We conducted a randomized, open-label, multicenter phase 2 trial at 29 academic medical centers in the United States to assess the safety and efficacy of anti-influenza plasma with hemagglutination inhibition (HAI) antibody titers of ≥ 1:80 to the infecting strain. Hospitalized children and adults (including pregnant women) with severe influenza A or B (defined as hypoxia or tachypnea) were randomly assigned to receive either 2 units (or pediatric equivalent) of antiinfluenza plasma plus standard care (P+S), versus standard care alone (S), and were followed for 28 days. The primary endpoint was time to normalization of patients' respiratory status (respiratory rate of ≤ 20 for adults or age defined thresholds of 20-38 for children), and a room air saturation of oxygen ≥ 93%. ClinicalTrials.gov Identifier: NCT01052480Findings-Between January 13, 2011 and March 2, 2015, 113 participants were screened, and 98 were randomized. Of the participants with confirmed influenza, 28 of 42 (67%) of P+S participants normalized their respiratory status by Day 28, as compared to 24 of 45 (53%) of S participants (p=0·069). The estimated hazard ratio comparing P+S to S was 1·71 (95% CI: 0·96 to 3·06). Six participants died, 1 (2%) and 5 (10%) from the P+S and S arms respectively (p=0·093). P+S participants had non-significant reductions in days in hospital (median 6 vs. 11 days, p=0·13) and days on mechanical ventilation (median 0 vs. 3 days, p=0·14), and significantly improved clinical status at Day 7 (p=0·020). Fewer P+S participants experienced SAEs compared to S recipients (20% vs. 38%, p= 0·041), the most frequent of which were acute respiratory distress syndrome (1 [2%] vs 2 [4%]) and stroke (1 [2%] vs 2 [4%]).Interpretation-Results from this Phase II randomized trial of immune plasma for the treatment of severe influenza provides support for a possible benefit of immunotherapy across the primary and secondary endpoints. A Phase III randomized trial is now underway to further evaluate this intervention.Beigel et al. Abstract The majority of emerging zoonoses originate in wildlife, and many are caused by viruses. However, there are no rigorous estimates of total viral diversity (here termed "virodiversity") for any wildlife species, despite the utility of this to future surveillance and control of emerging zoonoses. In this case study, we repeatedly sampled a mammalian wildlife host known to harbor emerging zoonotic pathogens (the Indian Flying Fox, Pteropus giganteus) and used PCR with degenerate viral family-level primers to discover and analyze the occurrence patterns of 55 viruses from nine viral families. We then adapted statistical techniques used to estimate biodiversity in vertebrates and plants and estimated the total viral richness of these nine families in P. giganteus to be 58 viruses. Our analyses demonstrate proof-of-concept of a strategy for estimating viral richness and provide the first statistically supported estimate of the number of undiscovered viruses in a mammalian host. We used a simple extrapolation to estimate that there are a minimum of 320,000 mammalian viruses awaiting discovery within these nine families, assuming all species harbor a similar number of viruses, with minimal turnover between host species. We estimate the cost of discovering these viruses to be~$6.3 billion (or~$1.4 billion for 85% of the total diversity), which if annualized over a 10-year study time frame would represent a small fraction of the cost of many pandemic zoonoses. IMPORTANCE Recent years have seen a dramatic increase in viral discovery efforts. However, most lack rigorous systematic design, which limits our ability to understand viral diversity and its ecological drivers and reduces their value to public health intervention. Here, we present a new framework for the discovery of novel viruses in wildlife and use it to make the first-ever estimate of the number of viruses that exist in a mammalian host. As pathogens continue to emerge from wildlife, this estimate allows us to put preliminary bounds around the potential size of the total zoonotic pool and facilitates a better understanding of where best to allocate resources for the subsequent discovery of global viral diversity. Lipkin WI. 2013 . A strategy to estimate unknown viral diversity in mammals. mBio 4(5): e00598-13. Abstract The broad range and diversity of interferon-stimulated genes (ISGs) function to induce an antiviral state within the host, impeding viral pathogenesis. While successful respiratory viruses overcome individual ISG effectors, analysis of the global ISG response and subsequent viral antagonism has yet to be examined. Employing models of the human airway, transcriptomics and proteomics datasets were used to compare ISG response patterns following highly pathogenic H5N1 avian influenza (HPAI)IMPORTANCE This work combines systems biology and experimental validation to identify and confirm strategies used by viruses to control the immune response. Using a novel screening approach, specific comparison between highly pathogenic influenza viruses and coronaviruses revealed similarities and differences in strategies to control the interferon and innate immune response. These findings were subsequently confirmed and explored, revealing both a common pathway of antagonism via type I interferon (IFN) delay as well as a novel avenue for control by altered histone modification. Together, the data highlight how comparative systems biology analysis can be combined with experimental validation to derive novel insights into viral pathogenesis. Abstract The intracellular transport of newly synthe- Abstract The seventh novel human infecting Betacoronavirus that causes pneumonia (2019 novel coronavirus, 2019-nCoV) originated in Wuhan, China. The evolutionary relationship between 2019-nCoV and the other human respiratory illness-causing coronavirus is not closely related. We sought to characterize the relationship of the translated proteins of 2019-nCoV with other species of Orthocoronavirinae. A phylogenetic tree was constructed from the genome sequences. A cluster tree was developed from the profiles retrieved from the presence and absence of homologs of ten 2019-nCoV proteins. The combined data were used to characterize the relationship of the translated proteins of 2019-nCoV to other species of Orthocoronavirinae. Our analysis reliably suggests that 2019-nCoV is most closely related to BatCoV RaTG13 and belongs to subgenus Sarbecovirus of Betacoronavirus, together with SARS coronavirus and Bat-SARS-like coronavirus. The phylogenetic profiling cluster of homolog proteins of one annotated 2019-nCoV protein against other genome sequences revealed two clades of ten 2019-nCoV proteins. Clade 1 consisted of a group of conserved proteins in Orthocoronavirinae comprising Orf1ab polyprotein, Nucleocapsid protein, Spike glycoprotein, and Membrane protein. Clade 2 comprised six proteins exclusive to Sarbecovirus and Hibecovirus. Two of six Clade 2 nonstructural proteins, NS7b and NS8, were exclusively conserved among 2019-nCoV, BetaCoV_RaTG, and BatSARS-like Cov. NS7b and NS8 have previously been shown to affect immune response signaling in the SARS-CoV experimental model. Thus, we speculated that knowledge of the functional changes in the NS7b and NS8 proteins during evolution may provide important information to explore the human infective property of 2019-nCoV. Abstract Drug Design, Development and Therapy Dovepress submit your manuscript | www.dovepress.com Dovepress 2255 O r i g i n a l r e s e a r c h open access to scientific and medical research Open access Full Text article http://dx.The thiazolidinedione class peroxisome proliferator-activated receptor gamma (PPARγ) agonists are restricted in clinical use as antidiabetic agents because of side effects such as edema, weight gain, and heart failure. The single and selective agonism of PPARγ is the main cause of these side effects. Multitargeted PPARα/γ/δ pan agonist development is the hot topic in the antidiabetic drug research field. In order to identify PPARα/γ/δ pan agonists, a compound database was established by core hopping of rosiglitazone, which was then docked into a PPARα/γ/δ active site to screen out a number of candidate compounds with a higher docking score and better interaction with the active site. Further, absorption, distribution, metabolism, excretion, and toxicity prediction was done to give eight compounds. Molecular dynamics simulation of the representative Cpd#1 showed more favorable binding conformation for PPARs receptor than the original ligand. Cpd#1 could act as a PPARα/γ/δ pan agonist for novel antidiabetic drug research. Abstract Objective: To report clinical, virological, and epidemiological features of the first death caused by a H5N6 avian influenza virus in Yunnan Province, China. Method: The case was described in clinical expression, chest radiography, blood test and treatment. Real-time RT-PCR was used to detect H5N6 virus RNA in clinical and environment samples. Epidemiological investigation was performed including case exposure history determinant, close contacts follow up, and environment sample collection. Results: The patient initially developed sore throat and coughs on 27 January 2015. The disease progressed to severe pneumonia, multiple organ dysfunction syndrome, and acute respiratory distress syndrome. And the patient died on 6 February. A highly pathogenic avian influenza A H5N6 virus was isolated from the tracheal aspirate specimen of the patient. The viral genome analyses revealed that the H5 hemmagglutinin gene belongs to 2.3.4.4 clade. Epidemiological investigation showed that the patient had exposure to wild bird. All close contacts of the patient did not present the same disease in seven consecutive days. A high H5 positive rate was detected in environmental samples from local live poultry markets.The findings suggest that studies on the source of the virus, transmission models, serologic investigations, vaccines, and enhancing surveillance in both humans and birds are necessary. Abstract Bovine viral diarrhea (BVD) is a chronic disease of cattle caused by infection with BVD virus (BVDV) and can result in economic losses within the livestock industry. In Japan, the test and culling policy is a basic control measure, and implementation of an adequate vaccination program is recommended as a national policy. In addition, optional control measures, including compulsory testing of introduced animals and bulk tank milk (BTM) testing as a mass screening method, are used in several provinces, but their efficacy has not been completely assessed. We evaluated these control measures using the scenario tree model of BVD in Japan, developed in the previous study. The model outputs indicated that compulsory testing of all introduced cattle, rather than only heifers and/or non-vaccinated cattle, was cost effective and reduced the risk of BVDV introduction due to animal movement and that BTM testing could effectively monitor most part of the cattle population. Vaccination coverage and BVDV prevalence among introduced cattle could also affect the cost effectiveness of compulsory testing of targeted cattle, particularly under low vaccination coverage or high BVDV prevalence. However, even with the implementation of a highly effective monitoring scheme for many years, BVD risk could not be eliminated; it instead converged at a very low level (0.02%). Disease models with a cost-effective output could be a powerful tool in developing a control scheme for chronic animal diseases, including BVD, with the consent of relevant stakeholders. Abstract Powered by advances in electron tomography, recent studies have extended our understanding of how viruses construct "replication factories" inside infected cells. Their function, however, remains an area of speculation with important implications for human health. It is clear from these studies that whatever their purpose, organelle structure is dynamic (M. Ulasli, M. H. Verheije, C. A. de Haan, and F. Reggiori, Cell. Microbiol. 12:844-861, 2010) and intricate (K. Knoops, M. Kikkert, S. H. Worm, J. C. Zevenhoven-Dobbe, Y. van der Meer, et al., PLOS Biol. 6:e226, 2008). But by concentrating on medically important viruses, these studies have failed to take advantage of the genetic variation inherent in a family of viruses that is as diverse as the archaea, bacteria, and eukaryotes combined (C. Lauber, J. J. Goeman, M. del Carmen Parquet, P. T. Nga, E. J. Snijder, et al., PLOS Pathog. 9:e1003500, 2013). In this climate, Maier et al. (H. J. Maier, P. C. Hawes, E. M. Cottam, J. Mantell, P. Verkade, et al., mBio 4:e00801-13, 2013) explored the replicative structures formed by an avian coronavirus that appears to have diverged at an early point in coronavirus evolution and shed light on controversial aspects of viral biology. Citation Neuman BW. 2013. How the double spherules of infectious bronchitis virus impact our understanding of RNA virus replicative organelles. mBio 4(6):e00987-13. Abstract Previous results suggested that, after intranasal inoculation, mouse hepatitis virus (MHV), a neurotropic coronavirus, entered the central nervous system (CNS) via the olfactory and trigeminal nerves. To prove this hypothesis, the effect of interruption of the olfactory pathway on spread of the virus was studied using in situ hybridization . Unilateral surgical ablation of this pathway prevented spread of the virus via the olfactory tract on the side of the lesion . MHV RNA could be detected, however, at distal sites on the operated side, indicating that the virus spread via well-described circuits involving the anterior commissure from the control (intact) side of the brain . Viral transport via the trigeminal nerve was not affected by removal of the olfactory bulb, showing that the surgical procedure was specific for the olfactory pathway. These results prove conclusively that MHV gains entry to the CNS via a transneuronal route, and spreads to additional sites in the brain via known neuroanatomic pathways. Abstract A novel avian-origin H7N9 influenza A virus (IAV) emerged in China in 2013, causing mild to lethal human respiratory infections. H7N9 originated with multiple reassortment events between avian viruses and carries genetic markers of human adaptation. Determining whether H7N9 induces a host response closer to that with human or avian IAV is important in order to better characterize this emerging virus. Here we compared the human lung epithelial cell response to infection with A/Anhui/ 01/13 (H7N9) or highly pathogenic avian-origin H5N1, H7N7, or human seasonal H3N2 IAV. The transcriptomic response to H7N9 was highly specific to this strain but was more similar to the response to human H3N2 than to that to other avian IAVs. H7N9 and H3N2 both elicited responses related to eicosanoid signaling and chromatin modification, whereas H7N9 specifically induced genes regulating the cell cycle and transcription. Among avian IAVs, the response to H7N9 was closest to that elicited by H5N1 virus. Host responses common to H7N9 and the other avian viruses included the lack of induction of the antigen presentation pathway and reduced proinflammatory cytokine induction compared to that with H3N2. Repression of these responses could have an important impact on the immunogenicity and virulence of H7N9 in humans. Finally, using a genome-based drug repurposing approach, we identified several drugs predicted to regulate the host response to H7N9 that may act as potential antivirals, including several kinase inhibitors, as well as FDA-approved drugs, such as troglitazone and minocycline. Importantly, we validated that minocycline inhibited H7N9 replication in vitro, suggesting that our computational approach holds promise for identifying novel antivirals. IMPORTANCE Whether H7N9 will be the next pandemic influenza virus or will persist and sporadically infect humans from its avian reservoir, similar to H5N1, is not known yet. High-throughput profiling of the host response to infection allows rapid characterization of virus-host interactions and generates many hypotheses that will accelerate understanding and responsiveness to this potential threat. We show that the cellular response to H7N9 virus is closer to that induced by H3N2 than to that induced by H5N1, reflecting the potential of this new virus for adaptation to humans. Importantly, dissecting the host response to H7N9 may guide host-directed antiviral development.Citation Josset L, Zeng H, Kelly SM, Tumpey TM, Katze MG. 2014. Transcriptomic characterization of the novel avian-origin influenza A (H7N9) virus: specific host response and responses intermediate between avian (H5N1 and H7N7) and human (H3N2) viruses and implications for treatment options. mBio 5(1):e01102-13. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.Systems Vaccinology Identifies an Early Innate Immune Signature as a Correlate of Antibody Responses to the Ebola Vaccine rVSV-ZEBOV Graphical Abstract Highlights d 5 early innate markers correlate with antibody response to Ebola vaccine rVSV-ZEBOV d IP-10 on day 3 after vaccination is an independent correlate of antibody induction d RNA-seq analysis identifies early gene expression signature linked to IP-10 Abstract To substantiate psychological symptoms following humidifier disinfectant (HD) disasters, counseling records of 26 victims and 92 family members of victims (45 were bereaved) were analyzed retrospectively. Among the victims, 34.6% had Clinical Global Impression-Severity scores of over 4, which meant they were moderately ill. While anxiety/fear and depression with respiratory symptoms were frequently observed in victims and family members, chronic psychological distress such as alcohol/smoking abuse and insomnia was relatively high in bereaved family members. In conclusion, it is important to provide mental health support for victims and their families, focusing on the characteristic symptoms of each group as well as monetary compensation. Abstract The synthesis and characterization of isotopomer tandem nucleic acid mass tag-peptide nucleic acid (TNT-PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens. Abstract Aerial spraying of products to kill larvae or adult mosquitoes is a public health measure used to control vectorborne diseases. In some outbreaks, the intervention has evoked controversy and community resistance. This study evaluated how local opinion leaders in US localities affected by Zika think about community engagement in public health policies for outbreak response. In December 2017 through March 2018, 4 focus groups were convened in Houston, TX, New Orleans, LA, Miami, FL, and Brooklyn, NY. They discussed a hypothetical scenario that featured vector control by aerial spraying. Participants (N = 20) more readily accepted this vector control method under 4 conditions: They were informed of alternatives, benefits, and risks for human health and the environment. Public health claims were backed by objective evidence and an authority figure genuinely working in the community's interests. They received timely notice about how to mitigate toxin exposure. And, aerial spraying helped to protect vulnerable individuals. The community engagement requirements of the local opinion leaders resonate with core principles of recent public health ethics frameworks: namely, personal autonomy, transparency, reasonableness, and solidarity. Participants foresaw problems with community consent in an era of growing social media use and mistrust in governmental and scientific authority. They also debated whether health authorities should use moral-based arguments, in addition to science-based ones, to communicate aerial spraying's risks and benefits. Abstract Two topics that have attracted recent attention in the field of autophagy concern the source of the membrane that is used to form the autophagosome during macroautophagy and the role of noncanonical autophagic pathways. The 2 topics may converge when considering the intersection of autophagy with viral infection. We suggest that noncanonical autophagy, which is sensitive to treatment with brefeldin A, may converge with the infectious cycles of certain DNA and RNA viruses that utilize membrane from the ER and cis-Golgi. Abstract In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV-and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF). Abstract Objective: To describe the current use of obstetric practice Web sites to disseminate Zika virus information to patients. Design: Review of 913 randomly selected practice Web sites and associated social media accounts in January and August 2016. Setting: Obstetric practice Web sites and associated social media accounts, United States of America. Participants: N/A. Main Outcome Measures: Proportion of obstetric practice Web sites and linked social media accounts providing Zika virus information.Results: Twenty-five percent and 35% of obstetric practice Web sites had information posted about Zika virus in January 2016 and August 2016, respectively. Between the 2 time points, the proportion of practices posting Zika virus content on Facebook and Twitter declined (Facebook: 15% in January, 9% in August; Twitter: 12% in January, 8% in August). In August, the most frequently observed Zika virus-related content themes were the use of insect repellent (14%) and travel advisories (14%). At both time points, practices affiliated with large university hospitals were more likely to have posted information on Zika virus than independent OB/GYN-only practices: January: odds ratio (OR) (95% confidence interval [CI]) = 5.68 (3.50-9.20); August: OR (95% CI) = 8.37 (5.31-13.17). Similarly, practices associated with nonuniversity hospitals were more likely to have posted information than independent OB/GYN-only practices: January: OR (95% CI) = 2.71 (1.88-3.92); August: OR (95% CI) = 6.75 (4.75-9.60). Conclusion: Obstetric care practices are not fully utilizing their practice Web sites to relay Zika virus information to their patients. Since practitioner-sponsored Web sites have the capacity to directly reach the populations at greatest risk for Zika virus complications, public health professionals should consider adapting their materials and provider outreach campaigns to more easily accommodate Web site-based information dissemination during this type of public health emergency. There must be greater recognition of the value information gains in the eyes of the patient when it is validated by their own provider, especially when that patient is part of the highest-risk population for a given emergency. Public health organizations should strive to minimize the burden it takes for providers to relay useful resources to patients in order to maximize the impact that those resources can have. Abstract A dataset of 103 SARS-CoV isolates (101 human patients and 2 palm civets) was investigated on different aspects of genome polymorphism and isolate classification. The number and the distribution of single nucleotide variations (SNVs) and insertions and deletions, with respect to a "profile", were determined and discussed ("profile" being a sequence containing the most represented letter per position). Distribution of substitution categories per codon positions, as well as synonymous and non-synonymous substitutions in coding regions of annotated isolates, was determined, along with amino acid (a.a.) property changes. Similar analysis was performed for the spike (S) protein in all the isolates (55 of them being predicted for the first time). The ratio Ka/Ks confirmed that the S gene was subjected to the Darwinian selection during virus transmission from animals to humans. Isolates from the dataset were classified according to genome polymorphism and genotypes. Genome polymorphism yields to two groups, one with a small number of SNVs and another with a large number of SNVs, with up to four subgroups with respect to insertions and deletions. We identified three basic nine-locus genotypes: TTTT/TTCGG, CGCC/TTCAT, and TGCC/TTCGT, with four subgenotypes. Both classifications proposed are in accordance with the new insights into possible epidemiological spread, both in space and time. Abstract Systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. In this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model pathogen. We utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. This unbiased approach produced a high-priority list of 4 genes in one pathway out of over 3,500 genes that were differentially expressed following SARS-CoV infection. With these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following SARS-CoV infection in mice. We validated the importance of the urokinase pathway for SARS-CoV disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. The results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as SARS-CoV., and acute respiratory distress syndrome (ARDS) leading to pulmonary fibrosis and death. We identified sets of differentially expressed genes that contribute to ALI and ARDS using lethal and sublethal SARS-CoV infection models. Mathematical prioritization of our gene sets identified the urokinase and extracellular matrix remodeling pathways as the most enriched pathways. By infecting Serpine1-knockout mice, we showed that the urokinase pathway had a significant effect on both lung pathology and overall SARS-CoV pathogenesis. These results demonstrate the effective use of unbiased modeling techniques for identification of high-priority host targets that regulate disease outcomes. Similar transcriptional signatures were noted in 1918 and 2009 H1N1 influenza virus-infected mice, suggesting a common, potentially treatable mechanism in development of virus-induced ALI. TO, Law GL, Katze MG, McWeeney S, Baric RS. 2013. Mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury. mBio 4(4):e00271-13. Abstract African swine fever virus (ASFV) infection induces apoptosis in the infected cell; however, the consequences of this activation on virus replication have not been defined. In order to identify the role of apoptosis in ASFV infection, we analyzed caspase induction during the infection and the impact of caspase inhibition on viral production. Caspases 3, 9 and 12 were activated from 16 h post-infection, but not caspase 8. Indeed, caspase 3 activation during the early stages of the infection appeared to be crucial for efficient virus exit. In addition, the inhibition of membrane blebbing reduced the release of virus particles from the cell. ASFV uses the endoplasmic reticulum (ER) as a site of replication and this process can trigger ER stress and the unfolded protein response (UPR) of the host cell. In addition to caspase 12 activation, indicators of ER stress include the upregulation of the chaperones calnexin and calreticulin upon virus infection. Moreover, ASFV induces transcription factor 6 signaling pathway of the UPR, but not the protein kinase-like ER kinase or the inositol-requiring enzyme 1 pathways. Thus, the capacity of ASFV to regulate the UPR may prevent early apoptosis and ensure viral replication. Abstract A. Monthly number of emergency room visits and deaths within 7 days of myocardial infarction.MonthNumber of deaths within 7 days (n) Ratio of deaths in 2015 (%) Abstract This nationwide, prospective cohort study evaluated pulmonary function and radiological sequelae according to infection severity in 73 survivors from the 2015 Middle East respiratory syndrome (MERS) outbreak in Korea. Patients with severe pneumonia in MERS-coronavirus infection had more impaired pulmonary function than those with no or mild pneumonia at the 1-year follow-up, which was compatible with the radiological sequelae. Severe pneumonia significantly impairs pulmonary function and makes long radiological sequelae in MERS.The 2015 outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection in the Republic of Korea developed from a traveler returning from the Middle East, 1 which is the largest outbreak outside of the Arabian Peninsula to date. This unprecedented nationwide outbreak resulted in 186 laboratory-confirmed cases with 38 fatalities and > 16,000 individuals being quarantined. 2-4 During the outbreak, a comprehensive screening test including MERS-CoV real-time reverse transcription polymerase chain reaction (rRT-PCR) was performed in all possible contactors to prevent further spread of the disease. Positive MERS-CoV rRT-PCR findings were observed in patients with no or mild symptoms, who were also subjected to epidemiological investigation and follow-up. 1There have been many reports of long-term sequelae of severe acute respiratory syndrome (SARS), 5-10 but to the best of our knowledge no report has addressed long-term sequelae in Abstract Background: Molecular detection of Middle East respiratory syndrome coronavirus (MERS-CoV) using real-time reverse transcription (rRT)-PCR assays is the method of choice for diagnosis of MERS. We evaluated the performance of the PowerChek MERS (upE & ORF1a) real-time PCR Kit (PowerChek MERS assay; Kogene Biotech, Korea) a one-step rRT-PCR assay for the qualitative detection of MERS-CoV.We evaluated PowerChek MERS assay performance in comparison with nested RT-PCR and sequencing of the RNA-dependent RNA polymerase (RdRp) and N genes. To evaluate diagnostic sensitivity and specificity, 100 clinical specimens (50 positive and 50 negative for MERS-CoV) were simultaneously tested by using the PowerChek MERS and sequencing assays. Assay performance, including limit of detection and precision, was evaluated in vitro by using MERS-CoV RNA transcripts. Analytical specificity was evaluated with a diverse collection of 16 respiratory virus-positive clinical specimens and 14 respiratory bacterial isolates.Results: The 95% limits of detection of the PowerChek MERS assay for the upE and the open rading frame (ORF)1a were 16.2 copies/μL and 8.2 copies/μL, respectively. No crossreactivity was observed. The diagnostic sensitivity and specificity of the PowerChek MERS assay were both 100% (95% confidence interval, 91.1-100%).PowerChek MERS assay is a straightforward and accurate assay for detecting MERS-CoV RNA. The assay will be a useful tool for the rapid diagnosis of MERS and could prove especially important for MERS outbreak control. Abstract The FilmArray Respiratory Panel (FA-RP) is an FDA certified multiplex PCR that can detect 17 viruses and 3 bacteria responsible for upper respiratory tract infections, thus it is potentially useful to the assessment of the age-related prevalence of these pathogens.In this observational study, we retrospectively analyzed the results of all the respiratory samples, which had been processed during 1 year-period (November 2015 to November 2016) with the FA-RP, in the Central Laboratories of Hygeia & Mitera General Hospitals of Athens, Greece. In order to have an age-related distribution, the following age groups were implemented: (<2), (≥2, <5), (≥5, <10), (≥10, <18), (≥18, <45), (≥45, <65), and (≥65) years old.Among 656 respiratory samples tested, 362 (55%) were from male and 294 (45%) from female patients, while 356 (54.3%) were positive and 300 (45.7%) negative. In the first age-group (<2), 41/121 samples (33.9%) revealed human rhinovirus/enterovirus (HRV) and 16 (13.2%) adenovirus (Adv), followed by respiratory syncytial virus (RSV), coronavirus, human metapneumovirus (Hmpv), and parainfluenza viruses (PIV). In the age-group (≥2, <5), Adv predominated with 37/147 samples (25.2%), followed by HRV, RSV, coronavirus (all types), and influenza, Hmpv and PIV. In the age-group (≥5, <10), HRV was identified in 25/80 samples (31.3%), Adv in 18 (22.5%), influenza in 11 (13.8%), and Hmpv in 6 (7.5%). Influenza predominated in the age-group (≥10, <18), with 4/22 samples (18.2%), while in the remaining age-groups (≥18), HRV was the commonest isolated pathogen, 33/286 (11.5%), followed by influenza with 20 (7%) (influenza A H1-2009, 11/20).In our patient series, HRV seemed to prevail in most age-groups, followed by Adv, although Influenza was the second most frequent pathogen isolated in the age-groups (≥18). Moreover, increasing age corresponded to increasing possibility of having a negative sample, indicating that FilmArray may be more useful before adolescence. Abbreviations: Adv = adenovirus, FA-RP = FilmArray Respiratory Panel, HCoV = coronaviruses, Hmpv = human metapneumovirus, HRSV, RSV = (human) respiratory syncytial virus, HRV = human rhinovirus/enterovirus, PIV = parainfluenza virus, Rs = respiratory samples, URIs = upper respiratory tract infections. Abstract Posttranslational modification of yeast glycoproteins with od,3-1inked mannose is initiated within a Golgi compartment analogous to the medial Golgi cisternae of higher eukaryotes. We have characterized the synthesis, posttranslational modification, and localization of the yeast od,3 mannosyltransferase (Mnnlp) using antibodies prepared against a segment of this protein expressed in bacteria. Mnnlp is initially synthesized as a 98.5-kD, type II integral membrane glycoprotein that is modified with both N-and O-linked oligosaccharides. It is subject to a slow, incremental increase in molecular mass that is dependent upon protein transport to the Golgi complex. Self-modification of Mnnlp with od,3 mannose epitopes, primarily on O-linked oligosaccharides, is at least partly responsible for the incremental increase in molecular mass. Abstract In the present study, the whole human genome oligo microarray was employed to investigate the gene expression profile in symptomatic pulmonary embolism (PE). METHODS: Twenty patients with PE and 20 age and gender matched patients without PE as controls were enrolled into the present study in the same period. The diagnosis of PE was based on the clinical manifestations and findings on imaging examinations. Acute arterial and/or venous thrombosis was excluded in controls. The whole human genome oligo microarray was employed for detection. Statistical analysis was performed with t test following analysis of very small samples of repeated measurements and Gene Ontology (GO) analysis. RESULTS: Genomic data showed no damage to vascular endothelial cells in PE patients. Genomic data only found increased mRNA expression of a small amount of coagulation factors in PE patients. In the PE group, anticoagulant proteins, Fibrinolytic system and proteins related to platelet functions only played partial roles in the pathogenesis of PE. In addition, the mRNA expressions of a fraction of adhesion molecules were markedly up-regulated. Gene Ontology analysis showed the genes with down-regulated expressions mainly explain the compromised T cell immunity. Symptomatic VTE patients have compromised T cell immunity. CONCLUSION: The damage to vascular endothelial cells is not necessary in the pathogenesis of VTE, and only a fraction of factors involved in the shared coagulation cascade are activated. Genomic results may provide a new clue for clinical diagnosis, treatment and prevention of VTE. Abstract !3 ()132786%8) A7 -223:%8-:) %4463%', -2 *%'-0-8%8-2+ -2*36 1%8-32 7,%6-2+ %132+ " *)()6%0 %+)2'-)7 (96-2+ 8,) 6)')28 398 &6)%/7 3* -2*09)2>% %2( 3# Abstract Objective The clinical characteristics and chest imaging findings of viral pneumonia and several interstitial lung diseases (ILDs) overlap, and viral pneumonia may be underrecognized and misdiagnosed as certain ILDs. To clarify the frequency of viral pneumonia among patients with acute progressive clinical courses that required a differential diagnosis between ILDs and pneumonia, and to determine the most frequent ILDs misdiagnosed in cases of viral pneumonia.We retrospectively analyzed patients hospitalized from 2010 to 2017 with an acute clinical course (! 30 days) who underwent bronchoalveolar lavage (BAL) for the differential diagnosis of infection and ILDs. We performed a multiplex PCR for respiratory viruses using the patients' preserved BAL fluid. The final diagnosis was made by a multidisciplinary approach and after considering the PCR results. The diagnosis at discharge was compared to the final diagnosis. Results Among the 109 patients, 53 were diagnosed with viral pneumonia. Viral pneumonia and other diseases showed some differences in symptoms and laboratory data; however, the differences were small or overlapped. Viral pneumonia was misdiagnosed on discharge as acute fibrinous organizing pneumonia, cryptogenic organizing pneumonia, or chronic eosinophilic pneumonia (AFOP/COP/CEP) (n=22), acute interstitial pneumonia (n=5), connective tissue disease-related ILDs (n=3), unclassifiable interstitial pneumonia (n=2), drug-induced ILD (n=1), and pneumonia (n=20). Conclusion Approximately half of the patients who underwent BAL had viral pneumonia. The most common ILD-related misdiagnoses were AFOP/COP/CEP. Differences in symptoms and laboratory findings between viral pneumonia and other diseases were small, and viral pneumonia should be included in the differential diagnosis when physicians encounter cases in which the abovementioned ILDs are suspected. Abstract Basal cell carcinoma (BCC) is the most common type of human skin cancer, which is driven by the aberrant activation of Hedgehog signaling. Previous evidence indicated that sex determining region Y-box 2 (SOX2) is associated with the tumor metastasis. However, the expression and role of SOX2 in BCC remain unknown. Therefore, the aim of the current study was to analyze the possible mechanism of SOX2 in the progression of BCC. The levels of SOX2 in BCC cells were detected by reverse transcription-quantitative polymerase chain reaction. Transwell assays were also used to determine the migration and invasion of BCC cells. Immunoblotting and immunofluorescence were used for analyzing the role of SOX2 knockdown in the serine-arginine protein kinase 1 (SRPK1)-mediated phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway in BCC cells. The results demonstrated that SOX2 is overexpressed in BCC tissues and cells. In addition, SOX2 knockdown inhibited the migration and invasion of BCC cells, and the epithelial-mesenchymal transition (EMT) progress of BCC cells. It was also observed that SOX2 knockdown decreased SRPK1 expression, which further led to the downregulation of PI3K and AKT expression levels in BCC cells. Furthermore, SRPK1 transfection or PI3K/AKT pathway activation abolished the inhibitory effects of SOX2 knockdown on the migration, invasion and EMT progress of BCC cells. In conclusion, these results indicated that SOX2 may potentially serve as a target for BCC therapy by targeting the SRPK1-mediated PI3K/AKT signaling pathway. Abstract Rhinoviruses (RV's) are common human pathogens of the respiratory tract being the most frequent cause of mild diseases of the upper respiratory tract (common cold) but more importantly they are a major initiator of acute exacerbations of chronic airway diseases. Infections can be life threatening in the latter context however RV -induced common colds have an associated economic cost from loss of productivity due to absence from work or school. There are no appropriate antiviral therapies available and vaccine strategies have failed because of the large number of viral serotypes and the lack of cross-serotype protection generated. Here, approaches past and present for development of a vaccine to these widespread human pathogens are highlighted. Abstract The West African sub-continent is currently experiencing its first, and ironically, the largest and longest Ebola viral diseases (EVD) outbreak ever documented in modern medical history. The current outbreak is significant in several ways, including longevity, magnitude of morbidity and mortality, occurrence outside the traditional niches, rapid spread and potential of becoming a global health tragedy. The authors provided explicit insights into the current and historical background, drivers of the epidemic, societal impacts, status of vaccines and drugs development and proffered recommendations to halt and prevent future occurrences. The authors reviewed mainly five databases and a hand search of key relevant literature. We reviewed 51 articles that were relevant up until the 18 th of August 2014. The authors supplemented the search with reference list of relevant articles and grey literature as well as relevant Internet websites. Article searches were limited to those published either in English or French. There are strong indications that the EVD may have been triggered by increased human activities and encroachment into the forest ecosystem spurred by increasing population and povertydriven forest-dependent local economy. Containment efforts are being hampered by weak and fragile health systems, including public health surveillance and weak governance, certain socio-anthropological factors, fast travels (improved transport systems) and globalization. The societal impacts of the EBV outbreak are grave, including economic shutdown, weakening of socio-political systems, psychological distress, and unprecedented consumption of scarce health resources. The research and development (R&D) pipeline for product against EBV seems grossly insufficient. The outbreak of Ebola and the seeming difficulty to contain the epidemic is simply a reflection of the weak health system, poor surveillance and emergency preparedness/ response, poverty and disconnect between the government and the people in many West African countries. Although interventions by the United Nations and other international development agencies could ultimately halt the epidemic, local communities must be engaged to build trust and create demand for the public health interventions being implemented in the Ebola-ravaged populations. In the intermediate and long term, post-Ebola rehabilitation should focus on strengthening of health systems, improving awareness about zoonosis and health behaviors, alleviating poverty and mitigating the impact of triggering factors. Finally, national governments and international development partners should mobilize huge resources and investments to spur or facilitate R&D of disease control tools for emerging and pernicious infectious diseases (not limited to EVD). Abstract Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), a condition that threatens the sustainability of the livestock industry. A fluorescent loop-mediated isothermal amplification (fLAMP) assay targeting BLV env sequences was developed and used to evaluate 100 bovine blood samples. Compared with a conventional real-time PCR (rPCR) assay, the fLAMP assay achieved 87.3% (62/71) sensitivity and 100% (29/29) specificity. The rPCR assay took 65 min, while the fLAMP assay took 8 min to 30 min from the beginning of DNA amplification to final judgement with a comparable limit of detection. The fLAMP is a potential tool for the rapid and simple diagnosis of BLV infection to supplement ELISA testing and can be used by local laboratories and slaughterhouses without special equipment.KEY WORDS: BLV, bovine leukemia virus, enzootic bovine leucosis, fLAMP, fluorescent loopmediated isothermal amplification Bovine leukemia virus (BLV), a member of the Retroviridae family and the Deltaretrovirus genus, causes enzootic bovine leukosis (EBL). Although BLV-infections are mainly asymptomatic, within several years approximately 30% and 0.1-10% of BLV-infected cattle develop persistent lymphocytosis and fatal lymphosarcoma, respectively [19, 23] . In the European Union (EU), BLV infections have been eradicated, and several nations are officially recognized as free from BLV infection [13, 23] . In contrast, increases in BLV infection and EBL have occurred in Japan [9] [10] [11] . In Japan, cattle diagnosed with lymphosarcoma, which are disapproved for meat consumption by legal regulations, cause vast economic losses to the Japanese livestock industry. Accurate and reliable diagnosis of BLV infection is therefore crucial to accelerate efforts to implement an effective eradication program.Serological tests that detect persistent anti-BLV antibodies, such as agar-gel immunodiffusion and enzyme linked immunosorbent assays (ELISAs) are routinely used worldwide for the effective screening of BLV-infected cattle [9, 11, 16] . However, these tests are insufficient to correctly diagnose the early stages of infection [5, 9, 16] . Therefore, supplemental use of a rapid and sensitive assay for the detection of BLV-genomic DNA is required to reinforce accurate diagnosis.Although nested PCR assays detect BLV-genomic DNA with high sensitivity [1, 14, 23], they require time-consuming and tedious electrophoretic analysis and are plagued by laboratory contamination. Although real-time PCR assays are more rapid and easier to perform compared with nested PCR assays and achieve equivalent analytical sensitivity (limit of detection, LOD), the assays require expensive equipment [3, 16] . Further, detection of at least two distinct genomic targets is desirable to prevent including false-negatives [20] .Loop-mediated isothermal amplification (LAMP) is a promising candidate diagnostic tool for the rapid and sensitive detection of BLV genomic DNA. The LAMP assay involves isothermal amplification at 60-67°C within 60 min and one-step detection of a target gene using simple analysis of turbidity (turbidimetric LAMP or tLAMP) [5, 14, 15, 24] . Therefore, the assay is faster and easier to perform than nested PCR and real-time PCR assays with equivalent analytical sensitivity [5, 12, 14, 15, 24] . tLAMP assays afford rapid diagnosis of viral diseases [15, 24] , including BLV detection through targeting the long terminal repeat (LTR) Abstract Background. Efficacy endpoints in influenza clinical trials may include clinical symptoms and virological measurements, although virology cannot serve as the primary endpoint. We investigated the relationship between influenza A RNA copy number and quantity of infectious viruses in hospitalized influenza patients.Methods. One hundred fifty influenza-infected, hospitalized patients were included in this prospective cohort study spanning the 2012-2013 influenza season. Daily nasopharyngeal samples were collected during hospitalization, and influenza A RNA copy number and infectious viral titer were monitored.Results. The decay rate for 50% tissue culture infectious dose (TCID 50 ) was 0.51 ± 0.14 log 10 TCID 50 /mL per day, whereas the RNA copy number decreased at a rate of 0.41 ± 0.04 log 10 copies/mL per day (n = 433). The log ratio of the RNA copy number to the infectious viral titer within patient changes significantly with −0.25 ± 0.09 units per day (P = .0069). For a 12-day observation period, the decay corresponds to a decline of this ratio of 3 log influenza RNA copies.Conclusions. Influenza RNA copy number in nasal swabs is co-linear with culture, although the rate of decay of cell culture-based viral titers was faster than that observed with molecular methods. The study documented a clear decreasing log ratio of the RNA copy number to the infectious viral titer of the patients over time. Abstract Objective: To assess the feasibility and validity of unsupervised participant-collected nasal swabs to detect respiratory pathogens in a low-income, urban minority population. Methods: This project was conducted as part of an ongoing community-based surveillance study in New York City to identify viral etiologies of acute respiratory infection. In January 2014, following sample collection by trained research assistants, participants with acute respiratory infection from 30 households subsequently collected and returned a self-collected/ parent-collected nasal swab via mail. Self/parental swabs corresponding with positive reverse transcription polymerase chain reaction primary research samples were analyzed. Results: Nearly all (96.8%, n=30/31) households agreed to participate; 100% reported returning the sample and 29 were received (median time: 8 days). Most (18; 62.1%) of the primary research samples were positive. For eight influenza-positive research samples, seven (87.5%) self-swabs were also positive. For ten other respiratory pathogen-positive research samples, eight (80.0%) self-swabs were positive. Sensitivity of self-swabs for any respiratory pathogen was 83.3% and 87.5% for influenza, and specificity for both was 100%. There was no relationship between level of education and concordance of results between positive research samples and their matching participant swab. Conclusion: In this pilot study, self-swabbing was feasible and valid in a low-income, urban minority population. Abstract Yeast Sec12p is a type II transmembrane Abstract The origin of viruses remains mysterious because of their diverse and patchy molecular and functional makeup. Although numerous hypotheses have attempted to explain viral origins, none is backed by substantive data. We take full advantage of the wealth of available protein structural and functional data to explore the evolution of the proteomic makeup of thousands of cells and viruses. Despite the extremely reduced nature of viral proteomes, we established an ancient origin of the "viral supergroup" and the existence of widespread episodes of horizontal transfer of genetic information. Viruses harboring different replicon types and infecting distantly related hosts shared many metabolic and informational protein structural domains of ancient origin that were also widespread in cellular proteomes. Phylogenomic analysis uncovered a universal tree of life and revealed that modern viruses reduced from multiple ancient cells that harbored segmented RNA genomes and coexisted with the ancestors of modern cells. The model for the origin and evolution of viruses and cells is backed by strong genomic and structural evidence and can be reconciled with existing models of viral evolution if one considers viruses to have originated from ancient cells and not from modern counterparts. Abstract Diffuse alveolar damage (DAD) is a histological change in lung tissue, and is generally caused by an acute lung injury, which is characterized by bilateral and widespread damages. Localized DAD occurs very rarely. The causes for DAD are numerous, but the chief cause is acute interstitial pneumonia or acute exacerbation of idiopathic interstitial pneumonia, in cases of idiopathic manifestation. The 82-year-old patient, in this case study, showed a DAD lesion in only 1 lobe. The patient was otherwise healthy, with no previous symptoms of DAD. He was admitted to our medical center owing to localized infiltration, observed on his chest radiograph. Laboratory studies showed no signs of infections. DAD was confirmed by a surgical lung biopsy. The patient received corticosteroid treatment and had gradually improved. We report the case of a patient with localized, idiopathic DAD that cannot be classified as acute interstitial pneumonia or acute exacerbation of idiopathic interstitial pneumonia. Abstract The Ebola virus disease outbreak in West Africa and the Middle East Respiratory Syndrome outbreak in the Republic of Korea have given huge impacts in different aspects. Health security is no more a new coinage. Global health security became more realistic in its practical application. In the perspective of global health, it will be helpful to peruse lessons learned from the Ebola outbreak in West Africa and MERS outbreak in Korea.Global Health Security S27 Abstract Objectives The common cold is the main cause of medical time loss in elite sport. Rapid diagnosis has been a challenge that may be amenable to molecular point-ofcare testing (POCT). Methods We performed a prospective observational study of the common cold in Team Finland during the 2018 Winter Olympic Games. There were 44 elite athletes and 68 staff members. The chief physician recorded the symptoms of the common cold daily on a standardised form. Two nasal swabs were taken at the onset of symptoms. One swab was analysed within 45 min using a molecular POCT for respiratory syncytial virus and influenza A and B viruses. After the Games, the other swab was tested for 16 possible causative respiratory viruses using PCR in laboratory-based testing. Results 20 out of 44 (45%) athletes and 22 out of 68 (32%) staff members experienced symptoms of the common cold during a median stay of 21 days. Eleven (26%) samples tested virus-positive using POCT. All subjects with influenza (n=6) and 32 close contacts were treated with oseltamivir. The aetiology of the common cold was finally detected in 75% of the athletes and 68 % of the staff members. Seven virus clusters were identified. They were caused by coronaviruses 229E, NL63 and OC43, influenza B virus, respiratory syncytial virus A, rhinovirus and human metapneumovirus. The virus infections spread readily within the team, most commonly within the same sport discipline. Conclusions The cold was indeed a common illness in Team Finland during the Winter Olympic Games. POCT proved to be clinically valuable, especially for influenza. The aetiology of the common cold was identified in most cases. Abstract We present a unique and informative instance of respiratory syncytial virus (RSV) infection associated with antiphospholipid syndrome (APS), and discuss this case in the context of the literature addressing the immunopathogenesis of APS associated with diverse infections. We describe the case of a 43-yearold man with no significant past medical history who presented with the acute onset of fever, hemoptysis, and extensive bullous, ecchymotic lesions in both lower extremities. Punch biopsy of the lesion demonstrated thrombotic vasculopathy. Further evaluation revealed serum antiphospholipid antibodies as well as a positive RSV PCR in a nasal swab specimen. Clinical manifestations, positive laboratory and pathological findings were strongly suggestive of APS associated with a recent RSV infection. When an infectious etiology is considered for APS, RSV should also be included in the differential diagnosis.2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).We present what, to our knowledge, is the first reported case of antiphospholipid syndrome (APS) associated with Respiratory Syncytial Virus (RSV) infection. APS is a disorder characterized by vascular thrombosis, or pregnancy-related morbidity, in the presence of antiphospholipid (aPL) antibodies [1]. APS is classified as either primary or secondary, and secondary APS is associated with autoimmune disease, infection, medication or malignancy [2] . RSV is a common causative agent of acute viral upper and lower respiratory tract infection throughout all age groups. Despite the common occurrence of RSV respiratory infections, an association or possible causal relationship between RSV and APS has not to our knowledge been discussed previously. We report a case where progressive, extensive ecchymoses appeared to be the initial manifestation of APS occurring in temporal association with, and seemingly triggered by, a recent episode of RSV infection.A 43-year-old male with no significant past medical history presented during the month of December with a 4-day history of fever (maximum temperature 38.8 C), shaking chills, hemoptysis and worsening dyspnea. He had experienced intermittent night sweats, pain and swelling of both wrists and ankle joints over the last several months. He also reported a 10 kg weight loss during this time, but never sought medical attention. He had noted a painful ecchymotic lesion on his left calf one day prior to the admission. Over the subsequent 24 h, the patient developed similar lesions on his abdomen, back, right upper arm and right calf. The patient's 3 year old son had recently had a respiratory illness.On physical examination, the patient had a temperature of 35.3 C, blood pressure of 117/75 mmHg, pulse rate of 112 beats/ min, respiratory rate of 29 breaths/min, and an oxygen saturation of 96% on room air. There were bilateral lung wheezes, and multiple painful ecchymosis with several hemorrhagic bullae over his trunk and extremities (Fig. 1) . CBC on admission showed WBC 9000/mm3 (87% neutrophils) and Hemoglobin 13.4 g/dL, and comprehensive metabolic panel including electrolytes, liver and renal function was all within normal range. The significant laboratory data included high lactate dehydrogenase of 799 IU/L, erythrocyte sedimentation rate of 81 mm/h, and C-reactive protein level of 38.5 mg/dL. The Xray and computed tomography (CT) of the * Corresponding author. Present address: Azumabashi 1-23-30, River Pier Azumabashi Room 2707, Sumida-ku, Abstract Purpose: In May 2015, South Korea experienced an epidemic of Middle East respiratory syndrome (MERS). This study investigated the impacts of MERS epidemic on emergency care utilization and mortality in South Korea. Materials and Methods: A natural experimental study was conducted using healthcare utilization and mortality data of the entire Korean population. The number of monthly emergency room (ER) visits was investigated to identify changes in emergency care utilization during the MERS epidemic; these trends were also examined according to patients' demographic factors, disease severity, and region. Deaths within 7 days after visiting an ER were analyzed to evaluate the impact of the reduction in ER visits on mortality. Results: The number of ER visits during the peak of the MERS epidemic (June 2015) decreased by 33.1% compared to the average figures from June 2014 and June 2016. The decrease was observed in all age, sex, and income groups, and was more pronounced for low-acuity diseases (acute otitis media: 53.0%; upper respiratory infections: 45.2%) than for high-acuity diseases (myocardial infarctions: 14.0%; ischemic stroke: 16.6%). No substantial changes were detected for the highest-acuity diseases, with increases of 3.5% for cardiac arrest and 2.4% for hemorrhagic stroke. The number of deaths within 7 days of an ER visit did not change significantly. Conclusion: During the MERS epidemic, the number of ER visits decreased in all age, sex, and socioeconomic groups, and decreased most sharply for low-acuity diseases. Nonetheless, there was no significant change in deaths after emergency care. Abstract Background A/H1N1, more commonly referred to as swine flu, emerged in Mexico in spring 2009. It rapidly spread across the world and was classed as a global pandemic on 11 June 2009. Objective To analyse UK newsprint coverage of the swine flu pandemic. Methods Content analysis of 2374 newsprint articles published in eight UK national newspapers between 1 Abstract A virtual screening analysis of our library of phytochemical structures with dengue virus protein targets has been carried out using a molecular docking approach. A total of 2194 plant-derived secondary metabolites have been docked. This molecule set comprised of 290 alkaloids (68 indole alkaloids, 153 isoquinoline alkaloids, 5 quinoline alkaloids, 13 piperidine alkaloids, 14 steroidal alkaloids, and 37 miscellaneous alkaloids), 678 terpenoids (47 monoterpenoids, 169 sesquiterpenoids, 265 diterpenoids, 81 steroids, and 96 triterpenoids), 20 aurones, 81 chalcones, 349 flavonoids, 120 isoflavonoids, 74 lignans, 58 stilbenoids, 169 miscellaneous polyphenolic compounds, 100 coumarins, 28 xanthones, 67 quinones, and 160 miscellaneous phytochemicals. Dengue virus protein targets examined included dengue virus protease (NS2B-NS3pro), helicase (NS3 helicase), methyltransferase (MTase), RNA-dependent RNA polymerase (RdRp), and the dengue virus envelope protein. Polyphenolic compounds, flavonoids, chalcones, and other phenolics were the most numerous of the strongly docking ligands for dengue virus protein targets. Abstract Individual patients with life-threatening or severely debilitating diseases can petition the U.S. Food and Drug Administration (FDA) through their physicians to have expanded access (EA) to drugs that are in clinical trials but have not reached full FDA approval (the "single-patient" investigational new drug [IND] application). Additionally, recent state and federal laws-so-called "right to try legislation"-allow patients to approach drug companies directly for access prior to FDA approval. While these pathways provide potential access for individual patients to investigational drugs, different EA pathways permit entire groups of certain patients to access investigational drugs prior to FDA approval. This review focuses on special categories of EA INDs intended for multiple patients-the intermediate-group IND and the widespreadtreatment IND-as well as emergency authorization for use of investigational drugs and biological products (e.g., Abstract DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid highresolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the wholegenome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive singlenucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%. Abstract Long non-coding RNAs (lncRNAs) are found not only in mammals but also in other organisms, including viruses. Recent findings suggest that lncRNAs play various regulatory roles in multiple major biological and pathological processes. During viral life cycles, lncRNAs are involved in a series of steps, including enhancing viral gene expression, promoting viral replication and genome packaging, boosting virion release, maintaining viral latency and assisting viral transformation; additionally, lncRNAs antagonize host antiviral innate immune responses. In contrast to proteins that function in viral infection, lncRNAs are expected to be novel targets for the modulation of all types of biochemical processes due to their broad characteristics and profound influence. This review highlights our current understanding of the regulatory roles of lncRNAs during viral infection processes with an emphasis on the potential usefulness of lncRNAs as a target for viral intervention strategies, which could have therapeutic implications for the application of a clinical approach for the treatment of viral diseases. Abstract Continuously emerging highly pathogenic human coronaviruses (HCoVs) remain a major threat to human health, as illustrated in past SARS-CoV and MERS-CoV outbreaks. The development of a drug with broad-spectrum HCoV inhibitory activity would address this urgent unmet medical need. Although previous studies have suggested that the HR1 of HCoV spike (S) protein is an important target site for inhibition against specific HCoVs, whether this conserved region could serve as a target for the development of broad-spectrum pan-CoV inhibitor remains controversial. Here, we found that peptide OC43-HR2P, derived from the HR2 domain of HCoV-OC43, exhibited broad fusion inhibitory activity against multiple HCoVs. EK1, the optimized form of OC43-HR2P, showed substantially improved pan-CoV fusion inhibitory activity and pharmaceutical properties. Crystal structures indicated that EK1 can form a stable six-helix bundle structure with both short -HCoV and long -HCoV HR1s, further supporting the role of HR1 region as a viable pan-CoV target site. Abstract Japanese encephalitis (JE) is neuroinflammation characterized by uncontrolled infiltration of peripheral leukocytes into the central nervous system (CNS). We previously demonstrated exacerbation of JE following CD11c hi dendritic cell (DC) ablation in CD11c-DTR transgenic mice. Moreover, CD11c hi DC ablation led to abnormal differentiation of CD11b + Ly-6C hi monocytes and enhanced permeability of the blood-brain barrier (BBB), resulting in promoting the progression of JE. Here, we examined changes in lymphoid and myeloid-derived leukocyte subpopulations associated with pro-and anti-inflammation during JE progression. The analyses of this study focused on regulatory CD4 + Foxp3 + regulatory T cells (Tregs), IL-17 + CD4 + Th17 cells, and CD11b + Ly-6C hi and Ly-6C lo monocytes. CD11c hi DC ablation resulted in the accumulation of IL-17 + CD4 + Th17 cells in the CNS, thereby leading to lower ratio of Tregs to Th17 cells. This result was corroborated by the higher expression levels of IL-17 and RORgT in CD4 + T cells from the brains of CD11c hi DC-ablated mice. In addition, CD11c hi DC-ablated mice showed higher frequency and total number of inflammatory CD11b + Ly-6C hi monocytes, whereas CD11b + Ly-6C lo monocytes were detected with lower frequency and total number in CD11c hi DC-ablated mice. Furthermore, CD11c hi DC ablation altered the phenotype and function of CD11b + Ly-6C lo monocytes, resulting in lower levels of activation marker and anti-inflammatory cytokine (IL-10 and TGF-b) expression. Collectively, these results indicate that CD11c hi DC ablation caused an imbalance in CD4 + Th17/Treg cells and CD11b + Ly-6C hi /Ly-6C lo monocytes in the lymphoid tissue and CNS during JE progression. This imbalanced orchestration of pro-and anti-inflammatory leukocytes following CD11c hi DC ablation may contribute to the exacerbation of JE. [Immune Network 2017;17(3):192-200] Abstract The West African Ebola epidemic has set in motion a collective endeavour to conduct accelerated clinical trials, testing unproven but potentially lifesaving interventions in the course of a major public health crisis. This unprecedented effort was supported by the recommendations of an ad hoc ethics panel convened in August 2014 by the WHO. By considering why and on what conditions the exceptional circumstances of the Ebola epidemic justified the use of unproven interventions, the panel's recommendations have challenged conventional thinking about therapeutic development and clinical research ethics. At the same time, unanswered ethical questions have emerged, in particular: (i) the specification of exceptional circumstances, (ii) the specification of unproven interventions, (iii) the goals of interventional research in terms of individual versus collective interests, (iv) the place of adaptive trial designs and (v) the exact meaning of compassionate use with unapproved interventions.Examination of these questions, in parallel with empirical data from research sites, will help build pragmatic foundations for disaster research ethics. Furthermore, the Ebola clinical trials signal an evolution in the current paradigms of therapeutic research, beyond the case of epidemic emergencies. Abstract RNA-dependent RNA polymerase (RdRp) is essential to viral replication and is therefore one of the primary targets of countermeasures against these dangerous infectious agents. Development of broad-spectrum therapeutics targeting polymerases has been hampered by the extreme sequence variability of these sequences. RdRps range in length from 400-800 residues, yet contain only $20 residues that are conserved in most species. In this study, we made structure-based comparisons that are independent of sequence composition using a recently developed algorithm. We identified residue-to-residue correspondences of multiple protein structures and created (two-dimensional) structure-based alignment maps of 37 polymerase structures that provide both sequence and structure details. Using these maps, we determined that $75% of each polymerase species consists of seven protein segments, each of which has high structural similarity to segments in other species, though they are widely divergent in sequence composition and order. We define each of these segments as a 'homomorph', and each includes (though most are much larger than) the well-known conserved polymerase motifs. All homomorphs contact the template tunnel or nucleoside triphosphate (NTP) entry tunnel and the exterior of the protein, suggesting they constitute a structural and functional skeleton common among the polymerases. Abstract The etiology of Porcine respiratory disease complex is complicated by infections with multiple pathogens, and multiple infections increase the difficulty in identifying the causal pathogen. In this present study, we developed a detection system of microbes from porcine respiratory by using TaqMan real-time PCR (referred to as Dempo-PCR) to screen a broad range of pathogens associated with porcine respiratory diseases in a single run. We selected 17 porcine respiratory pathogens (Actinobacillus pleuropneumoniae, Boldetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida, Pasteurella multocida toxin, Streptococcus suis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynovie, porcine circovirus 2, pseudorabies virus, porcine cytomegalovirus, swine influenza A virus, porcine reproductive and respiratory virus US strain, EU strain, porcine respiratory coronavirus and porcine hemagglutinating encephalomyelitis virus) as detection targets and designed novel specific primer-probe sets for seven of them. In sensitivity test by using standard curves from synthesized DNA, all primer-probe sets showed high sensitivity. However, porcine reproductive and respiratory virus is known to have a high frequency of genetic mutations, and the primer and probe sequences will need to be checked at a considerable frequency when performing Dempo-PCR from field samples. A total of 30 lung samples from swine showing respiratory symptoms on six farms were tested by the Dempo-PCR to validate the assay's clinical performance. As the results, 12 pathogens (5 virus and 7 bacteria) were detected and porcine reproductive and respiratory virus US strain, Mycoplasma hyorhinis, Haemophilus parasuis, and porcine cytomegalovirus were detected at high frequency. These results suggest that Dempo-PCR assay can be applied as a screening system with wide detection targets. Abstract Nucleotide deprivation and imbalance present detrimental conditions for animals and are thus expected to trigger cellular responses that direct protective changes in metabolic, developmental, and behavioral programs, albeit such mechanisms are vastly underexplored. Following our previous finding that Caenorhabditis elegans shut down germ cell proliferation in response to pyrimidine deprivation, we find in this study that endonuclease ENDU-2 regulates nucleotide metabolism and germ cell proliferation in response to nucleotide imbalance and other genotoxic stress, and that it affects mitotic chromosomal segregation in the intestine and lifespan. ENDU-2 expression is induced by nucleotide imbalance and genotoxic stress, and ENDU-2 exerts its function in the intestine, mostly by inhibiting the phosphorylation of CTPS-1 through repressing the PKA pathway and histone deacetylase HDA-1. Human EndoU also affects the response to genotoxic drugs. Our work reveals an unknown role of ENDU-2 in regulating nucleotide metabolism and animals' response to genotoxic stress, which may link EndoU function to cancer treatment.Jia et al. show that an endonuclease plays a critical role in a nucleotide response system in nematodes and possibly mammalian cells. It affects nucleotide metabolism and reproductive development in response to nucleotide imbalance or other stresses, and it does so by inhibiting the phosphorylation of CTP synthase in the gut.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Abstract During the past decade several notable viruses have suddenly emerged from obscurity or anonymity to become serious global health threats, provoking concern regarding their sustained epidemic transmission in immunologically naive human populations. With each new threat comes the call for rapid vaccine development. Indeed, vaccines are considered a critical component of disease prevention for emerging viral infections because, in many cases, other medical options are limited or non-existent, or that infections result in such a rapid clinical deterioration that the effectiveness of therapeutics is limited. While classic approaches to vaccine development are still amenable to emerging viruses, the application of molecular techniques in virology has profoundly influenced our understanding of virus biology, and vaccination methods based on replicating, attenuated and non-replicating virus vector approaches have become useful vaccine platforms. Together with a growing understanding of viral disease emergence, a range of vaccine strategies and international commitment to underpin development, vaccine intervention for new and emerging viruses may become a possibility. Abstract Transchromosomic bovines (Tc-bovines) adaptively produce fully human polyclonal immunoglobulin (Ig)G antibodies after exposure to immunogenic antigen(s). The National Interagency Confederation for Biological Research and collaborators rapidly produced and then evaluated anti-Ebola virus IgG immunoglobulins (collectively termed SAB-139) purified from Tc-bovine plasma after sequential hyperimmunization with an Ebola virus Makona isolate glycoprotein nanoparticle vaccine. SAB-139 was characterized by several in vitro production, research, and clinical level assays using wild-type Makona-C05 or recombinant virus/antigens from different Ebola virus variants. SAB-139 potently activates natural killer cells, monocytes, and peripheral blood mononuclear cells and has high-binding avidity demonstrated by surface plasmon resonance. SAB-139 has similar concentrations of galactose-α-1,3-galactose carbohydrates compared with human-derived intravenous Ig, and the IgG1 subclass antibody is predominant. All rhesus macaques infected with Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 and treated with sufficient SAB-139 at 1 day (n = 6) or 3 days (n = 6) postinfection survived versus 0% of controls. This study demonstrates that Tc-bovines can produce pathogen-specific human Ig to prevent and/or treat patients when an emerging infectious disease either threatens to or becomes an epidemic. Abstract The authors carried out a multistage stratified survey of 3,541 households representative of the population in four regions of China. They classified households as being "well prepared" for an emergency based on possession or affirmation of nine of fourteen "indicators" in a standard checklist. Fewer than 10% of households were considered "well prepared," while many professed ignorance or lack of interest in preparedness. Preparedness was associated with participation in emergency training and knowledge, and was negatively associated with reliance on external rescuers as well as a fatalistic outlook.Comments. Overall this is a well-designed and informative study. The text is well-written and unfolds logically, and the data support the overall conclusions. Considering the size and importance of China as the world's most populous country, with several regions especially prone to natural disasters such as floods and earthquakes, the study makes an important contribution to the literature on disaster preparedness, and could have a considerable public health impact in China.Nevertheless, there are a number of details that are somewhat sketchy, for which additional information and analysis would be helpful. Abstract Since the 2006 Pandemic Influenza Preparedness and Response Plan according to the World Health Organization's recommendation, the Republic of Korea has prepared and periodically evaluated the plan to respond to various public health crises including pandemic influenza. Korea has stockpiled 13,000,000 doses of antiviral drugs covering 26% of the Korean population and runs 519 isolated beds in 16 medical institutions. The division of public health crisis response in Korea Centers for Disease Control and Prevention are in charge of responding to public health crises caused by emerging infectious diseases including severe acute respiratory syndrome, avian influenza human infection, and pandemic influenza. Its job description includes preparing for emerging infectious diseases, securing medical resources during a crisis, activating the emergency response during the crisis, and fortification of capabilities of public health personnel. It could evolve into a comprehensive national agency to deal with public health crisis based on the experience of previous national emerging infectious diseases. Abstract A survey of Turkey Coronavirus (TCoV) and Astrovirus (TAstV-2) prevalence was carried out from February to December during 2006 year in semiarid region of Brazil, from a turkey producer area, localized in South Eastern of Brazil. To asses the risk factor related to clinical material, climatic condition and type of RT-PCR applied, cloacal swabs (CS), faeces, sera, bursa of Fabricius (BF), thymus (TH) and spleen (SP) and ileumcaeca region were collected from 30-day-old poults suffering of enteritis episode characterized as poult enteritis mortality syndrome (PEMS). The PEMS clinical features were characterized by watery to foamy faeces, light brown-yellow in colour and low mortality rate. Meteorological data (rainfall and relative humidity) observed during along the study presented monthly average temperature ranging from 39.3 and 31.2ºC, precipitation in rainy season from 40 to 270.3 mm/month, and no rain during dry season. Simplex RT-PCR gave odds ratio (OR) values suggesting that ileum-caeca region is at higher chance (OR=1.9; p=0.9741) to have both viral RNA than faeces (OR=1.5; p=0.7319). However, multiplex RT-PCR showed 3.98 (p=0.89982) more chance to give positive results in faeces than CS at dry season. The major risk factors seem to be low rate of humidity and high temperatures at winter, probably responsible for spread, easily, the TCoV and TAstv-2 among the flocks. The positive results of both virus suggested that they can play an important role in enteric disorders, associated to low humidity and high temperatures frequently found in tropical countries. Abstract Despite increasing clinical relevance of Chikungunya virus (CHIKV) infection, caused by a rapidly emerging pathogen, recommended guidelines for its inactivation do not exist. In this study, we investigated the susceptibility of CHIKV to inactivation by heat and commercially available hand, surface, and World Health Organization-recommended disinfectants to define CHIKV prevention protocols for healthcare systems. Abstract OBJECTIVES: Herpes zoster has been widely described in the context of different systemic autoimmune diseases but not dermatomyositis/polymyositis. Therefore, we analyzed the prevalence, risk factors and herpes zoster outcomes in this population. METHOD: A retrospective cohort study of herpes zoster infections in dermatomyositis/polymyositis patients was performed. The patients were followed at a tertiary center from 1991 to 2012. For the control group, each patient with herpes zoster was paired with two patients without herpes zoster. Patients were matched by gender and the type of myositis, age at myositis onset and disease duration. RESULTS: Of 230 patients, 24 (10.4%) had a histories of herpes zoster (19 with dermatomyositis and five with polymyositis, two-thirds female). The mean age of the patients with herpes zoster was 44.6¡16.8 years. No difference between the groups was found regarding cumulative clinical manifestations. Disease activity, autoantibody, muscle and leukogram parameters were also comparable between the groups. No differences in immunosuppressive (alone or in association with other immunosuppressive therapies) or glucocorticoid (current use, medium dose and cumulative dose in the last two months) therapies were found between patients with and without herpes zoster. However, a higher proportion of patients in the herpes zoster group received chloroquine diphosphate compared to the control group. All of the patients received acyclovir; 58.3% of patients had postherpetic neuralgia and no cases of recurrence were reported. Furthermore, individuals who were taking high prednisone doses at the time of the herpes zoster diagnosis had reduced levels of postherpetic neuralgia. CONCLUSIONS: These data suggest that chloroquine diphosphate could predispose patients with dermatomyositis/polymyositis to developing herpes zoster, particularly women and dermatomyositis patients. Abstract So far it has been demonstrated that the signal sequence of proteins which are made at the ER functions both at the level of protein targeting to the ER and in initiation of chain translocation across the ER membrane. However, its possible role in completing the process of chain transfer (see Singer, S. J., P. A. Maher, and M. P. Yaffe. Proc. Natl. Acad. Sci. USA. 1987. 84:1015-1019) has remained elusive. In this work we show that the p62 protein of Semliki Forest virus contains an uncleaved signal sequence at its NH2-terminus and that this becomes glycosylated early during synthesis and translocation of the p62 polypeptide. As the glycosylation of the signal sequence most likely occurs after its release from the ER membrane our results suggest that this region has no role in completing the transfer process. Abstract In many countries, vaccines are used for the prevention of foot-and-mouth disease (FMD). However, because there is no protection against FMD immediately after vaccination, research and development on antiviral agents is being conducted to induce protection until immunological competence is produced. This study tested whether well-known chemicals used as RNA virus treatment agents had inhibitory effects on FMD viruses (FMDVs) and demonstrated that ribavirin showed antiviral effects against FMDV in vitro/in vivo. In addition, it was observed that combining the administration of the antiviral agents orally and complementary therapy with vaccines synergistically enhanced antiviral activity and preserved the survival rate and body weight in the experimental animals. Antiviral agents mixed with an adjuvant were inoculated intramuscularly along with the vaccines, thereby inhibiting virus replication after injection and verifying that it was possible to induce early protection against viral infection prior to immunity being achieved through the vaccine. Finally, pigs treated with antiviral agents and vaccines showed no clinical signs and had low virus excretion. Based on these results, it is expected that this combined approach could be a therapeutic and preventive treatment for early protection against FMD. Abstract Introduction: Chronic obstructive pulmonary disease (COPD) is a leading cause of disability and death. The most common cause of COPD is smoking. There is evidence suggesting that genetic factors influence COPD susceptibility and variants in several candidate genes have been significantly associated with COPD. In this study, we aimed to investigate the possible association of the TNF-α -308, SPB+1580, IL-13 -1055 gene polymorphisms and latent adenovirus C infection with COPD in an Egyptian population. Material and methods: Our study included 115 subjects (75 smokers with COPD, 25 resistant smokers and 15 non-smokers) who were subjected to spirometric measurements, identification of adenovirus C and genotyping of TNF-α -308G/A, SP-B+1580 C/T and IL-13 -1055 C/T polymorphisms by real-time PCR. Results: The adenovirus C gene was identified in all subjects. The distribution of TNF-α genotypes showed no significant differences between different groups. However, homozygous A genotype was associated with a significant decrease in FEV 1 , FEV 1 /FVC and FEF25/75% of predicted in COPD (p < 0.05). As regards SP-B genotypes, resistant smokers had a significantly higher homozygous T genotype frequency compared to COPD and non smokers (p = 0.005). Interleukin 13 genotypes showed no significant difference between different groups. There was a significant decrease in FEF25/75% of predicted in T allele carriers in COPD patients (p = 0.001). Conclusions: The COPD is a disease caused by the interaction of combined genes and environmental influences, in the presence of smoking and latent adenovirus C infection, TNF-α -308A, SPB +1580 T and IL-13 -1055 T polymorphisms predispose to the development of COPD. Abstract Within the last 15 years, two related coronaviruses (Severe Acute Respiratory Syndrome [SARS]-CoV and Middle East Respiratory Syndrome [MERS]-CoV) expanded their host range to include humans, with increased virulence in their new host. Coronaviruses were recently found to have little intrinsic disorder compared with many other virus families. Because intrinsically disordered regions have been proposed to be important for rewiring interactions between virus and host, we investigated the conservation of intrinsic disorder and secondary structure in coronaviruses in an evolutionary context. We found that regions of intrinsic disorder are rarely conserved among different coronavirus protein families, with the primary exception of the nucleocapsid. Also, secondary structure predictions are only conserved across 50-80% of sites for most protein families, with the implication that 20-50% of sites do not have conserved secondary structure prediction. Furthermore, nonconserved structure sites are significantly less constrained in sequence divergence than either sites conserved in the secondary structure or sites conserved in loop. Avoiding regions symptomatic of conformational flexibility such as disordered sites and sites with nonconserved secondary structure to identify potential broad-specificity antiviral targets, only one sequence motif (five residues or longer) remains from the >10,000 starting sites across all coronaviruses in this study. The identified sequence motif is found within the nonstructural protein (NSP) 12 and constitutes an antiviral target potentially effective against the present day and future coronaviruses. On shorter evolutionary timescales, the SARS and MERS clades have more sequence motifs fulfilling the criteria applied. Interestingly, many motifs map to NSP12 making this a prime target for coronavirus antivirals. Abstract The Virus Pathogen Database and Analysis Resource (ViPR, www.ViPRbrc.org) is an integrated repository of data and analysis tools for multiple virus families, supported by the National Institute of Allergy and Infectious Diseases (NIAID) Bioinformatics Resource Centers (BRC) program. ViPR contains information for human pathogenic viruses belonging to the Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Flaviviridae, Filoviridae, Hepeviridae, Herpesviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae, Rhabdoviridae and Togaviridae families, with plans to support additional virus families in the future. ViPR captures various types of information, including sequence records, gene and protein annotations, 3D protein structures, immune epitope locations, clinical and surveillance metadata and novel data derived from comparative genomics analysis. Analytical and visualization tools for metadatadriven statistical sequence analysis, multiple sequence alignment, phylogenetic tree construction, BLAST comparison and sequence variation determination are also provided. Data filtering and analysis workflows can be combined and the results saved in personal 'Workbenches' for future use. ViPR tools and data are available without charge as a service to the virology research community to help facilitate the development of diagnostics, prophylactics and therapeutics for priority pathogens and other viruses. Abstract We aimed to compare the therapeutic efficacy of prolonged macrolide (PMC), corticosteroids (CST), doxycycline (DXC), and levofloxacin (LFX) against macrolideunresponsive Mycoplasma pneumoniae (MP) pneumonia in children and to evaluate the safety of the secondary treatment agents. Methods: We retrospectively analyzed the data of patients with MP pneumonia hospitalized between January 2015 and April 2017. Macrolide-unresponsiveness was clinically defined with a persistent fever of ≥ 38.0°C at ≥ 72 hours after macrolide treatment. The cases were divided into four groups: PMC, CST, DXC, and LFX. We compared the time to defervescence (TTD) after secondary treatment and the TTD after initial macrolide treatment in each group with adjustment using propensity score-matching analysis. Results: Among 1,165 cases of MP pneumonia, 190 (16.3%) were unresponsive to macrolides. The proportion of patients who achieved defervescence within 48 hours in CST, DXC, and LFX groups were 96.9% (31/33), 85.7% (12/14), and 83.3% (5/6), respectively. The TTD after initial macrolide treatment did not differ between PMC and CST groups (5.1 vs. 4.2 days, P = 0.085), PMC and DXC groups (4.9 vs. 5.7 days, P = 0.453), and PMC and LFX groups (4.4 vs. 5.0 days, P = 0.283). No side effects were observed in the CST, DXC, and LFX groups. Conclusion: The change to secondary treatment did not show better efficacy compared to PMC in children with macrolide-unresponsive MP pneumonia. Further studies are needed to guide appropriate treatment in children with MP pneumonia. Abstract The converging epidemics of tuberculosis (TB) and end-stage renal disease (ESRD) have generated a significant public health burden, however, previous studies have been limited to a small number of patients. This nationwide cohort study aimed to assess the rate of developing active TB among patients receiving dialysis for ESRD. Methods: The Korean national health insurance database was used to identify patients receiving dialysis for new-onset ESRD during 2004-2013, who were propensity score matched to an equivalent number of non-dialysis subjects from the general population. The incidences of active TB in the ESRD and control cohorts were calculated for 2004-2013, and multivariable Cox proportional hazards model was used to evaluate the ESRD-related risk of active TB. Results: During 2004-2013, 59,584 patients received dialysis for newly diagnosed ESRD. In the dialysis and control cohorts, 457 (0.8%) and 125 (0.2%) cases of active TB were detected, respectively. Patients with ESRD were associated with a significantly higher risk of active TB compared to the controls (incidence rate ratio, 4.80). The ESRD cohort had an independently elevated risk of active TB (adjusted hazard ratio, 4.39; 95% confidence interval, 3.60-5.37). Conclusion: We found that patients receiving dialysis for ESRD had an elevated risk of active TB. These results highlight the need for detailed and well-organised guidelines for active TB screening among patients with ESRD. Abstract Hematopoietic stem cell transplantation (HSCT) is a treatment for hematologic malignancies, immune deficiencies, or genetic diseases, ect. Recently, the number of HSCTs performed in Korea has increased and the outcomes have improved. However, infectious complications account for most of the morbidity and mortality after HSCT. Post-HSCT infectious complications are usually classified according to the time after HSCT: pre-engraftment, immediate post-engraftment, and late post-engraftment period. In addition, the types and risk factors of infectious complications differ according to the stem cell source, donor type, conditioning intensity, region, prophylaxis strategy, and comorbidities, such as graft-versushost disease and invasive fungal infection. In this review, we summarize infectious complications after HSCT, focusing on the Korean perspectives. Abstract To investigate the utility of cerebrospinal fluid (CSF) anti-feline coronavirus (FCoV) antibody test for diagnosis of feline infectious peritonitis (FIP), the antibody titers were tested in CSF and sera from 271 FIP-suspected neurological cats. CSF antibody was detected in 28 cats, which were divided into 2 groups; 15 with CSF titer of 1:80 or lower and 13 with CSF titer of 1:640 or higher. In the latter group, reciprocal serum titer/reciprocal CSF titer was 8 or lower, which is extremely lower than normal range (256-2048), and FCoV RNA was detected in all of 11 CSF samples assayed by RT-PCR. Our findings indicate that CSF titer of 1:640 or higher may be served as a candidate for the index for diagnosing FIP. Abstract OBJECTIVES: To determine the viruses and risk factors associated with hospital and intensive care unit (ICU) admissions in infants with acute bronchiolitis.INTRODUCTION: Bronchiolitis is a major cause of morbidity in infants. Widespread use of molecular-based methods has yielded new insights about its etiology, but the impact of viral etiologies on early outcomes is still unclear.METHODS: Seventy-seven infants with bronchiolitis who were under two years of age and visited an emergency unit were included. Using molecular-based methods, samples were tested for 12 different respiratory viruses. Logistic regression models were used to identify clinical and virological variables associated with the main endpoints: hospital admission and ICU admission.We identified at least one virus in 93.5% of patients, and coinfections were found in nearly 40% of patients. RSV was the most common pathogen (63.6%), followed by rhinovirus (39%). Identification of RSV was only associated with an increased risk of hospital admission in the univariate model. Younger age and enterovirus infection were associated with an increased risk of hospital admission, while atopy of a first-degree relative showed a protective effect. Prematurity was associated with an increased risk of admission to the ICU. Coinfections were not associated with worse outcomes.CONCLUSIONS: Molecular-based methods resulted in high rates of viral identification but did not change the significant role of RSV in acute bronchiolitis. Younger age and enterovirus infection were risk factors for hospital admission, while prematurity appeared to be a significant risk factor for admission to the ICU in acute viral bronchiolitis. Abstract A novel bat-origin coronavirus emerged in Wuhan, China in December 2019 and continues to spread across China and the world. At the time of writing, a massive global response has been implemented to control the disease as it spreads from person to person. Yet the high-risk human-wildlife interactions and interfaces that led to the emergence of SARS-CoV and of 2019-nCoV continue to exist in emerging disease hotspots globally. To prevent the next epidemic and pandemic related to these interfaces, we call for research and investment in three areas: 1) surveillance among wildlife to identify the high-risk pathogens they carry; 2) surveillance among people who have contact with wildlife to identify early spillover events; and 3) improvement of market biosecurity regarding the wildlife trade. As the emergence of a novel virus anywhere can impact the furthest reaches of our connected world, international collaboration among scientists is essential to address these risks and prevent the next pandemic. Abstract Along with tRNAs, enzymes that modify anticodon bases are a key aspect of translation across the tree of life. tRNA modifications extend wobble pairing, allowing specific ("target") tRNAs to recognize multiple codons and cover for other ("nontarget") tRNAs, often improving translation efficiency and accuracy. However, the detailed evolutionary history and impact of tRNA modifying enzymes has not been analyzed. Using ancestral reconstruction of five tRNA modifications across 1093 bacteria, we show that most modifications were ancestral to eubacteria, but were repeatedly lost in many lineages. Most modification losses coincided with evolutionary shifts in nontarget tRNAs, often driven by increased bias in genomic GC and associated codon use, or by genome reduction. In turn, the loss of tRNA modifications stabilized otherwise highly dynamic tRNA gene repertoires. Our work thus traces the complex history of bacterial tRNA modifications, providing the first clear evidence for their role in the evolution of bacterial translation. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 575-583 h t t p : / / w w w . b j m i c r o b i o l . c o m . b r / Abstract This study aimed to describe real-life microbiological testing of adults hospitalised with community-acquired pneumonia (CAP) and to assess concordance with the Abstract The expression of any gene must be precisely controlled for appropriate function. This expression can be controlled at various levels. This includes epigenetic regulation through DNA methylation or histone modifications. At the posttranscriptional level, regulation can be via alternative splicing or controlling messenger RNA (mRNA) stability. RNA cleavage is one way to control mRNA stability. For example, microRNA (miRNA)-induced mRNA cleavage has long been recognised in plants. RNA cleavage also appears to be widespread in other kingdoms of life, and it is now clear that mRNA cleavage plays critical functions in animals. Although miRNA-induced mRNA cleavage can occur in animals, it is not a widespread mechanism. Instead, mRNA cleavage can be induced by a range of other mechanisms, including by endogenous short inhibitory RNAs (endo-siRNAs), as well as the Ribonuclease III (RNase III) enzymes Drosha and Dicer. In addition, RNA cleavage induced by endo-siRNAs and PIWI-interacting RNAs (piRNAs) is important for genome defence against transposons. Moreover, several RNase has been identified as important antiviral mediators. In this review, we will discuss these various RNA endonucleolytic cleavage mechanisms utilised by animals to regulate the expression of genes and as a defence against retrotransposons and viral infection. Abstract The El glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex . In animal cells expressing the protein from cDNA, the El protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990 . Proc. Natl. Acad. Sci. USA. 87:6944-6948) . We show that the first of the three membrane-spanning domains of the El protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alphasubunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when 19 Abstract Health functional food is referred to a food prepared or processed from specific components or ingredients for functionality beneficial to the body through extraction, concentration, purification, blending and other methods. The demand for health functional foods is steadily increasing, and red ginseng is the most demanded food among women in the 50s, followed by multivitamin, omega-3, glucosamine and aloe. To date, there is insufficient evidence on the effect of red ginseng on exercise capacity, somatic symptom and cognitive performance in healthy individuals. Moreover, evidence is insufficient that a nutritional dose of vitamin or mineral reduces the incidence of cardiovascular disease and cancer, or mortality rate. A steady intake of oily fish is recommended to prevent the incidence of cardiovascular disease for postmenopausal women. Consumption of omega-3 fatty acids is expected to prevent cardiovascular disease in postmenopausal women with almost no intake of oily fish and those not taking statins. It still remains controversial whether glucosamine is effective in the treatment of osteoarthritis. Hence, physicians should fully inform patients with all controversial information about the effectiveness of glucosamine when prescribing glucosamine for patients with osteoarthritis. (J Menopausal Med 2015;21:12-18) Abstract An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-qTOF-MS/MS) method was developed and validated for the simultaneous determination of glycyrrhizin and glycyrrhetic acid. These analytes were separated on a reverse phase C18 column using a mobile phase of acetonitrile:2% acetic acid in water (75:25, v/v) with a flow rate of 200 mL/min. The qTOF-MS was operated under multiple reaction monitoring (MRM) mode using the electrospray ionization (ESI) technique with positive ion polarity. A comparison of three different extraction techniques i.e. accelerated solvent extraction (ASE), extraction under ultrasonic waves (USW) and the classical extraction by percolation (CE) method was done and quantification of these extracts was also carried out by the proposed method. Abstract Background: Development an effective vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) is urgent and limited information is available on vaccination in nonhuman primate (NHP) model. We herein report of evaluating a recombinant receptor-binding domain (rRBD) protein vaccine in a rhesus macaque model. Methods: Nine monkeys were randomly assigned to high-dose, low-dose and mock groups,which were immunized with different doses of rRBD plus alum adjuvant or adjuvant alone at different time points (0, 8, 25 weeks). Immunological analysis was conducted after each immunisation. Monkeys were challenged with MERS-CoV at 14 days after the final immunisation followed by observation for clinical signs and chest X-rays. Nasal, oropharyngeal and rectal swabs were also collected for analyses. Monkeys were euthanized 3 days after challenge and multiple specimens from tissues were collected for pathological, virological and immunological tests. Conclusion: Robust and sustained immunological responses (including neutralisation antibody) were elicited by the rRBD vaccination. Besides, rRBD vaccination alleviated pneumonia with evidence of reduced tissue impairment and clinical manifestation in monkeys. Furthermore, the rRBD vaccine decreased viral load of lung, trachea and oropharyngeal swabs of monkeys. These data in NHP paves a way for further development of an effective human vaccine against MERS-CoV infection. Abstract In the era of the "sickest first" policy, patients with very high model for end-stage liver disease (MELD) scores have been increasingly admitted to the intensive care unit with the expectation that they will receive a liver transplant (LT) in the absence of improvement on supportive therapies. Such patients are often admitted in a context of acute-on-chronic liver failure with extrahepatic failures. Sequential assessment of scores or classification based on organ failures within the first days after admission help to stratify the risk of mortality in this population. Although the prognosis of severely ill cirrhotic patients has recently improved, transplant-free mortality remains high. LT is still the only curative treatment in this population. Yet, the increased relative scarcity of graft resource must be considered alongside the increased risk of losing a graft in the initial postoperative period when performing LT in "too sick to transplant" patients. Variables associated with poor immediate post-LT outcomes have been identified in large studies. Despite this, the performance of scores based on these variables is still insufficient. Consideration of a patient's comorbidities and frailty is an appealing predictive approach in this population that has proven of great value in many other diseases. So far, local expertise remains the last safeguard to LT. Using this expertise, data are accumulating on favourable post-LT outcomes in very high MELD populations, particularly when LT is performed in a situation of stabilization/improvement of organ failures in selected candidates. The absence of "definitive" contraindications and the control of "dynamic" contraindications allow a "transplantation window" to be defined. This window must be identified swiftly after admission given the poor short-term survival of patients with very high MELD scores. In the absence of any prospect of LT, withdrawal of care could be discussed to ensure respect of patient life, dignity and wishes. Abstract Case summary Herein a drug-resistant IgG-lambda-type multiple myeloma associated with probable phaeochromocytoma in a cat is described. A 12-year-old cat presented with weakness, weight loss, progressive blindness and open-mouth breathing, in addition to polyuria and polydipsia of 2 months' duration. Abdominal ultrasonography revealed a left adrenal mass. Phaeochromocytoma was suspected on the basis of cytology and was associated with systemic hypertension. Biochemistry showed hyperproteinaemia. Serum protein electrophoresis revealed a narrow spike in the gamma region, identified as IgG lambda type at immunoelectrophoresis. Bone marrow cytology revealed an infiltrate with numerous mature plasma cells. The cat was resistant to two different drugs for multiple myeloma and was euthanased 6 months later because of anorexia and persistent poor general condition. Relevance and novel information This is the first clinical description of multiple myeloma associated with a suspected phaeochromocytoma in a cat. Abstract Thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. However, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. Here, we propose five "standard" categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. We also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit tradeoffs associated with different levels of finishing. Our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques. Abstract The ability of the infectious bronchitis H120 (a Massachusetts strain) and CR88 (a 793B strain) live attenuated vaccine viruses to protect from two Middle East infectious bronchitis virus isolates, IS/885/00-like (IS/885) and IS/1494/06-like (IS/1494) in broiler chicks was investigated. Day-old chicks were separated into three groups, (I) vaccinated with H120 at day-old followed by CR88 at 14 days-old, (II) vaccinated with H120 and CR88 simultaneously at day-old and again with CR88 at 14 days-old, (III) control unvaccinated. At 30 days-old, each of the groups was challenged with virulent IS/885 or IS/1494. Protection was evaluated based on the clinical signs, tracheal and kidney gross lesions and tracheal ciliostasis. Results showed that administering combined live H120 and CR88 vaccines simultaneously at day-old followed by CR88 vaccine at 14 days-old gave more than 80 per cent tracheal ciliary protection from both of the Middle East isolates. In addition, this programme conferred 100 per cent protection from clinical signs and tracheal or kidney lesions. The other vaccination programme, H120 at day-old followed by CR88 at 14 days-old, the tracheal ciliary protection conferred were 60 per cent and 80 per cent from IS/885/00-like and IS/1494/06like, respectively. Awad F, et al. Vet Rec Open 2015;2:e000111. Abstract Initiation of translation of the full-length messenger RNA of HIV-1, which generates the viral structural proteins and enzymes, is cap-dependent but can also use an internal ribosome entry site (IRES) located in the 5 0 untranslated region. Our aim was to define, through a mutational analysis, regions of HIV-1 IRES that are important for its activity. A dualluciferase reporter construct where the Renilla luciferase (Rluc) translation is cap-dependent while the firefly luciferase (Fluc) translation depends on HIV-1 IRES was used. The Fluc/Rluc ratio was measured in lysates of Jurkat T cells transfected with the dual-luciferase plasmid bearing either the wild-type or a mutated IRES. Deletions or mutations in three regions decreased the IRES activity but deletion or mutations of a stem-loop preceding the primer binding site increased the IRES activity. The wild-type IRES activity, but not that of an IRES with a mutated stem-loop, was increased when cells were treated with agents that induce oxidative stress. Such stress is known to be caused by HIV-1 infection and we propose that this stem-loop is involved in a switch that stimulates the IRES activity in cells infected with HIV-1, supporting the suggestion that the IRES activity is up-regulated in the course of HIV-1 replication cycle. Abstract Background/purpose: The licensed dentists in Taiwan should join the postgraduate year training program for dentists (PGYD) since 2010. This study aimed to analyze the project types and the geographical distribution of the PGYD training institutions in Taiwan from 2010 to 2018. Materials and methods: From 2010 to 2018, 735 hospitals and clinics participated in four types of PGYD project including hospital as the single training institution (project A), clinic as the single training institution (project B), hospital as the main training institution in the joint training group (project C), and clinic as the main training institution in the joint training group (project D). The project types and the geographical distribution of the training institutions were analyzed. Abstract Spatio-temporal aspects in the propagation of infectious pathogens of humans are reviewed. Mathematical modelling of these issues using metapopulation models is presented. Abstract H igh-mobilit y-g roup -box ch romosoma l protein 1 (HMGB1) is a ubiquitous and abundant nuclear protein in eukaryotic cells. Nuclear HMGB1 serves an important role in maintaining nuclear stability under stress. However, extracellular HMGB1 exerts actions, which are distinctly different compared with these intracellular functions. HMGB1, when released extracellularly, is a potent innate signal, which initiates host defense mechanisms or tissue regeneration. HMGB1 has two DNA-binding domains: HMG A box and B box. The HMGB1 A box exhibits an antagonistic, anti-inflammatory effect, and is a potential therapeutic target, however, the large-scale expression and purification of the HMGB1 A box with high efficiency remains to be reported. In the present study, a SUMO-fusion expression system was used to express and purify high levels of functional HMGB1 A box to meet the requirements of therapeutic protein production. Abstract Over the years, a vast array of information concerning the interactions of viruses with their hosts has been collected. However, recent advances in proteomics and other system biology techniques suggest these interactions are far more complex than anticipated. One particularly interesting and novel aspect is the analysis of cellular proteins incorporated into mature virions. Though sometimes considered purification contaminants in the past, their repeated detection by different laboratories suggests that a number of these proteins are bona fide viral components, some of which likely contribute to the viral life cycles. The present mini review focuses on cellular proteins detected in herpesviruses. It highlights the common cellular functions of these proteins, their potential implications for host-pathogen interactions, discusses technical limitations, the need for complementing methods and probes potential future research avenues. Abstract Word count: Main Text word count: 3393;We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service or company that could be construed as influencing the position presented in our study. Our study sponsor: Professor Yanhua Hao(corresponding) Organized surveyors to conduct field research for getting the first-hand survey data nonprofitable, then her graduate students to input these data into computer software Epidata, analyzing the results of this research only for academic research, finally, we decide to submit the report for publication.Objective This study aimed to assess household preparedness for emergency events and its determinants in China. Design: A cross-sectional questionnaire survey was conducted on 3,541 households in China in 2015. Participants: Households were selected using a stratified cluster sampling strategy, representing central, eastern, western and southern regions of China. The designed questionnaires were administered through face-to-face interviews. Outcome Measures: Household emergency preparedness was measured with 14 indicators, tapping into the supply of nine emergency necessities (food and water, extra batteries,battery-powered radio, battery-operated flashlight, first aid kit, gas mask, fire extinguisher, escape ropes, whistle), coverage of accident insurance, knowledge of local emergency response systems (emergency numbers, exit routes and shelters), and availability of a household evacuation plan. If an individual acted on nine of the 14 indicators, they were deemed well-prepared. Logistic regression models were established to identify predictors of well-preparedness based on 3,541 returned questionnaires containing no missing values. Results: Only 9.9% of households were well-prepared for emergencies: 53.6% did not know what to do and 31.6% did not want to think about it. A higher level of preparedness was found in the respondents who participated in emergency training activities (AOR=2.458), had better emergency knowledge (AOR=2.278), reported less fate-submissiveness (AOR=1.622) and more self-reliance (AOR=1.376), had a higher income (AOR=1.400), and held more positive attitudes toward preparedness (AOR=1.295).Household preparedness for emergency events is poor in China. lack of motivation, negative attitude to preparedness and knowledge shortfall are major but remediable barriers for household preparedness. Abstract Zoonoses represent a public health risk recently pointed out by the spreading of previously unknown human infectious diseases emerging from animal reservoirs such as severe acute respiratory syndrome and avian influenza caused by H5N1-virus. These outbreaks have shown that animal breeding activities can pose a significant public health risk. Until now, the risk of zoonoses has probably been underestimated, particularly in occupational settings. The emergence or re-emergence of bacterial (Mycobacterium bovis and Brucella spp) or viral (hepatitis E virus) infections shows that zoonoses should be considered as emerging risks in agricultural and animal breeding and should be addressed by specific preventive interventions. Close cooperation and interaction between veterinarians, occupational health physicians and public health operators is necessary, for a worldwide strategy to ex pand interdisciplinary collaborations and communications in all aspects of health care for humans, animals and the environment. This is what the One Health Approach was intended to be. Abstract Conidiobolomycosis is a rare fungal disease of both humans and animals, occurring mainly in tropical and subtropical climates. We describe a disseminated fungal infection in a young, apparently immunocompetent dog who initially presented for antibiotic resistant pneumonia. Histopathology and mycology identified a Conidiobolus sp., further confirmed as Conidiobolus incongruus through DNA sequencing of D1/D2 regions. This is the first report of this species causing disease in dogs and the fifth reported infection in animals. Abstract Globally, there are a number of emerging pathogens. For most, there are no licensed vaccines available for human use, although there is ongoing research and development. However, given the extensive and increasing list of emerging pathogens and the investment required to bring vaccines into clinical use, the task is huge. Overlaid on this task is the risk of anti-microbial resistance (AMR) acquisition by micro-organisms which can endow a relatively harmless organism with pathogenic potential. Furthermore, climate change also introduces a challenge by causing some of the insect vectors and environmental conditions prevalent in tropical regions to begin to spread out from these traditional areas, thus increasing the risk of migration of zoonotic disease.Vaccination provides a defence against these emerging pathogens. However, vaccines for pathogens which cause severe, but occasional, disease outbreaks in endemic pockets have suffered from a lack of commercial incentive for development to a clinical standard, encompassing Phase III clinical trials for efficacy. An alternative is to develop such vaccines to request US Emergency Use Authorization (EUA), or equivalent status in the United States, Canada and the European Union, making use of a considerable number of regulatory mechanisms that are available prior to licensing. This review covers the status of vaccine development for some of the emerging pathogens, the hurdles that need to be overcome to achieve EUA or an equivalent regional or national status and how these considerations may impact vaccine development for the future, such that a more comprehensive stockpile of promising vaccines can be achieved. Abstract Overlapping genes in viruses maximize the coding capacity of their genomes and allow the generation of new genes without major increases in genome size. Despite their importance, the evolution and function of overlapping genes are often not well understood, in part due to difficulties in their detection. In addition, most bioinformatic approaches for the detection of overlapping genes require the comparison of multiple genome sequences that may not be available in metagenomic surveys of virus biodiversity. We introduce a simple new method for identifying candidate functional overlapping genes using single virus genome sequences. Our method uses randomization tests to estimate the expected length of open reading frames and then identifies overlapping open reading frames that significantly exceed this length and are thus predicted to be functional. We applied this method to 2548 reference RNA virus genomes and find that it has both high sensitivity and low false discovery for genes that overlap by at least 50 nucleotides. Notably, this analysis provided evidence for 29 previously undiscovered functional overlapping genes, some of which are coded in the antisense direction suggesting there are limitations in our current understanding of RNA virus replication. Abstract It is well known that the local renin-angiotensin system (RAS) is activated in the diabetic state, which results in an increase in the level of oxidative stress injury to pancreatic β cells. The angiotensin-converting enzyme 2 (ACE2)/angiotensin (1-7) [Ang (1-7)]/Mas axis is a negative regulator of the classical renin-angiotensin system. In order to investigate the antioxidant effect of Ang (1-7) on pancreatic β cells, INS-1 cells were cultured and oxidative stress was induced by treatment with H 2 O 2 . Glucose-stimulated insulin secretion (GSIS), the generation of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and glucose-stimulated calcium (GSCa) responses in β cells were determined following treatment with Ang (1-7) . It was observed that H 2 O 2 significantly impaired the insulin secreting function, increased the production of ROS, and also decreased the levels of GSCa and MMP. Pre-treatment with Ang (1-7) alleviated these effects and treatment with A779 [antagonist of Ang (1-7)] prevented the effects of Ang (1-7). Based on these findings, it was concluded that Ang (1-7) can protect pancreatic β cells from oxidative injury and such protection can be blocked by its antagonist A779. Abstract Cell culture and mTOR inhibition. HeLa (ATCC: CCL-2), HEK293T (ATCC: CRL-326), primary human foreskin fibroblasts (ATCC: SCRC-1041), TZM-bl (NIH AIDS Reagent Resource: 8129) and murine embryonic fibroblasts (a gift from M. Diamond) and any derivatives produced in this study were cultivated at 37°C and 5% CO2 in DMEM (Gibco) complemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Gibco) and regularly passaged with the aid of Trypsin-EDTA (0.05%). Rapamycin, Torin 1, bafilomycin A1, MG-132, cycloheximide (Sigma) and EGA (Calbiochem) were reconstituted in DMSO and stored in aliquots at -20°C. Earle's Balanced Salt Solution (EBSS) without calcium, magnesium, or phenol red was purchased from Gibco.Immunofluorescence confocal microscopy. Cells were seeded (15,000/well) in Lab-Tek II chamber slides (Thermo Fisher) overnight and treated with DMSO, rapamycin (20 uM), or rapamycin and bafilomycin A1 (1uM) for four hours followed by fixation/permeabilization with Cytofix/Cytoperm (BD) for 20 minutes. Primary antibodies were diluted in Odyssey blocking buffer in PBS (Li-Cor) and incubated with cells for 30 minutes at room temperature. Cells were washed and incubated with Alexa Fluor-labeled secondary antibodies for 30 minutes at room temperature. Abstract Human embryo lung cellular protein interacting with severe acute respiratory syndrome-coronavirus nonstructural protein-10 (HEPIS) is expressed at varying levels in multiple organs and breast cancer cell lines. However, its expression and function in breast cancer cells has yet to be studied. Therefore, RNA in situ hybridization was used to detect the expression of HEPIS in breast cancer and cancer-adjacent normal breast tissue. HEPIS was expressed at lower levels in breast cancer compared with that in adjacent normal tissue. Subcellular localization of HEPIS was mainly found in the cytoplasm of HeLa cells. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine cell proliferation assays were used to investigate the role of HEPIS in cancer cell proliferation. Ectopic expression of HEPIS in MCF-7 cells was found to significantly inhibit cell proliferation. In contrast, knockdown of HEPIS by RNA interference exhibited the opposite effect. Furthermore, a dual-luciferase reporter assay was performed and HEPIS overexpression specifically inhibited the activity of the NF-κB reporter gene. Results of the gene chip assay revealed that 2,231 genes were differentially expressed in HEPIS-overexpressing cells. Results of the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that these genes were enriched in the 'mitogen-activated protein kinase signaling pathway', 'JAK-STAT signaling pathway' and 'focal adhesion'. Reverse transcription-quantitative PCR was used to confirm the expression levels of the differentially expressed genes interleukin 2 receptor subunit α (IL2RA), interferon α and β receptor subunit 2 (IFNAR2) and IFα8 (IFNA8). In conclusion, the results of the present study indicated that HEPIS may function as a potential repressor of breast cancer. Abstract # Contributed equally J o u r n a l P r e -p r o o f Highlights We reviewed discovery and development process of broad-spectrum antiviral agents. We summarized the information on 119 safe-in-man agents in freely accessible database. Further studies will increase the number of broad-spectrum antivirals, expand spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections.: Viral diseases are one of the leading causes of morbidity and mortality in the world. Virus-specific vaccines and antiviral drugs are the most powerful tools to combat viral diseases. However, broad-spectrum antiviral agents (BSAAs, i.e. compounds targeting viruses belonging to two or more viral families) could provide additional protection of general population from emerging and reemerging viral diseases reinforcing the arsenal of available antiviral options. Here, we reviewed discovery and development of BSAAs and summarized the information on 119 safe-in-man agents in freely accessible database (https://drugvirus.info/). Future and ongoing pre-clinical and clinical studies will increase the number of BSAAs, expand spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections as well as co-infections. J o u r n a l P r e -p r o o f 2015). Antiviral drugs and vaccines are used to fight viral infections in human (De Clercq and Li, 2016; Marston et al., 2014). Previously, there has been a focus on "one drug, one virus" dogma, which relied on targeting virus-specific factors. A counterpoint to this is "one drug, multiple viruses" paradigm, which came with the discovery of broad-spectrum antiviral agents (BSAAs), small-molecules that inhibit a wide range of human viruses (Bekerman and Einav, 2015; de Clercq and Montgomery, 1983; Debing et al., 2015; Ianevski et al., 2019; Rada and Dragun, 1977; Sidwell et al., 1972). This paradigm was based on the observation that different viruses utilize similar pathways and host factors to replicate inside a cell (Bosl et al., 2019). Although the concept of BSAAs has been around for almost 50 years, the field received a new impetus with recent outbreaks of Ebola, Zika, Dengue, influenza and other viral infections, the discovery of novel host-directed agents as well as development of drug repositioning methodology. Drug repurposing, also called repositioning, redirecting, reprofiling, is a strategy for generating additional value from an existing drug by targeting disease other than that for which it was originally intended (Nishimura and Hara, 2018; Pushpakom et al., 2019). This has significant advantages over new drug discovery since chemical synthesis steps, manufacturing processes, reliable safety, and pharmacokinetic properties in pre-clinical (animal model) and early clinical developmental phases (phase 0, I and IIa) are already available (Figure 1). Therefore, repositioning of launched or even failed drugs to viral diseases provides unique translational opportunities, including a substantially higher probability of success J o u r n a l P r e -p r o o f De Clercq, E., Li, G., 2016. Approved Antiviral Drugs over the Past 50 Years. Clin Microbiol Rev 29, 695-747. de Clercq, E., Montgomery, J.A., 1983. Broad-spectrum antiviral activity of the carbocyclic analog of 3-deazaadenosine. Antiviral Res 3, 17-24. de Graaf, M., Herfst, S., Schrauwen, E.J., van den Hoogen, B.G., Osterhaus, A.D., Fouchier, R.A., 2007. An improved plaque reduction virus neutralization assay for human metapneumovirus. Abstract Case-based learning and problem-based learning have demonstrated great promise in reforming science education. Yet an instructor, in newly considering this suite of interrelated pedagogical strategies, faces a number of important instructional choices. Different features and their related values and learning outcomes are profiled here, including: the level of student autonomy; instructional focus on content, skills development, or nature-of-science understanding; the role of history, or known outcomes; scope, clarity, and authenticity of problems provided to students; extent of collaboration; complexity, in terms of number of interpretive perspectives; and, perhaps most importantly, the role of applying versus generating knowledge. Abstract Infectious disease during an emergency condition can raise the death rate 60 times in comparison to other causes including trauma. An epidemic, or outbreak, can occur when several aspects of the agent (pathogen), population (hosts), and the environment create an ideal situation for spread. Overcrowding, poor regional design and hygiene due to poverty, dirty drinking water, rapid climate changes, and natural disasters, can lead to conditions that allow easier transmission of disease. Once it has been established that an emergency condition exists, there must be a prompt and thorough response for communicable disease control. A camp should be created, and the disease managed rapidly. The overall goals are rapid assessment, prevention, surveillance, outbreak control, and disease management.Other safeguards such as airborne droplet, regular droplet, and contact precautions are necessary to prevent spread of unique vectors. Airborne particles are small (less than or Turk J Emerg Med 2015;15(Suppl 1):20-26 Abstract A 12-year-old spayed female domestic shorthair cat was evaluated for a 3-week history of abdominal distension. Chyloabdomen secondary to pancreatic carcinoma was diagnosed. The cat was palliatively managed using rutin and a low-fat diet. The etiology, diagnosis and management of chyloabdomen are discussed. Abstract Emodin-induced hepatotoxicity in vivo and in vitro has been gaining increasing attention. However, the exact molecular pathways underlying these effects remain poorly clarified. The aim of the present study was to evaluate the cytotoxic effect of emodin on HepaRG cells and to define the underlying mechanism. The results demonstrated that emodin evidently inhibited HepaRG cell growth in a dose-and time-dependent manner by blocking cell cycle progression in the S and G2/M phase and by inducing apoptosis. Emodin treatment also resulted in generation of reactive oxygen species (ROS), which abrogated mitochondrial membrane potential (MMP). The above effects were all suppressed by antioxidants, such as N-acetylcysteine (NAC). Further studies by western blot analysis howed that emodin upregulated p53, p21, Bax, cyclin E, cleaved caspase-3, 8 and 9, and cleaved poly(ADP-ribose)polymerase (PARP). However, the protein expression of Bcl-2, cyclin A and CDK2 was downregulated. Taken together, our results suggest that emodin induces apoptosis via the mitochondrial apoptosis pathway through cell cycle arrest and ROS generation in HepaRG cells. Abstract The accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b, 9b and ORF14), predicted unknown proteins (PUPs) encoded by the genes, are considered to be unique to the severe acute respiratory syndrome coronavirus (SARS-CoV) genome. These proteins play important roles in various biological processes mediated by interactions with their partners. However, very little is known about the interactions among these accessory proteins. Here, a EYFP (enhanced yellow fluorescent protein) bimolecular fluorescence complementation (BiFC) assay was used to detect the interactions among accessory proteins. 33 out of 81 interactions were identified by BiFC, much more than that identified by the yeast two-hybrid (Y2H) system. This is the first report describing direct visualization of interactions among accessory proteins of SARS-CoV. These findings attest to the general applicability of the BiFC system for the verification of protein-protein interactions. Abstract Global climate change is expected to affect the frequency, intensity and duration of extreme water-related weather events such as excessive precipitation, floods, and drought. We conducted a systematic review to examine waterborne outbreaks following such events and explored their distribution between the different types of extreme water-related weather events. Four medical and meteorological databases (Medline, Embase, GeoRef, PubMed) and a global electronic reporting system (ProMED) were searched, from 1910 to 2010. Eighty-seven waterborne outbreaks involving extreme water-related weather events were identified and included, alongside 235 ProMED reports. Heavy rainfall and flooding were the most common events preceding outbreaks associated with extreme weather and were reported in 55 . 2 % and 52 . 9% of accounts, respectively. The most common pathogens reported in these outbreaks were Vibrio spp. (21 . 6%) and Leptospira spp. (12 . 7 %). Outbreaks following extreme water-related weather events were often the result of contamination of the drinking-water supply (53 . 7 %). Differences in reporting of outbreaks were seen between the scientific literature and ProMED. Extreme water-related weather events represent a risk to public health in both developed and developing countries, but impact will be disproportionate and likely to compound existing health disparities.The online version of this article is published within an Open Access environment subject to the conditions of the Creative Commons Attribution-NonCommercial-ShareAlike licence . The written permission of Cambridge University Press must be obtained for commercial re-use. Abstract Korean Red Ginseng (KRG) is a functional food and has been well known for keeping good health due to its anti-fatigue and immunomodulating activities. However, there is no data on Korean red ginseng for its preventive activity against acute respiratory illness (ARI). The study was conducted in a randomized, double-blinded, placebo-controlled trial in healthy volunteers (Clinical Trial Number: NCT01478009). Our primary efficacy end point was the number of ARI reported and secondary efficacy end point was severity of symptoms, number of symptoms, and duration of ARI. A total of 100 volunteers were enrolled in the study. Fewer subjects in the KRG group reported contracting at least 1 ARI than in the placebo group (12 [24.5%] vs 22 [44.9%], P = 0.034), the difference was statistically significant between the two groups. The symptom duration of the subjects who experienced the ARI, was similar between the two groups (KRG vs placebo; 5.2 ± 2.3 vs 6.3 ± 5.0, P = 0.475). The symptom scores were low tendency in KRG group (KRG vs placebo; 9.5 ± 4.5 vs 17.6 ± 23.1, P = 0.241). The study suggests that KRG may be effective in protecting subjects from contracting ARI, and may have the tendency to decrease the duration and scores of ARI symptoms. Abstract Bluetongue virus (BTV) encodes a single capping protein, VP4, which catalyzes all reactions required to generate cap1 structures on nascent viral transcripts. Further, structural analysis by X-ray crystallography indicated each catalytic reaction is arranged as a discrete domain, including a nucleoside-2 0 -O-methyltransferase (2 0 -O MTase). In this study, we have exploited the structural information to identify the residues that are important for the catalytic activity of 2 0 -O MTase of VP4 and their influence on BTV replication. The effect of these mutations on GMP binding, guanylyltransferase (GTase) and methylase activities were analysed by a series of in vitro biochemical assays using recombinant mutant proteins; subsequently their effects on virus replication were assessed by introducing the same mutations in replicating viral genome using a reverse genetics system. Our data showed that single substitution mutations in the catalytic tetrad K-D-K-E were sufficient to abolish 2 0 -O MTase activity in vitro and to completely abrogate BTV replication in cells; although these mutants retained the upstream GMP binding, GTase and guanine-N7-methyltransferase activities. Mutations of the surrounding substrate-binding pocket (predicted to recruit cap0) had variable effects on in vitro VP4 capping activity. Only triple but not single substitution mutations of these residues in genome resulted in reduced virus replication kinetics. This is the first report investigating the importance of 2 0 -O MTase function for any member of the Reoviridae and highlights the significance of K-D-K-E tetrad and surrounding residues for the efficiency of 2 0 -O MTase activity and in turn, for virus fitness. Abstract The recent Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak has originated from a failure in the national quarantine system in the Republic of Korea as most basic role of protecting the safety and lives of its citizens. Furthermore, a number of the Korean healthcare system's weaknesses seem to have been completely exposed. The MERS-CoV outbreak can be considered a typical public health crisis in that the public was not only greatly terrorized by the actual fear of the disease, but also experienced a great impact to their daily lives, all in a short period of time. Preparedness for and an appropriate response to a public health crisis require comprehensive systematic public healthcare measures to address risks comprehensively with an all-hazards approach. Consequently, discussion regarding establishment of post-MERS-CoV improvement measures must focus on the total reform of the national quarantine system and strengthening of the public health infrastructure. In addition, the Korea Centers for Disease Control and Prevention must implement specific strategies of action including taking on the role of "control tower" in a public health emergency, training of Field Epidemic Intelligence Service officers, establishment of collaborative governance between central and local governments for infection prevention and control, strengthening the roles and capabilities of community-based public hospitals, and development of nationwide crisis communication methods. Abstract Salmonella Typhimurium is one of the most important zoonotic pathogens worldwide and a major cause of economic losses in the pig production chain. The emergence of multi-drug resistant strains over the past years has led to considerations about an enhanced surveillance of bacterial food contamination. Currently, ELISA is the method of choice for high throughput identification of S. Typhimurium. The sensitivity and specificity of this assay might be improved by application of new diagnostic antibodies. We focused on plant-based expression of candidate diagnostic TM43-E10 antibodies discovered using as antigen the S. Typhimurium OmpD protein. The scFv-TM43-E10 and scFv-Fc-TM43-E10 antibody derivatives have been successfully produced in N. benthamiana using a deconstructed movementdeficient PVX vector supplemented with the gb silencing suppressor from Poa semilatent virus. The plant-made antibodies showed the same antigen-binding specificity as that of the microbial/mammalian cell-produced counterparts and could recognize the OmpD antigen in S. Typhimurium infected plant samples. Abstract Background: Feline infectious peritonitis (FIP) is the most common infectious central nervous system (CNS) disease in the cat and is invariably fatal. Improved means of antemortem diagnosis is required to facilitate clinical decision making. Information regarding the magnetic resonance imaging (MRI) findings of neurologic FIP currently is limited, resulting in the need for better descriptions to optimize its use as a diagnostic tool.Objective: To describe the clinicopathologic features and MRI findings in cases of confirmed neurologic FIP. Animals: Twenty-four client-owned cats with histopathologic confirmation of neurologic FIP. Methods: Archived records from 5 institutions were retrospectively reviewed to identify cases with confirmed neurologic FIP that had undergone antemortem MRI of the CNS. Signalment, clinicopathologic, MRI, and histopathologic findings were evaluated.Results: Three distinct clinical syndromes were identified: T3-L3 myelopathy (3), central vestibular syndrome (7), and multifocal CNS disease (14). Magnetic resonance imaging abnormalities were detected in all cases, including meningeal contrast enhancement (22), ependymal contrast enhancement (20), ventriculomegaly (20), syringomyelia (17), and foramen magnum herniation (14). Cerebrospinal fluid was analysed in 11 cases; all demonstrated a marked increase in total protein concentration and total nucleated cell count. All 24 cats were euthanized with a median survival time of 14 days (range, 2-115) from onset of clinical signs. Histopathologic analysis identified perivascular pyogranulomatous infiltrates, lymphoplasmacytic infiltrates, or both affecting the leptomeninges (16), choroid plexuses (16), and periventricular parenchyma (13).Conclusions and Clinical Importance: Magnetic resonance imaging is a sensitive means of detecting neurologic FIP, particularly in combination with a compatible signalment, clinical presentation, and CSF analysis. Abstract Because many diseases are multifactorial disorders, the scientific progress in genomics and genetics should be taken into consideration in public health research. In this context, genomic databases will constitute an important source of information. Consequently, it is important to identify and characterize the State's role and authority on matters related to public health, in order to verify whether it has access to such databases while engaging in public health genomic research. We first consider the evolution of the concept of public health, as well as its core functions, using a comparative approach (e.g. WHO, PAHO, CDC and the Canadian province of Quebec). Following an analysis of relevant Quebec legislation, the precautionary principle is examined as a possible avenue to justify State access to and use of genomic databases for research purposes. Finally, we consider the Influenza pandemic plans developed by WHO, Canada, and Quebec, as examples of key tools framing public health decision-making process. We observed that State powers in public health, are not, in Quebec, well adapted to the expansion of genomics research. We propose that the scope of the concept of research in public health should be clear and include the following characteristics: a commitment to the health and well-being of the population and to their determinants; the inclusion of both applied research and basic research; and, an appropriate model of governance (authorization, follow-up, consent, etc.). We also suggest that the strategic approach version of the precautionary principle could guide collective choices in these matters. Abstract Computational methods for determining the secondary structure of RNA sequences from given alignments are currently either based on thermodynamic folding, compensatory base pair substitutions or both. However, there is currently no approach that combines both sources of information in a single optimization problem. Here, we present a model that formally integrates both the energy-based and evolution-based approaches to predict the folding of multiple aligned RNA sequences. We have implemented an extended version of Pfold that identifies base pairs that have high probabilities of being conserved and of being energetically favorable. The consensus structure is predicted using a maximum expected accuracy scoring scheme to smoothen the effect of incorrectly predicted base pairs. Parameter tuning revealed that the probability of base pairing has a higher impact on the RNA structure prediction than the corresponding probability of being single stranded. Furthermore, we found that structurally conserved RNA motifs are mostly supported by folding energies. Other problems (e.g. RNA-folding kinetics) may also benefit from employing the principles of the model we introduce. Our implementation, PETfold, was tested on a set of 46 well-curated Rfam families and its performance compared favorably to that of Pfold and RNAalifold. Abstract Background: Influenza viruses are among the major causes of serious human respiratory tract infection worldwide. In line with the high disease burden attributable to influenza, these viruses play an important, but often neglected, role in travel medicine. Guidelines and recommendations regarding prevention and management of influenza in travellers are scarce. Of special interest for travel medicine are risk populations and also circumstances that facilitate influenza virus transmission and spread, like travel by airplane or cruise ship and mass gatherings. Methods: We conducted a PUBMED/MEDLINE search for a combination of the MeSH terms Influenza virus, travel, mass gathering, large scale events and cruise ship. In addition we gathered guidelines and recommendations from selected countries and regarding influenza prevention and management in travellers. By reviewing these search results in the light of published knowledge in the fields of influenza prevention and management, we present best practice advice for the prevention and management of influenza in travel medicine. Results: Seasonal influenza is among the most prevalent infectious diseases in travellers. Known host-associated risk factors include extremes of age and being immune-compromised, while the most relevant environmental factors are associated with holiday cruises and mass gatherings. Conclusions: Pre-travel advice should address influenza and its prevention for travellers, whenever appropriate on the basis of the epidemiological situation concerned. Preventative measures should be strongly recommended for travellers at high-risk for developing complications. In addition, seasonal influenza vaccination should be considered for any traveller wishing to reduce the risk of incapacitation, particularly cruise ship crew and passengers, as well as those participating in mass gatherings. Besides advice concerning preventive measures and vaccination, advice on the use of antivirals may be considered for some travellers. Abstract All positive-stranded RNA viruses with genomes >$7 kb encode helicases, which generally are poorly characterized. The core of the nidovirus superfamily 1 helicase (HEL1) is associated with a unique N-terminal zinc-binding domain (ZBD) that was previously implicated in helicase regulation, genome replication and subgenomic mRNA synthesis. The high-resolution structure of the arterivirus helicase (nsp10), alone and in complex with a polynucleotide substrate, now provides first insights into the structural basis for nidovirus helicase function. A previously uncharacterized domain 1B connects HEL1 domains 1A and 2A to a long linker of ZBD, which further consists of a novel RING-like module and treble-clef zinc finger, together coordinating three Zn atoms. On substrate binding, major conformational changes were evident outside the HEL1 domains, notably in domain 1B. Structural characterization, mutagenesis and biochemistry revealed that helicase activity depends on the extensive relay of interactions between the ZBD and HEL1 domains. The arterivirus helicase structurally resembles the cellular Upf1 helicase, suggesting that nidoviruses may also use their helicases for post-transcriptional quality control of their large RNA genomes. Abstract Graphical Abstract Highlights d Several unique conformational states of an elongating RdRp exist d Only one conformation incorporates nucleotide analogs with therapeutic potential d An analog thought to be a chain terminator actually promotes RdRp backtracking d Distinctive behavior of backtrack-inducing analog on virus variants in cell culture SUMMARYRNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotidesubstrate-binding and catalytic sites. Conventional, quantitative, kinetic analysis of antiviral ribonucleotides monitors one or a few incorporation events.Here, we use a high-throughput magnetic tweezers platform to monitor the elongation dynamics of a prototypical RdRp over thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors. We observe multiple RdRp-RNA elongation complexes; only a subset of which are competent for analog utilization. Incorporation of a pyrazine-carboxamide nucleotide analog, T-1106, leads to RdRp backtracking. This analysis reveals a mechanism of action for this antiviral ribonucleotide that is corroborated by cellular studies. We propose that induced backtracking represents a distinct mechanistic class of antiviral ribonucleotides. Abstract The foot and mouth disease virus (FMDV) is sensitive to acids and can be inactivated by exposure to low pH conditions. Spraying animals at risk of infection with suspensions of acid-forming microorganisms has been identified as a potential strategy for preventing FMD. Kombucha is one of the most strongly acid-forming symbiotic probiotics and could thus be an effective agent with which to implement this strategy. Moreover, certain Chinese herbal extracts are known to have broad-spectrum antiviral effects. Chinese herbal kombucha can be prepared by fermenting Chinese herbal extracts with a kombucha culture. Previous studies demonstrated that Chinese herbal kombucha prepared in this way efficiently inhibits FMDV replication in vitro. To assess the inhibitory effects of Chinese herbal kombucha against FMDV in vitro, swine challenged by intramuscular injection with 1000 SID 50 of swine FMDV serotype O strain O/China/99 after treatment with Chinese herbal kombucha were partially protected against infection, as demonstrated by a lack of clinical symptoms and qRT-PCR analysis. In a large scale field trial, spraying cattle in an FMD outbreak zone with kombucha protected against infection. Chinese herbal kombucha may be a useful probiotic agent for managing FMD outbreaks. Abstract Background: Viruses are obligate parasites that depend on the cellular machinery of the host to regenerate and manufacture their proteins. Most antiviral drugs on the market today target viral proteins. However, the more recent strategies involve targeting the host cell proteins or pathways that mediate viral replication. This new approach would be effective for most viruses while minimizing drug resistance and toxicity. Methods: Cytomegalovirus replication, latency, and immune response are mediated by the intermediate early protein 2, the main protein that determines the effectiveness of drugs in cytomegalovirus inhibition. This review explains how intermediate early protein 2 can modify the action of cyclosporin A, an immunosuppressive, and antiviral drug. It also links all the pathways mediated by cyclosporin A, cytomegalovirus replication, and its encoded proteins. Results: Intermediate early protein 2 can influence the cellular cyclophilin A pathway, affecting cyclosporin A as a mediator of viral replication or anti-cytomegalovirus drug. Conclusion: Cyclosporin A has a dual function in cytomegalovirus pathogenesis. It has the immunosuppressive effect that establishes virus replication through the inhibition of T-cell function. It also has an anti-cytomegalovirus effect mediated by intermediate early protein 2. Both of these functions involve cyclophilin A pathway. Abstract Background: Respiratory viruses are the leading cause of respiratory tract infections among children and are responsible for causing morbidity and mortality worldwide. This study was performed to detect viruses in children with respiratory infections and describe their epidemiology and clinical characteristics.Methods: In this descriptive cross sectional study, throat swabs and wash specimens from 202 children younger than six years of age with diagnosis of a respiratory tract infection from a total of 897 specimens were evaluated using multiplex PCR method.Results: Respiratory viruses were detected in 92 children: respiratory synsytial virus, 16.8%; influenza virus, 5.4%; parainfluenza virus, 8.4%; adenovirus, 14.4% and human metapneumo virus 0.49% with male predominance and higher distribution in children younger than 1 year of age with preference in the cold months of year. The clinical presentations of all detected viruses were almost similar.Conclusion: In the present study, nine different respiratory viruses were detected. RSV causes the great majority of respiratory virus infections in children. There was no significant difference in epidemiologic patterns of these viruses in comparison to other studies. Abstract Protein quality control (PQC) is required to ensure cellular health. PQC is recognized for targeting the destruction of defective polypeptides, whereas regulated protein degradation mechanisms modulate the concentration of specific proteins in concert with physiological demands. For example, ion channel levels are physiologically regulated within tight limits, but a system-wide approach to define which degradative systems are involved is lacking. We focus on the Kir2. Abstract Objective: Established preclinical disease models are essential for not only studying aetiology and/or pathophysiology of the relevant diseases but more importantly also for testing prevention and/or treatment concept(s). The present study proposed and established a detailed induction and assessment protocol for a unique and cost-effective preclinical steroid-associated osteonecrosis (SAON) in rats with pulsed injections of lipopolysaccharide (LPS) and methylprednisolone (MPS). Methods: Sixteen 24-week-old male SpragueeDawley rats were used to induce SAON by one intravenous injection of LPS (0.2 mg/kg) and three intraperitoneal injections of MPS (100 mg/kg) with a time interval of 24 hour, and then, MPS (40 mg/kg) was intraperitoneally injected three times a week from week 2 until sacrifice. Additional 12 rats were used as normal controls. Two and six weeks after induction, animals were scanned by metabolic dual energy Xray absorptiometry for evaluation of tissue composition; serum was collected for bone turnover markers, Microfil perfusion was performed for angiography, the liver was collected for histopathology and bilateral femora and bilateral tibiae were collected for histological examination. Results: Three rats died after LPS injection, i.e., with 15.8% (3/19) mortality. Histological evaluation showed 100% incidence of SAON at week 2. Dual energy X-ray absorptiometry showed significantly higher fat percent and lower lean mass in SAON group at week 6. Micro-computed tomography (Micro-CT) showed significant bone degradation at proximal tibia 6 weeks after SAON induction. Angiography illustrated significantly less blood vessels in the Journal of Orthopaedic Translation (2018) 13, 13e24 proximal tibia and significantly more leakage particles in the distal tibia 2 weeks after SAON induction. Serum amino-terminal propeptide of type I collagen and osteocalcin were significantly lower at both 2 and 6 weeks after SAON induction, and serum carboxy-terminal telopeptide was significantly lower at 6 weeks after SAON induction. Histomorphometry revealed significantly lower osteoblast surface and higher marrow fat fraction and oedema area in SAON group. Hepatic oedema appeared 2 weeks after SAON induction, and lipid accumulation appeared in the liver of SAON rats 6 weeks after SAON induction. Conclusion: The present study successfully induced SAON in rats with pulsed injection of LPS and MPS, which was well simulating the clinical feature and pathology. Apart from available large animal models, such as bipedal emus or quadrupedal rabbits, our current SAON small model in rats could be a cost-effective preclinical experimental model to study body metabolism, molecular mechanism of SAON and potential drugs developed for prevention or treatment of SAON. The translational potential of this article: The present study successfully induced SAON in a small animal model in rats with pulsed injection of LPS and MPS. The evaluation protocols with typical histopathologic ON features and advanced evaluation approaches to identify the metabolic disorders of SAON could be used in future rat SAON studies. The SAON rat model is a suitable and cost-effective animal model to study molecular mechanism of SAON and potential drugs developed for prevention and treatment of SAON. Abstract In this study the toxicity of antimalarial drug chloroquine (CQ) on certain enzymological (GOT, GPT and LDH) and histopathological alterations (Gill, liver and kidney) of a freshwater fish Cyprinus carpio was studied after acute (96 h) and sublethal (35 days) exposure. The median lethal concentration (96 h) of CQ was 31.62 mg/ml. During acute treatment (CQ at 31.62 mg/ml) the treated fish groups showed a significant increase in GOT and GPT activities in blood plasma; whereas LDH activity was decreased when compare to control groups. To analyse the effects of drug at the lowest concentration, the fish were exposed to 3.16 mg/ml (1/10th of 96 h LC50 value) for 96 h. In sublethal treatment (3.16 mg/ml) GOT activity increased up to 14th day and decreased during the rest of the exposure period (21, 28 and 35th day). A biphasic response in GPT activity was observed. LDH activity was found to be increased throughout the study period (35 days) compare to control groups. The alterations in enzyme activities in blood plasma were found to be significant at p < 0.05 (DMRT). Many histopathological changes in vital organs such as gill, liver and kidney of fish were observed in CQ treated group (acute and sub-lethal) compare to normal group. The alterations in the enzymological and histopathological study in the present investigation indicate that the drug CQ has toxic effects on non-target organisms. We conclude that the alterations in enzymological parameters and histopathological changes can be used as biomarker to assess the health of the aquatic organism/environment. Further data on molecular studies are needed to define the mode of action and toxicity of these emerging pollutants. Abstract Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions. Abstract Neurotropic coronavirus induces an acute encephalomyelitis accompanied by focal areas of demyelination distributed randomly along the spinal column. The initial areas of demyelination increase only slightly after the control of infection. These circumscribed focal lesions are characterized by axonal sparing, myelin ingestion by macrophage/microglia, and glial scars associated with hypertrophic astrocytes, which proliferate at the lesion border. Accelerated virus control in mice lacking the anti-inflammatory cytokine IL-10 was associated with limited initial demyelination, but low viral mRNA persistence similar to WT mice and declining antiviral cellular immunity. Nevertheless, lesions exhibited sustained expansion providing a model of dysregulated white matter injury temporally remote from the acute CNS insult. Expanding lesions in the absence of IL-10 are characterized by sustained microglial activation and partial loss of macrophage/microglia exhibiting an acquired deactivation phenotype. Furthermore, IL-10 deficiency impaired astrocyte organization into mesh like structures at the lesion borders, but did not prevent astrocyte hypertrophy. The formation of discrete foci of demyelination in IL-10 sufficient mice correlated with IL-10 receptor expression exclusively on astrocytes in areas of demyelination suggesting a critical role for IL-10 signaling to astrocytes in limiting expansion of initial areas of white matter damage. Abstract Case series summary This case series discusses novel characteristics identified in two cases of cowpox. One presented with upper airway signs, and was identified to have a focal laryngeal lesion. The other had central neurological signs at the terminal stages, with intracytoplasmic inclusion bodies identified within the cerebral hemispheres on histopathology. Relevance and novel information Currently, cowpox would be an unlikely consideration in patients with neurological signs or upper respiratory noise. These cases both document novel presentations of cowpox infection, which clinicians should be aware of and consider as differential diagnoses in patients with these atypical presentations. Abstract Golgi a-mannosidase II (G1cNAc transferase 1-dependent a1,3[a1,6] mannosidase, EC 3.2.1.114) catalyzes the final hydrolytic step in the N-glycan maturation pathway acting as the committed step in the conversion of high mannose to complex type structures . We have isolated overlapping clones from a murine cDNA library encoding the full length a-mannosidase II open reading frame and most of the 5' and 3' untranslated region . The coding sequence predicts a type II transmembrane protein with a short cytoplasmic tail (five amino acids), a single transmembrane domain (21 amino acids), and a large COOH-terminal catalytic domain (1,124 amino acids) . This domain organization which is shared with the Golgi glycosyltransferases suggests that the common structural motifs may have a functional role in Golgi enzyme function or localization .Three sets of polyadenylated clones were isolated extending 3' beyond the open reading frame by as N AND O-GLYCAN structures are increasingly being found to contribute to biological recognition events during development, oncogenic transformation, and cell adhesion (3, 10, 11, 37, 38, 40) . The enzymes involved in the maturation of cell surface and intracellular N-glycans are found in the endoplasmic reticulum and Golgi complex where they act upon newly synthesized glycoproteins to generate an array of different structures from a common oligosaccharide precursor (18). The N-glycan processing pathway consists of three stages : (a) the initial synthesis of a dolichol-linked precursor oligosaccharide and the en bloc transfer of the oligosaccharide to newly synthesized polypeptide Asn-X-Ser/Thr sequons on the lumenal face of the ER; (b) the trimming of the high mannose structures by a-glucosidases and a-mannosidases in the ER and Golgi complex; and (c) the elaboration of the branched oligosaccharide chains by Golgi glycosyltransferases . The trimming phase of the pathway is accomplished by a-glucosidases I and II as well as a collection of processing a1,2-mannosidases (29) in the ER and Golgi complex. The resulting Man5GlcNAc2 structure is then modified by the addition of much as 2,543 bp . Northern blots suggest that these polyadenylated clones totaling 6.1 kb in length correspond to minor message species smaller than the full length message. The largest and predominant message on Northern blots (7.5 kb) presumably extends another N1.4-kb downstream beyond the longest of the isolated clones. Transient expression of the a-mannosidase II cDNA in COS cells resulted in 8-12-fold overexpression of enzyme activity, and the appearance of crossreactive material in a perinuclear membrane array consistent with a Golgi localization . A region within the catalytic domain of the a-mannosidase II open reading frame bears a strong similarity to a corresponding sequence in the rat liver endoplasmic reticulum a-mannosidase and the vacuolar a-mannosidase of Saccharomyces cerevisiae. Partial human a-mannosidase II cDNA clones were also isolated and the gene was localized to human chromosome 5. a single G1cNAc by G1cNAc transferase I (GnT 1),' before the final hydrolytic steps in the pathway are accomplished by a-mannosidase II (Man 11), catalyzing the removal of a1,3and a1,6-mannosyl residues (50) . The trimming and elongation phases ofthe pathway overlap at the GnT I/Man II steps, with each reaction being obligatory for further processing steps. GnT I is essential for processing to hybrid or complex type structures (34), while the absence of Man II activity, either by inhibition with the alkaloid, swainsonine (52), or in the human autosomal genetic disease hereditary erythroblastic multinuclearity associated with positive acidified serum (HEMPAS), characterized by the reduced expression of Man 11(12), results in the accumulation of Asn-linked hybrid oligosaccharides in lieu of the standard array of complex type structures . The cleavage of glycoprotein processing in-Dr. Moremerfs present address is Abstract McCrate -S2Additional parameters for non-standard bases were incorporated in the Cornell et al. forcefield(1). Partial atomic charges were calculated for ms 2 t 6 A, mcm 5 s 2 U and ψ ( Figure S1 ), derived by fitting Hartree-Fock (2) restricted electrostatic potentials (RESP) (3) using the 6-31G* basis set (4-6) and normalizing the charges to have the same net charge as the corresponding unmodified base adenine or uridine. All electronic structure calculations were carried out using Gaussian 03M, Revision A.6 (7).Atom types were chosen from those already present in the Cornell et al. (1) or Generalized AMBER (8) Force Field ( Figure S2 ). In a few cases, additional force constants were assigned (Table S1 ). However, since similar connectivities were already present in the forcefield, the force constants for those connectivities were employed.All-atom root mean square deviations (RMSD) from starting structures were found to fluctuate around constant values ( Figure S3 ). Convergence of helical parameters was monitored over the 4.0 ns production run for all simulations. The moving averages of the average value over the duplex region for three helical parameters base-pair twist, base-pair inclination and xdisplacement form the global axis are shown in Figures S4-S6 .McCrate -S3Hydrogen bonding data for water to ψ39 are presented in Table S2 . The water molecules bridged between A38 O2P, ψ39 NH3, and ψ39 O1P in a classic configuration. However, sometimes the water molecule oscillated back and forth between A38 O2P and O1P while maintaining a constant hydrogen bond to ψ39 NH3. Residence lifetimes of water molecules are also present in Table S2 . Abstract Human bocavirus (HBoV) 1, predominantly found in the respiratory tract; and, HBoV2, 3 and 4, mainly detected in stool and associated with gastroenteritis. Despite worldwide occurrence, human bocavirus infection remains poorly understood, and the comprehension of many aspects of these viruses' biology (i.e. , taxonomy, phylogenetic relationships with other viruses, epidemiology, molecular mechanisms of interaction with human cells, association with other pathogens, etc. ) is necessary to clarify whether they are harmless passengers or true pathogens. Development of new diagnostic tools for detection of human bocaviruses will support this type of research. Abstract Polymorphism of angiotensin converting enzyme (ACE) gene is reported to be associated with ischemic heart disease, hypertrophic cardiomyopathy, and idiopathic dilated cardiomyopathy. In this study, we investigated the relationship between Kawasaki disease and insertion/deletion polymorphism of ACE gene. Fifty five Kawasaki disease patients and 43 healthy children were enrolled. ACE genotype was evaluated from each of the subjects' DNA fragments through polymerase chain reaction (PCR). Frequencies of ACE genotypes (DD, ID, II) were 12.7%, 60.0%, 27.3% in Kawasaki group, and 41.9%, 30.2%, 27.9% in control group respectively, indicating low rate of DD and high rate of ID genotype among Kawasaki patients (p<0.01). Comparing allelic (I, D) frequencies, I allele was more prevalent in Kawasaki group than in control group (57.3% vs. 43.0%, p<0.05). In Kawasaki group, both genotype and allelic frequencies were not statistically different between those with coronary dilatations and those without. ACE gene I/D polymorphism is thought to be associated with Kawasaki disease but not with the development of coronary dilatations. Abstract Use of pecan shell fiber in human food is presently limited, but could increase pending demonstration of safety. In a 91-day rat study, pecan shell fiber was administered at dietary concentrations of 0 (control), 50 000, 100 000 or 150 000 ppm. There was no effect of the ingredient on body weight of males or females or food consumption of females. Statistically significant increases in food consumption were observed throughout the study in 100 000 and 150 000 ppm males, resulting in intermittent decreases in food efficiency (150 000 ppm males only) that were not biologically relevant. All animals survived and no adverse clinical signs or functional changes were attributable to the test material. There were no toxicologically relevant changes in hematology, clinical chemistry or urinalysis parameters or organ weights in rats ingesting pecan shell fiber. Any macroscopic or microscopic findings were incidental, of normal variation and/or of minimal magnitude for test substance association. Pecan shell fiber was non-mutagenic in a bacterial reverse mutation test and non-clastogenic in a mouse peripheral blood micronucleus test. Based on these results, pecan shell fiber has an oral subchronic (13-week) no observable adverse effect level (NOAEL) of 150 000 ppm in rats and is not genotoxic at the doses analyzed. Abstract Infectious diseases have caused great catastrophes in human history, as in the example of the plague, which wiped out half of the population in Europe in the 14th century. Ebola virus and H7N9 avian influenza virus are 2 lethal pathogens that we have encountered in the second decade of the 21st century. Ebola infection is currently being seen in West Africa, and H7N9 avian flu appears to have settled in Southeast Asia. This article focuses on the current situation and the future prospects of these potential infectious threats to mankind. Abstract IL-35 is the newest member of IL-12 family. A dimeric protein consisting of two separate subunits has manifested suppressive actions on immune system, which is counterproductive in the context of cancers. Various reports have confirmed its inhibitory role on immune system which is carried out via formation of IL-35-producing regulatory T cells (iTr35), increased Treg development and suppressive Th17 cells growth. Although last decade has seen a great deal of scientific interest on this subject, the exact role, precise signal transduction and elaborative functions of IL-35 in tumor microenvironment (TME) remained elusive. Search for anti-IL-35 therapies have exhibited limited success in animal models. Contrarily, few studies have denied the idea that IL-35 plays a role in cancer. The purpose of this review is to analyze the reported scientific data on continuous symphony of IL-35 in cancers since the inception of former. Abstract This study aimed to compare the indicators (the rates of diagnosis, need for treatment, treatment initiation, and treatment completion) of management of latent tuberculosis infection (LTBI) in contacts and to identify the impact of active tuberculosis (TB) index case characteristics on the exposed population in congregated settings, such as schools, workplaces, and medical institutes. Methods: The data of 8,648 clusters in the TB epidemiological investigation database between 2013 and 2016 were extracted and analyzed to evaluate the indicators and perform multilevel logistic regression (MLR) analyses to identify the factors affecting each indicator. Results: The rates of total LTBI diagnosis, need for treatment, treatment initiation, and treatment completion were 15.2%, 10.2%, 69.4%, and 76.6%, respectively. After adjusting for other factors on MLR, the probability of diagnosis and need for treatment of latent TB in contacts was higher in most types of facilities than in schools. Conversely, treatment completion rates in these facilities were lower. Notably, the correctional institutions showed the highest odds ratio (OR) relative to school for LTBI diagnosis (OR, 6.37) and need for treatment (OR, 4.49) and the lowest OR for treatment completion (OR, 0.10). Conclusion: This study provided evidence for the implementation of latent TB control policies in congregated settings. Abstract Restoration of antigen-specific T cell immunity has the potential to clear persistent viral infection. T cell receptor (TCR) gene therapy can reconstitute CD8 T cell immunity in chronic patients. We cloned 10 virus-specific TCRs targeting 5 different viruses, causing chronic and acute infection. All 10 TCR genetic constructs were optimized for expression using a P2A sequence, codon optimization and the addition of a non-native disulfide bond. However, maximum TCR expression was only achieved after establishing the optimal orientation of the alpha and beta chains in the expression cassette; 9/10 TCRs favored the beta-P2A-alpha orientation over alpha-P2A-beta. Optimal TCR expression was associated with a significant increase in the frequency of IFN-gamma1 T cells. In addition, activating cells for transduction in the presence of Toll-like receptor ligands further enhanced IFN-gamma production. Thus, we have built a virus-specific TCR library that has potential for therapeutic intervention in chronic viral infection or virus-related cancers. Abstract Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cell associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our 1. Abbreviations used in this paper: EEV, extracellular enveloped virus; IBV, avian infectious bronchitis virus; IEV, intracellular enveloped virus; IMV, intracellular mature virus; INV, intracellular naked virus; MOI, multiplicity of infection; PDI, protein disulfide isomerase; PFU, plaque forming units; PI, postinfection; SLO, streptolysin O; VV, vaccinia virus. Abstract Accuracy in translation of the genetic code into proteins depends upon correct tRNA-mRNA recognition in the context of the ribosome. In human tRNA Lys;3 UUU three modified bases are present in the anticodon stem-loop-2-methylthio-N6-threonylcarbamoyladenosine at position 37 (ms 2 t 6 A37), 5-methoxycarbonylmethyl-2-thiouridine at position 34 (mcm 5 s 2 U34) and pseudouridine (c) at position 39-two of which, ms 2 t 6 A37 and mcm 5 s 2 U34, are required to achieve wild-type binding activity of wild-type human tRNA Lys;3 ]. Molecular dynamics simulations of nine tRNA anticodon stem-loops with different combinations of nonstandard bases were performed. The wild-type simulation exhibited a canonical anticodon stairstepped conformation. The ms 2 t 6 modification at position 37 is required for maintenance of this structure and reduces solvent accessibility of U36. Ms 2 t 6 A37 generally hydrogen bonds across the loop and may prevent U36 from rotating into solution. A water molecule does coordinate to c39 most of the simulation time but weakly, as most of the residence lifetimes are ,40 ps. Abstract Introduction: Asthma exacerbations may occur due to a variety of triggers including respiratory viruses. , Iran between 2014 and 2015. A nasopharyngeal aspirate or swab was obtained from each patient during admission. All samples were maintained at 4°C until submission to the virology laboratory and were tested for respiratory viruses by nucleic acid testing. Results: A total of 60 patients with asthma exacerbations were recruited for this study. Of the 60 samples collected from the patients with acute asthma exacerbations, rhinovirus was detected in 12 patients (20%), respiratory syncytial virus in 5 (8%), adenovirus in 5 (8%), and influenza virus in 1 (1.6%). Respiratory pathogens were not detected in 37 (61%) samples. All the samples investigated showed single viral infection. Conclusions: To conclude, the most common viruses detected were rhinovirus followed by respiratory syncytial virus (RSV) and adenovirus. RSV was more commonly associated with more severe attacks. Both the study design (e.g., time of sampling, age of the patients, etc.) and also the method used for viral detection influence the frequency of detection of the respiratory viruses. Abstract 2 The abbreviations used are: S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; MLV, murine leukemia virus; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-gubbing non-integrin, ACE2, argiotensin I conserting enzyme 2.FIGURE 4. Introduction of alanine residues in the S2 region modulates elastase-induced virus entry mediated by SARS-CoV S. A and B, pseudotyped virion infections were performed as described for Fig. 2. Infection was induced by treatment with elastase (A) or trypsin (B). Results are presented as relative light units (RLU). Error bars represent the S.E. of three independent experiments. C, cleavage of SARS-CoV by elastase. HEK293T cells expressing the wild type (WT)or mutant SARS-CoV spike protein were treated with 50 g/ml elastase. Cell surface proteins were biotinylated and precipitated, and cleavage products of the SARS-CoV spike protein were analyzed in Western blot with an antibody recognizing the C9 tag at the C terminus of the protein. Abstract Genome-wide association study (GWAS) is nowadays widely used to identify genes involved in human complex disease. The standard GWAS analysis examines SNPs/genes independently and identifies only a number of the most significant SNPs. It ignores the combined effect of weaker SNPs/genes, which leads to difficulties to explore biological function and mechanism from a systems point of view. Although gene set enrichment analysis (GSEA) has been introduced to GWAS to overcome these limitations by identifying the correlation between pathways/gene sets and traits, the heavy dependence on genotype data, which is not easily available for most published GWAS investigations, has led to limited application of it. In order to perform GSEA on a simple list of GWAS SNP P-values, we implemented GSEA by using SNP label permutation. We further improved GSEA (i-GSEA) by focusing on pathways/gene sets with high proportion of significant genes. To provide researchers an open platform to analyze GWAS data, we developed the i-GSEA4GWAS (improved GSEA for GWAS) web server. i-GSEA4GWAS implements the i-GSEA approach and aims to provide new insights in complex disease studies. i-GSEA4GWAS is freely available at Abstract This study aimed to analyze the epidemiology of pulmonary tuberculosis (PTB) and gained insight into the future TB control plan in China.We extracted epidemiological, clinical, and geographic data from TB prevention and control institutions in 6 cities of Shandong province, China, during 2005 to 2017.Among 224,480 diagnosed PTB, rural residents accounted for 93%, smear-positive PTB 52%, and new cases 92%. The incidence rate of overall PTB declined from 40.8 to 26.25 per 100,000 during 2005 to 2017. Except smear-negative PTB (7.57-19.87 per 100,000), the incidence of smear-positive PTB and all that stratified by age, sex, and treatment history decreased. With 80% reduction, the incidence of smear-positive PTB (6.38 per 100,000) and relapse cases (1.01 per 100,000) were already very low in 2017.With persistent efforts to combat TB, the disease burden had shifted from smear-positive PTB to smear-negative PTB. While new cases need continuous attention, further reducing the incidence of smear-negative PTB and elderly patients may have a greater impact on future TB control.Abbreviations: APC = annual percent change, CDC = Center for Disease Control and Prevention, CI = confidence interval, DOTS = directly observed treatment, short-course, DR TB = drug-resistant tuberculosis, DST = drug susceptibility testing, OR = odds ratio, PTB = pulmonary tuberculosis, SARS = severe acute respiratory syndrome, TB = tuberculosis. Abstract Adenoviral vectors are now being explored as vaccine carriers to prevent infectious diseases in humans and animals. There are two strategies aimed at the expression of a vaccine antigen by adenoviral vectors. The first includes an insertion of the foreign gene expression cassette into the E1 region. The second strategy is antigen incorporation into the viral capsid proteins. To extend this methodology, we have searched for new sites at the human adenovirus serotype 5 major capsid protein hexon for a vaccine antigen insertion. To this end, we utilized sites in the hexon hypervariable region (HVR) 7, 8 and 9 to display a 15-mer peptide containing the main neutralizing epitope of porcine reproductive and respiratory syndrome virus. However, we could not rescue the viruses with the insertions of the peptide into HVR 8 and 9, consistent with the viruses being unable to tolerate insertions at these sites. In contrast, the virus with the insertion of the peptide in HVR 7 was viable -growing well in cell culture and the inserted peptide was exposed on the virion surface. Abstract RNA pseudoknots are functional structure elements with key roles in viral and cellular processes. Prediction of a pseudoknotted minimum free energy structure is an NP-complete problem. Practical algorithms for RNA structure prediction including restricted classes of pseudoknots suffer from high runtime and poor accuracy for longer sequences. A heuristic approach is to search for promising pseudoknot candidates in a sequence and verify those. Afterwards, the detected pseudoknots can be further analysed using bioinformatics or laboratory techniques. We present a novel pseudoknot detection method called DotKnot that extracts stem regions from the secondary structure probability dot plot and assembles pseudoknot candidates in a constructive fashion. We evaluate pseudoknot free energies using novel parameters, which have recently become available. We show that the conventional probability dot plot makes a wide class of pseudoknots including those with bulged stems manageable in an explicit fashion. The energy parameters now become the limiting factor in pseudoknot prediction. DotKnot is an efficient method for long sequences, which finds pseudoknots with higher accuracy compared to other known prediction algorithms. DotKnot is accessible as a web server at Abstract Ownership of companion animals or pets is popular throughout the world. Unfortunately, such animals are susceptible to and potential reservoirs of zoonotic pathogens. Close proximity to and contact with pets can lead to human infections. The distribution of zoonotic diseases associated with companion animals such as dogs and cats is not uniform around the world, and moving animals between regions, countries, and continents carries with it the risk of relocating the pathogens they might harbor. Critical among these zoonotic diseases are rabies, echinococcosis, and leishmania. In addition, the protozoan parasites, Toxoplasma gondii and Giardia duodenalis, are also significant agents for human disease of pet origin. Considerable effort is applied to controlling movements of companion animals, particularly dogs, into the European Union. However, free movement of people and their pets within the European Union is a risk factor for the translocation of diseases and their vectors. This review considers the current distribution of some of these diseases, the risks associated with pet travel, and the controls implemented within Europe to prevent the free movement of zoonotic pathogens. Abstract An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the $30-kb severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNAdependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo-and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for coronavirus nsp8. Abstract C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-t~m frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2' 3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the 04 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendroeytes and type 2 astrocytes and react with the 04 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (04+ only) were increased in number at one week post viral inoculation 1. Abbreviations used in this paper: CNP, 2"3" ¢yclic-nucleotide 3' phosphohydrolase; CNS, central nervous system; GFAP, glial fibrillary acidic protein; WPI, weeks post inoculation. Abstract The monomer of influenza haemagglutinin is synthesized as a single polypeptide precursor that during maturation is cleaved by proteases into two active subunits. Other studies have demonstrated that the human Transmembrane Protease Serine 2 (TMPRSS2) can cleave the HA of human seasonal influenza viruses. Consequently, we have investigated the use of human Transmembrane Protease Serine 2 to produce high titre influenza haemmagglutinin (HA) lentiviral pseudotypes from Group 2 influenza viruses. Such pseudotypes represent powerful and safe tools to study viral entry and immune responses. Influenza pseudotype particles are obtained by co-transfecting human embryonic kidney HEK293T/17 cells using plasmids coding for the influenza HA, HIV gag-pol and a lentiviral vector incorporating firefly luciferase. However, in order to produce Group 2 pseudotypes, it was necessary to co-transfect a plasmid expressing the TMPRSS2 endoprotease, to achieve the necessary HA cleavage for infective particle generation. These lentiviral pseudotypes were shown to transduce HEK293T/17 cells with high efficiency. This demonstrates that TMPRSS2 is necessary for the functional activation, in vitro, of both the HA of human seasonal influenza and other Group 2 HA influenza strains. Additionally, we show that the Group 2 influenza pseudotype particles can be used as surrogate antigens in neutralization assays and are efficiently neutralized by corresponding influenza virus reference sera. These data demonstrate that the viral pseudotype system is a powerful method for serological surveillance of a wide range of influenza viruses. Abstract The possibility of performing fast and small-volume nucleic acid amplification and analysis on a single chip has attracted great interest. Devices based on this idea, referred to as micro total analysis, microfluidic analysis, or simply 'Lab on a chip' systems, have witnessed steady advances over the last several years. Here, we summarize recent research on chip substrates, surface treatments, PCR reaction volume and speed, architecture, approaches to eliminating cross-contamination and control and measurement of temperature and liquid flow. We also discuss product-detection methods, integration of functional components, biological samples used in PCR chips, potential applications and other practical issues related to implementation of lab-on-a-chip technologies.SUBSTRATES AND SURFACE TREATMENTS TO REDUCE BIOMOLECULE ADSORPTIONMost PCR microchambers or microchannels are fabricated from silicon (10-25) or glass (26-36) substrate. Polymers, such as polydimethylsiloxane (PDMS) (37-55), polycarbonate (PC) (56-63) and polymethylmethacrylate (PMMA) (64-68) have increasingly been utilized as alternative substrates. New substrates, such as SU-8 Abstract The present study was performed to investigate the seroprevalence and risk factors for Dirofilaria immitis infection in cats from Liaoning province, northeastern China. From October 2014 to September 2016, sera of 651 cats, including 364 domestic cats and 287 feral cats (332 females and 319 males) were assessed. They were tested for the presence of D. immitis antigen using SNAP Heartworm RT test kit. In this population, the average prevalence was 4.5%. Age and rearing conditions (feral or domestic) were found to be associated with the prevalence of D. immitis. The prevalence was significantly higher in feral cats compared with domestic cats (8.4% vs 1.4%, P < 0.01). There was no significant difference between males and females (4.7% vs 4.2%, P > 0.05), but older cats (≥ 3 years old) showed a statistically higher prevalence compared with younger cats (< 3 years old) in feral populations (16.8 vs 2.4%, P < 0.01), while the difference between the age groups was not statistically significant in domestic cats (2.4% vs 0.51%, P > 0.05), all these results suggest that outdoor exposure time may be one of the most important factors for D. immitis prevalence in cats. Results reveal that D. immitis are prevalence in domestic and feral cats in northeastern China, which indicates that appropriate preventive measures should be taken to decrease the incidence of feline heartworm disease in Liaoning province, northeastern China. Abstract This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact: reprints@medknow.com Cite this article as: Xu X, Liu X, Ou M, Xie C, Chen Y. Psychological flexibility of nurses in a cancer hospital: Preliminary validation of a chinese version of the work-related acceptance and action questionnaire. Asia Pac J Oncol Nurs 2018;5:83-90.Objective: To translate the English work-related acceptance and action questionnaire (WAAQ), make cross-cultural adaptations, and examine its psychometric properties when used by Chinese oncology nurses. Methods: After translation, the psychometric properties of the Chinese WAAQ were analyzed among 417 nurses, and content validity was determined by six experts. Results: Item-level content validity index (CVI) values were between 0.83 and 1.00; scale-level CVI/universal agreement (S-CVI/UA) and S-CVI/average were 0.86 and 0.98, respectively, which implicated a good content validity. The correlation of the Chinese WAAQ with AAQ-II (r s = −0.247, P < 0.001) suggested criterion validity, and those with General Health Questionnaire-12 (−0.250, <0.001) and general self-efficacy scale (0.491, <0.001) and Utrecht work engagement scale (UWES) (0.439, <0.001) suggested convergent validity. Exploratory factor analysis identified a seven-item, one-factor structure of WAAQ. The Chinese version of WAAQ had high internal consistency (Cronbach's α = 0.920), with an item-total correlation coefficient of 0.702-0.828 (P < 0.05), split-half reliability of 0.933, and test-retest reliability of 0.772. Conclusions: The Chinese WAAQ is a reliable and valid tool for assessing psychological flexibility in Chinese oncology nurses. Abstract In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lb pro , from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the Cterminal GlyGly motif, Lb pro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lb pro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lb pro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.ubiquitin | ISG15 | viral signaling | FMDV | structure Abstract Research has shown that obesity appears to spread through social ties. However, the association between other characteristics of social networks and obesity is unclear. This study aimed to identify the association between social network characteristics and body mass index (BMI, kg/m 2 ) in an elderly Korean population.: This cross-sectional study analyzed data from 657 Koreans (273 men, 384 women) aged 60 years or older who participated in the Korean Social Life, Health, and Aging Project. Network size is a count of the number of friends. Density of communication network is the number of connections in the social network reported as a fraction of the total links possible in the personal (ego-centric) network. Average frequency of communication (or meeting) measures how often network members communicate (or meet) each other. The association of each social network measure with BMI was investigated by multiple linear regression analysis.Results: After adjusting for potential confounders, the men with lower density (<0.71) and higher network size (4-6) had the higher BMI (β=1.089, p=0.037) compared to the men with higher density (>0.83) and lower size (1-2), but not in the women (p=0.393). The lowest tertile of communication frequency was associated with higher BMI in the women (β=0.885, p=0.049), but not in the men (p=0.140).Our study suggests that social network structure (network size and density) and activation (communication frequency and meeting frequency) are associated with obesity among the elderly. There may also be gender differences in this association. Abstract Ab 5 antibody; Ag 5 antigen; ALT 5alanine aminotransferase; AST 5aspartate aminotransferase; CFT 5 complement fixation test; EIA 5 enzyme-linked immunosorbent assay; GGT 5 gamma-glutamyl transferase; IFA 5 indirect fluorescent antibody; IgM 5 immunoglobulin M; WBC 5 white blood cell count. Abstract An explosion of knowledge and technology is revolutionizing medicine and patient care. Novel testing must be brought to the clinic with safety and accuracy, but also in a timely and cost-effective manner, so that patients can benefit and laboratories can offer testing consistent with current guidelines. Under the oversight provided by the Clinical Laboratory Improvement Amendments, laboratories have been able to develop and optimize laboratory procedures for use in-house. Quality improvement programs, interlaboratory comparisons, and the ability of laboratories to adjust assays as needed to improve results, utilize new sample types, or incorporate new mutations, information, or technologies are positive aspects of Clinical Laboratory Improvement Amendments oversight of laboratory-developed procedures. Laboratories have a long history of successful service to patients operating under Clinical Laboratory Improvement Amendments. A series of detailed clinical examples illustrating the quality and positive impact of laboratory-developed procedures on patient care is provided. These examples also demonstrate how Clinical Laboratory Improvement Amendments oversight ensures accurate, reliable, and reproducible testing in clinical laboratories. Abstract Backgrounds: Up to February 16, 2020, 355 cases have been confirmed as having COVID-19 infection on the Diamond Princess cruise ship. It is of crucial importance to estimate the reproductive number (R0) of the novel virus in the early stage of outbreak and make a prediction of daily new cases on the ship. Method: We fitted the reported serial interval (mean and standard deviation) with a gamma distribution and applied "earlyR" package in R to estimate the R0 in the early stage of COVID-19 outbreak. We applied "projections" package in R to simulate the plausible cumulative epidemic trajectories and future daily incidence by fitting the data of existing daily incidence, a serial interval distribution, and the estimated R0 into a model based on the assumption that daily incidence obeys approximately Poisson distribution determined by daily infectiousness. Results: The Maximum-Likelihood (ML) value of R0 was 2.28 for COVID-19 outbreak at the early stage on the ship. The median with 95% confidence interval (CI) of R0 values was 2.28 (2.06-2.52) estimated by the bootstrap resampling method. The probable number of new cases for the next ten days would gradually increase, and the estimated cumulative cases would reach 1514 (1384-1656) at the tenth day in the future. However, if R0 value was reduced by 25% and 50%, the estimated total number of cumulative cases would be reduced to 1081 (981-1177) and 758 (697-817), respectively. Conclusion: The median with 95% CI of R0 of COVID-19 was about 2.28 (2.06-2.52) during the early stage experienced on the Diamond Princess cruise ship. The future daily incidence and probable outbreak size is largely dependent on the change of R0. Unless strict infection management and control are taken, our findings indicate the potential of COVID-19 to cause greater outbreak on the ship. Abstract To assess the nurses' hand hygiene (HH) knowledge, perception, attitude, and self-reported performance in small-and medium-sized hospitals after Middle East Respiratory Syndrome outbreak.The structured questionnaire was adapted from the World Health Organization's survey. Data were collected between June 26 and July 14, 2017.Results: Nurses showed scores on knowledge (17.6±2.5), perception (69.3±0.8), self-reported HH performance of non-self (86.0± 11.0), self-reported performance of self (88.2±11.0), and attitude (50.5±5.5). HH performance rate of non-self was Y1 =36.678+ 0.555X1 (HH performance rate of self) (adjusted R 2 =0.280, p<0.001). The regression model for performance was Y4=18.302+0.247X41(peception)+0.232X42 (attitude)+0.875X42 (role model); coefficients were significant statistically except attitude, and this model significant statistically (adjusted R 2 =0.191, p<0.001).Conclusions: Advanced HH education program would be developed and operated continuously. Perception, attitude, role model was found to be a significant predictors of HH performance of self. So these findings could be used in future HH promotion strategies for nurses. Abstract Objective This study aimed to evaluate psychometric properties of the Munich ChronoType Questionnaire (MCTQ) in a sample of Korean older adults. Methods One-hundred ninety two participants aged 65 and over completed interview-based questionnaires about chronotype, insomnia, depression, and anxiety. Additionally, a small subset of subjects completed a 7-day sleep diary and actigraphy measurements. Results Morningness-Eveningness Questionnaire (MEQ) scores were significantly negatively correlated with Midpoint of sleep on free days corrected for sleep debt accumulated through weekdays (MSFsc) (r=-0.45, p<0.01) assessed by the MCTQ. MSFsc using the MCTQ was significantly positively correlated with MSFsc assessed by both the sleep diary (r=0.74, p<0.05) and actigraphy (r=0.76, p<0.05). Additionally, MSFsc assessed by the MCTQ was significantly positively correlated with insomnia (r=0.26, p<0.01), depression (r=0.25, p<0.01), and anxiety (r=0.18, p<0.05). Finally, based on MEQ scores, we derived a cut-off score for the MCTQ that distinguishes morning type and other types (intermediate/evening types) in older adults. Conclusion The results of these studies supported the validity of the MCTQ in Korean older adults. Additionally, while sleep rhythms in elder adults may be more advanced, eveningness tendency may be still important and indicative of sleep and psychological disturbance. Psychiatry Investig 2018;15(8):775-782 Abstract Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3Kdependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing. Abstract Surveillance of Middle East respiratory syndrome coronavirus (MERS-CoV) was conducted to explore the possible introduction and circulation of this novel virus in Catalonia, northeastern Spain. Five hundred and sixty-three samples from mild and severe respiratory infections collected between January 2012 and April 2013 were screened using real-time RT-PCR. All samples were negative, suggesting that MERS-CoV is not circulating silently in Catalonia. Abstract The initial cluster of severe pneumonia cases that triggered the COVID-19 epidemic was identified in Wuhan, China in December 2019. While early cases of the disease were linked to a wet market, human-to-human transmission has driven the rapid spread of the virus throughout China. The Chinese government has implemented containment strategies of city-wide lockdowns, screening at airports and train stations, and isolation of suspected patients; however, the cumulative case count keeps growing every day. The ongoing outbreak presents a challenge for modelers, as limited data are available on the early growth trajectory, and the epidemiological characteristics of the novel coronavirus are yet to be fully elucidated. We use phenomenological models that have been validated during previous outbreaks to generate and assess short-term forecasts of the cumulative number of confirmed reported cases in Hubei province, the epicenter of the epidemic, and for the overall trajectory in China, excluding the province of Hubei. We collect daily reported cumulative confirmed cases for the 2019-nCoV outbreak for each Chinese province from the National Health Commission of China. Here, we provide 5, 10, and 15 day forecasts for five consecutive days, February 5th through February 9th, with quantified uncertainty based on a generalized logistic growth model, the Richards growth model, and a sub-epidemic wave model. Our most recent forecasts reported here, based on data up until February 9, 2020, largely agree across the three models presented and suggest an average range of 7409e7496 additional confirmed cases in Hubei and 1128e1929 additional cases in other provinces within the next five days. Models also predict an average total cumulative case count between 37,415 and 38,028 in Hubei and 11,588e13,499 in other provinces by February 24, 2020. Mean estimates and uncertainty bounds for both Hubei and other provinces have remained relatively stable in the last three reporting dates (February 7th e 9th). We also observe that each of the models predicts that the epidemic has reached saturation in both Hubei and other provinces. Our findings suggest that the containment strategies implemented in China are successfully reducing transmission and that the epidemic growth has slowed in recent days. Abstract VERSION 1 -REVIEWPedro A Piedra Baylor College of Medicine Houston, TX, USA REVIEW RETURNED 13-Aug-2018This manuscript reports on a retrospective population-based cohort study on acute respiratory infections (ARIs) in the emergency department (ED) in Western Australia (WA) among Aboriginal and non-aboriginal children (0-17 years) residing in metropolitan, urban and rural settings from 2002 to 2012. Administrative ED datasets were linked to birth and perinatal records. The objectives were to compare overall and age-specific ED ARIs rates among Aboriginal and non-Aboriginal children in the different WA regions, monthly distribution and temporal trends, and to identify infant, maternal and socio-economic risk factors for ARI. The overall goal is to use such information to inform health service planning. Major findings. 26% of ED presentations were for ARIs. ARIs rates were highest in children <12 months for Aboriginal children (1,027.6 per 1000 child-years) and non-Aboriginal children (297.4 per 1000 child-years). Regional differences were observed, trend analysis revealed increase ARI rates for all children in rural and remote areas, and the strongest risk facts observed in Aboriginal and non-Aboriginal children were male sex, prematurity, caesarean delivery, birth in lower socio-economic areas and living in the Kimberley region. This manuscript provides strong ARI incidence rates in the ED for children residing in Western Australia, and highlights ARIs rates of 2 to 3 times higher in Aboriginal children compare to non-Aboriginal children as well as regional disparity. The root cause for these disparities were not address. Commenting on the density of physicians (per 1000 population) by metropolitan, urban and rural settings as well as by regions within WA is likely to be informative, and explain in part, the disparities observed between race and region. Areas with low density of health care providers is likely to have increased use of the ED for basic health care assessment impacting ED ARI rates. Abstract With over 3 billion airline passengers annually, the inflight transmission of infectious diseases is an important global health concern. Over a dozen cases of inflight transmission of serious infections have been documented, and air travel can serve as a conduit for the rapid spread of newly emerging infections and pandemics. Despite sensational media stories and anecdotes, the risks of transmission of respiratory viruses in an airplane cabin are unknown. Movements of passengers and crew may facilitate disease transmission. On 10 transcontinental US flights, we chronicled behaviors and movements of individuals in the economy cabin on single-aisle aircraft. We simulated transmission during flight based on these data. Our results indicate there is low probability of direct transmission to passengers not seated in close proximity to an infectious passenger. This data-driven, dynamic network transmission model of droplet-mediated respiratory disease is unique. To measure the true pathogen burden, our team collected 229 environmental samples during the flights. Although eight flights were during Influenza season, all qPCR assays for 18 common respiratory viruses were negative. airplane transportation | infectious disease transmission | influenza | Abstract The simultaneous electrochemical determination of myricetin and rutin remains a challenge due to their indistinguishable potentials. To solve this problem, we constructed a ternary platinum nanoparticle, reduced graphene oxide, multi-walled carbon nanotubes (Pt@r-GO@MWCNTs) nanocomposite via a facile one-pot synthetic method. Under the optimized conditions, the ternary Pt@r-GO@MWCNTs nanocomposite exhibited good electrocatalytic activity toward myricetin and rutin via solid phase extraction and excellent performance for the simultaneous determination of myricetin and rutin. The oxidation peak current of myricetin was proportional to its concentrations in the range of 0.05e50 mM with a detection limit of 0.01 mM (S/N ¼ 3). The linear range for rutin was 0.05e50 mM with a detection limit of 0.005 mM (S/N ¼ 3). The ternary nanocomposite sensor also exhibited good reproducibility and stability, and was successfully used for the simultaneous determination of myricetin and rutin in real orange juice samples with recoveries ranging between 100.57% and 108.46%. Abstract Oligonucleotide-directed mutagenesis was Abstract The rapid increase in the amount of protein sequence data available leads to an urgent need for novel computational algorithms to analyze and compare these sequences. This study is undertaken to develop an efficient computational approach for timely encoding protein sequences and extracting the hidden information.Methods: Based on two physicochemical properties of amino acids, a protein primary sequence was converted into a three-letter sequence, and then a graph without loops and multiple edges and its geometric line adjacency matrix were obtained. A generalized PseAAC (pseudo amino acid composition) model was thus constructed to characterize a protein sequence numerically.Results: By using the proposed mathematical descriptor of a protein sequence, similarity comparisons among β-globin proteins of 17 species and 72 spike proteins of coronaviruses were made, respectively. The resulting clusters agreed well with the established taxonomic groups. In addition, a generalized PseAAC based SVM (support vector machine) model was developed to identify DNA-binding proteins. Experiment results showed that our method performed better than DNAbinder, DNA-Prot, iDNA-Prot and enDNA-Prot by 3.29-10.44% in terms of ACC, 0.056-0.206 in terms of MCC, and 1.45-15.76% in terms of F1M. When the benchmark dataset was expanded with negative samples, the presented approach outperformed the four previous methods with improvement in the range of 2.49-19.12% in terms of ACC, 0.05-0.32 in terms of MCC, and 3.82-33.85% in terms of F1M.These results suggested that the generalized PseAAC model was very efficient for comparison and analysis of protein sequences, and very competitive in identifying DNA-binding proteins. Abstract within the receptor binding domain (RBD) of the MERS-CoV S protein. We used a complex consisting of the epitope peptide of the MERS-CoV S protein and CpG-DNA encapsulated in liposome complex to immunize mice, and produced the monoclonal antibodies 506-2G10G5 and 492-1G10E4E2. The western blotting data showed that both monoclonal antibodies detected the S protein and immunoprecipitated the native form of the S protein. Indirect immunofluorescence and confocal analysis suggested strong reactivity of the antibodies towards the S protein of MERS-CoV virus infected Vero cells. Furthermore, the 506-2G10G5 monoclonal antibody significantly reduced plaque formation in MERS-CoV infected Vero cells compared to normal mouse IgG and 492-1G10E4E2. Thus, we successfully produced a monoclonal antibody directed against the RBD domain of the S protein which could be used in the development of diagnostics and therapeutic applications in the future. [BMB Reports 2019; 52(6): 397-402] BMB Rep. 2019; 52(6): 397-402 www.bmbreports.org Abstract In the Anthropocene context, changes in climate, land use and biodiversity are considered among the most important anthropogenic factors affecting parasites-host interaction and wildlife zoonotic diseases emergence. Transmission of vector borne pathogens are particularly sensitive to these changes due to the complexity of their cycle, where the transmission of a microparasite depends on the interaction between its vector, usually a macroparasite, and its reservoir host, in many cases represented by a wildlife vertebrate. The scope of this paper focuses on the effect of some major, fast-occurring anthropogenic changes on the vectorial capacity for tick and mosquito borne pathogens. Specifically, we review and present the latest advances regarding two emerging vector-borne viruses in Europe: Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV). In both cases, variation in vector to host ratio is critical in determining the intensity of pathogen transmission and consequently infection hazard for humans. Forecasting vector-borne disease hazard under the global change scenarios is particularly challenging, requiring long term studies based on a multidisciplinary approach in a One-Health framework. Abstract One cross-sectional study describing the clinicopathologic abnormalities in 24 dogs seropositive to Bartonella vinsonii ssp. berkhoffi was identified. Three of 4 anemic dogs tested were Coombs'-positive, 1 yielding an IME value of 3.81.Bartonella spp. induce IMHA is low. As the range of pathogenesis for this organism is still emerging, further prospective, controlled studies are needed to address this question.Borrelia spp.One retrospective study described 1 dog with IMHA that was seropositive for B. burgdorferi, A. phagocytophilium, and R. rickettsii. 2 Owing to the co-exposure status, evidence for B. burgdorferi as a cause of IMHA could not be evaluated. In a separate study, a Coombs'positive anemia was documented in a dog with Borrelia turicatae spirochetemia, 3 yielding an IME value of 2.37.Based on isolated case reports, the overall evidence that Borrelia spp. infection induces IMHA is negligible. Considering that Borrelia infection in dogs is associated with other immune-mediated conditions (notably polyarthritis), 4 future prospective, Abstract Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesisIsolation of peripheral blood mononuclear cells (PBMC) Abstract Background. Paramyxoviruses include respiratory syncytial virus (RSV), parainfluenza virus (PIV), and human metapneumovirus (MPV), which may cause significant respiratory tract infectious disease (RTID) and mortality after allogeneic hematopoietic cell transplantation (HCT). However, clinical data regarding frequency and outcome are scarce.Methods. We identified all paramyxovirus RTIDs in allogeneic HCT recipients diagnosed by multiplex polymerase chain reaction between 2010 and 2014. Baseline characteristics of patients, treatment, and outcome of each episode were analyzed; ie, moderate, severe, and very severe immunodeficiency (verySID) according to HCT ≤6 months, T-or B-cell depletion ≤3 months, graft-versus-host disease, neutropenia, lymphopenia, or hypo-gammaglobulinemia. Results. One hundred three RTID episodes in 66 patients were identified (PIV 47% [48 of 103], RSV 32% [33 of 103], MPV 21% [22 of 103]). Episodes occurred in 85% (87 of 103) at >100 days post-HCT. Lower RTID accounted for 36% (37 of 103). Thirty-nine percent (40 of 103) of RTID episodes required hospitalization and more frequently affected patients with lower RTID. Six percent progressed from upper to lower RTID. Overall mortality was 6% and did not differ between paramyxoviruses. Sixty-one percent (63 of 103) of episodes occurred in patients with SID, and 20.2% (19 of 63) of episodes occurred in patients with verySID. Oral ribavirin plus intravenous immunoglobulin was administered in 38% (39 of 103) of RTIDs, preferably for RSV or MPV (P ≤ .001) and for SID patients (P = .001). Patients with verySID frequently progressed to lower RTID (P = .075), required intensive care unit transfer, and showed higher mortality.Conclusion. Paramyxovirus RTID remains a major concern in allogeneic HCT patients fulfilling SID and verySID, emphasizing that efficacious and safe antiviral treatments are urgently needed. Abstract The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS. Abstract The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30dependent changes in lymphocyte effector function. Thus, we conclude that receptor-ligand signaling interactions can be selectively regulated by an extracellular redox catalyst. Abstract Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H 2 O 2 ). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H 2 O 2 . However, H 2 O 2 -induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3-and 9.6fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans, which are possibly associated with enhanced oxidative stress and altered MAPK pathways, respectively. Abstract Few studies have assessed viral contamination in the rooms of hospital wards. This cross-sectional study evaluated the air and objects in patient-occupied rooms in pediatric wards for the presence of common respiratory viruses and Mycoplasma pneumoniae.Air samplers were placed at a short (60-80 cm) and long (320 cm) distance from the head of the beds of 58 pediatric patients, who were subsequently confirmed to be infected with enterovirus (n ¼ 17), respiratory syncytial virus (RSV) (n ¼ 13), influenza A virus (n ¼ 13), adenovirus (n ¼ 9), or M pneumoniae (n ¼ 6). Swab samples were collected from the surfaces of 5 different types of objects in the patients' rooms. All air and swab samples were analyzed via real-time quantitative polymerase chain reaction assay for the presence of the above pathogens.All pathogens except enterovirus were detected in the air, on the objects, or in both locations in the patients' rooms. The detection rates of influenza A virus, adenovirus, and M pneumoniae for the long distance air sampling were 15%, 67%, and 17%, respectively. Both adenovirus and M pneumoniae were detected at very high rates, with high concentrations, on all sampled objects.The respiratory pathogens RSV, influenza A virus, adenovirus, and M pneumoniae were detected in the air and/or on the objects in the pediatric ward rooms. Appropriate infection control measures should be strictly implemented when caring for such patients.(Medicine 95(14):e3014)Abbreviations: CAP = community-acquired pneumonia, PBS = phosphate-buffered saline, PCR = polymerase chain reaction, RSV = respiratory syncytial virus.Editor: Roman Leischik. Abstract Coronavirus disease 2019 (COVID-2019) has been recognized as a global threat, and several studies are being conducted using various mathematical models to predict the probable evolution of this epidemic. These mathematical models based on various factors and analyses are subject to potential bias. Here, we propose a simple econometric model that could be useful to predict the spread of COVID-2019. We performed Auto Regressive Integrated Moving Average (ARIMA) model prediction on the Johns Hopkins epidemiological data to predict the epidemiological trend of the prevalence and incidence of COVID-2019. For further comparison or for future perspective, case definition and data collection have to be maintained in real time. Abstract Porcine epidemic diarrhoea (PED) is a highly contagious and devastating enteric disease of pigs caused by porcine epidemic diarrhoea virus (PEDV), an enveloped, single-stranded RNA virus belonging to the Alphacoronavirus genus of the Coronaviridae family. The disease is clinically similar to other forms of porcine gastroenteritis. Pigs are the only known host of the disease, and the occurrence of PED in wild boars is unknown. The virus causes acute diarrhoea, vomiting, dehydration, and high mortality in suckling piglets reaching 100%. Heavy economic losses in the pig-farming industry were sustained in the USA between 2013 and 2015 when PEDV spread very quickly and resulted in epidemics. The loss in the US pig industry has been estimated at almost seven million pigs. The purpose of this review is a description of the current status of porcine epidemic diarrhoea in European pigs and the risk presented by the introduction of PEDV to Poland in comparison to the epidemics in the USA. Abstract Viruses are widely used as vectors for heterologous gene expression in cultured cells or natural hosts, and therefore a large number of viruses with exogenous sequences inserted into their genomes have been engineered. Many of these engineered viruses are viable and express heterologous proteins at high levels, but the inserted sequences often prove to be unstable over time and are rapidly lost, limiting heterologous protein expression. Although virologists are aware that inserted sequences can be unstable, processes leading to insert instability are rarely considered from an evolutionary perspective. Here, we review experimental work on the stability of inserted sequences over a broad range of viruses, and we present some theoretical considerations concerning insert stability. Different virus genome organizations strongly impact insert stability, and factors such as the position of insertion can have a strong effect. In addition, we argue that insert stability not only depends on the characteristics of a particular genome, but that it will also depend on the host environment and the demography of a virus population. The interplay between all factors affecting stability is complex, which makes it challenging to develop a general model to predict the stability of genomic insertions. We highlight key questions and future directions, finding that insert stability is a surprisingly complex problem and that there is need for mechanism-based, predictive models. Combining theoretical models with experimental tests for stability under varying conditions can lead to improved engineering of viral modified genomes, which is a valuable tool for understanding genome evolution as well as for biotechnological applications, such as gene therapy. Abstract Inclusion body disease (IBD) is an infectious fatal disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the disease is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the disease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novo assembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propagate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclusions in infected cells in culture and tissues from diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those of filoviruses than to those of other arenaviruses. These findings implicate these viruses as candidate etiologic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens.The cause of the disease has remained elusive, and no treatment exists. In addition to being important to pet owners, veterinarians, breeders, zoological parks, and aquariums, the study of animal disease is significant since animals are the source of virtually every emerging infectious human disease. We searched for candidate causative agents in snakes diagnosed with IBD and found a group of novel viruses distantly related mainly to arenaviruses but also to filoviruses, both of which can cause fatal hemorrhagic fevers when transmitted from animals to humans. In addition to providing evidence that strongly suggests that these viruses cause snake IBD, this discovery reveals a new and unanticipated domain of virus biology and evolution.Citation Stenglein MD, et al. 2012. Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: candidate etiological agents for snake inclusion body disease. mBio 3(4):e00180-12. Abstract This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation. Abstract The membrane-spanning and cytoplasmic domains of CD4 and CD8 were replaced by those of TGN38. After transient expression in HeLa cells, the location of the hybrid proteins was determined using immunofluorescence and quantitative immuno-electron microscopy, FACS analysis and metabolic labeling. The membrane-spanning domain was found to contain a signal that localized hybrid proteins to the TGN. This was in addition to the signal previously identified in the cytoplasmic domain (Bos, K., C. Wraight, and K. Abstract A stray female cat of unknown age, presenting bright red watery diarrhea, was submitted to the Animal and Plant Quarantine Agency for diagnosis. In the small intestines extracted from the necropsied cat, numerous white ovalshaped organisms were firmly embedded in the mucosa and there was thickening of intestinal wall. Histopathological analysis revealed severe necrotizing enteritis, together with atrophied intestinal villi, exfoliated enterocytes, and parasitic worms. Recovered worms were identified as Pharyngostomum cordatum by morphological observation and genetic analysis. Although P. cordatum is known to occur widely in Korea, this is the first clinical description of an infection by P. cordatum causing severe feline enteritis. Abstract Prior studies have shown that complement plays a critical role in controlling WNV infection in complement (C) 3 −/− and complement receptor 1/2 −/− mice. Here, we dissect the contributions of the individual complement activation pathways to the protection from WNV disease. Genetic defi ciencies in C1q, C4, factor B, or factor D all resulted in increased mortality in mice, suggesting that all activation pathways function together to limit WNV spread. In the absence of alternative pathway complement activation, WNV disseminated into the central nervous system at earlier times and was associated with reduced CD8 + T cell responses yet near normal anti-WNV antibody profi les.Our results suggest that individual pathways of complement activation control WNV infection by priming adaptive immune responses through distinct mechanisms. Abstract Background: It is important to have an estimate of the incidence of acute disseminated encephalomyelitis (ADEM) because the incidence of ADEM is unknown and the outcomes undefined in China.Objectives: This study attempts to describe ADEM incidence in large Chinese populations located in four geographically different and moderately distant areas of the same province. Methods: A retrospective investigation was conducted with ADEM patients in Nanjing, Nantong, Yancheng and Xuzhou. The survey was carried out in regions that might have received patients meeting the case definition of ADEM provided by the International Pediatric MS Study Group from 2008 to 2011. A total of 125 hospitals were included and 412 patients were identified through the hospital information systems (HIS). Results: The incidence of ADEM was 0.32/100,000/year. There are two peaks on the age-specific ADEM rates curve. One is 0.77/100,000/year among 0-to 9-year-olds, the other is 0.45/100,000/year in those aged 5059 years. The incidence rate found for ADEM in males was 0.34/100,000/year, and in females was 0.29/100,000/year. The highest incidence rate was in Nanjing (0.40/100,000/year). Conclusions: The average annual incidence of ADEM was 0.32/100,000/year. The peak age of onset was 5059 years old and 09 years old. The incidence among males was insignificantly higher than that among females. There was no significant difference in incidence by seasonal variation. Abstract Dengue virus serotype 2 (DENV2) is widespread and responsible for severe epidemics. While primary DENV2 infections stimulate serotype-specific protective responses, a leading vaccine failed to induce a similar protective response. Using human monoclonal antibodies (hMAbs) isolated from dengue cases and structure-guided design of a chimeric DENV, here we describe the major site on the DENV2 envelope (E) protein targeted by neutralizing antibodies. DENV2-specific neutralizing hMAb 2D22 binds to a quaternary structure epitope. We engineered and recovered a recombinant DENV4 that displayed the 2D22 epitope. DENV2 neutralizing antibodies in people exposed to infection or a live vaccine tracked with the 2D22 epitope on the DENV4/2 chimera. The chimera remained sensitive to DENV4 antibodies, indicating that the major neutralizing epitopes on DENV2 and -4 are at different sites. The ability to transplant a complex epitope between DENV serotypes demonstrates a hitherto underappreciated structural flexibility in flaviviruses, which could be harnessed to develop new vaccines and diagnostics. IMPORTANCE Dengue virus causes fever and dengue hemorrhagic fever. Dengue serotype 2 (DENV2) is widespread and frequently responsible for severe epidemics. Natural DENV2 infections stimulate serotype-specific neutralizing antibodies, but a leading DENV vaccine did not induce a similar protective response. While groups have identified epitopes of single monoclonal antibodies (MAbs), the molecular basis of DENV2 neutralization by polyclonal human immune sera is unknown. Using a recombinant DENV displaying serotype 2 epitopes, here we map the main target of DENV2 polyclonal neutralizing antibodies induced by natural infection and a live DENV2 vaccine candidate. Proper display of the epitope required the assembly of viral envelope proteins into higher-order structures present on intact virions. Despite the complexity of the epitope, it was possible to transplant the epitope between DENV serotypes. Our findings have immediate implications for evaluating dengue vaccines in the pipeline as well as designing next-generation vaccines. AM, Baric RS. 2015. A new quaternary structure epitope on dengue virus serotype 2 is the target of durable type-specific neutralizing antibodies. mBio 6(5):e01461-15. Abstract Objective: To assess the role of spatial proximity, defined as patients sharing bays, in the spread of norovirus during outbreaks in hospitals. Design: Enhanced surveillance of norovirus outbreaks between November 2009 and November 2011.Methods: Data were gathered during 149 outbreaks of norovirus in hospital wards from five hospitals in two major cities in England serving a population of two million. We used the time between the first two cases of each outbreak to estimate the serial interval for norovirus in this setting. This distribution and dates of illness onset were used to calculate epidemic trees for each outbreak. We then used a permutation test to assess whether proximity, for all outbreaks, was more extreme than would be expected by chance under the null hypothesis that proximity was not associated with transmission risk.Results: 65 outbreaks contained complete data on both onset dates and ward position. We estimated the serial interval to be 1.86 days (95% CI 1.6 to 2.2 days), and with this value found strong evidence to reject the null hypothesis that proximity was not significant ( p<0.001). Sensitivity analysis using different values of the serial interval showed that there was evidence to reject the null hypothesis provided the assumed serial interval was less than 2.5 days.Conclusions: Our results provide evidence that patients occupying the same bay as patients with symptomatic norovirus infection are at an increased risk of becoming infected by these patients compared with patients elsewhere in the same ward.Harris JP, Lopman BA, Cooper BS, et al. BMJ Open 2013;3:e003060. Abstract Ciliocytophthoria (CCP) defines a degenerative process of the ciliated cells consequent to viral infections, and it is characterized by typical morphological changes. We evaluated the distinct and characteristic phases of CCP, by means of the optical microscopy of the nasal mucosa (nasal cytology), in 20 patients (12 males and 8 females; aged between 18 and 40 years). Three phases of CCP by nasal cytology are detected. This outcome confirms that CCP represents a sign of suffering nasal epithelial cell. (www.actabiomedica.it) Abstract As a result of rapid economic growth over the previous three decades, China has become the second largest economy worldwide since 2010. However, as a developing country with the largest population, this rapid economic growth primarily based on excessive consumption and waste of resources. Thus, China has been facing particularly severe ecological and environmental problems in speeding up industrialization and urbanization. The impact of the health risk factors is complex and difficult to accurately predict. Therefore, it is critical to investigate potential threats in the context of the human-animal-environment interface to protect human and animal health. The ''One Health'' concept recognizes that human health is connected to animal and environmental health. This review primarily discusses specific health problems in China, particularly zoonoses, and explains the origin and development of the One Health approach, as well as the importance of a holistic approach in China. Abstract Peptides are secreted by different cell types and are trendy therapeutic agents that have attracted attention for the treatment of several diseases such as infections. Antimicrobial peptides exert various mechanisms such as changing cell membrane permeability which leads to inhibition or death of bacterial cells. mesenchymal stem cells (MSCs) are key to produce antimicrobial peptides and to inhibit the growth of pathogens. These cells have been shown to be capable of producing antimicrobial peptides upon exposure to different bacteria. As a result, antimicrobial peptides can be considered as novel agents for the treatment of infectious diseases. The purpose of this review was to investigate the targets and mechanisms of antimicrobial peptides secreted by MSCs. Abstract Pneumonia refers to lung inflammation caused by different pathogens or other factors, and is a common pediatric disease occurring in infants and young children. It is closely related to the anatomical and physiological characteristics of infants and young children and is more frequent during winter and spring, or sudden changes in temperature. Pneumonia is a serious disease that poses a threat to children's health and its morbidity and mortality rank first, accounting for 24.5-65.2% of pediatric inpatients. Due to juvenile age, severe illness and rapid changes, children often suffer acute heart failure, respiratory failure and even toxic encephalopathy at the same time. The concurrence in different stages of the process of emergency treatment tends to relapse, which directly places the lives of these children at risk. Severe pneumonia constitutes one of the main causes of infant mortality. In the process of nursing children with severe pneumonia, intensive care was provided, including condition assessment and diagnosis, close observation of disease, keeping the airway unblocked, rational oxygen therapy, prevention and treatment of respiratory and circulatory failure, support of vital organs, complications, and health education. The inflammatory response was proactively controlled, to prevent suffocation and reduce mortality. In summary, positive and effective nursing can promote the rehabilitation of children patients, which can be reinforced with adequate communication with the parents and/or caretakers. Abstract The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins are small and abundant membrane-bound proteins, whose specific interactions mediate membrane fusion during cell fusion or cellular trafficking. In this study, we report the use of a label-free method, called imaging ellipsometer to analyze the interactions among three SNAREs, namely Sec22p, Ykt6p and Sso2p. The SNAREs were immobilized on the silicon wafer and then analyzed in a pairwise mode with microfluidic array, leading us to discover the interactions between Ykt6p and Sso2p, Sec22p and Sso2p. Moreover, by using the real-time function of the imaging ellipsometer, we were able to obtain their association constants (K A ) of about 10 4 M 21 . We argue that the use of imaging ellipsometer coupled with microfluidic device will deepen our understanding of the molecular mechanisms underlying membrane fusion process. Abstract This review paper examines the growing implications of China's engagement in shaping innovative national initiatives against infectious diseases and poverty control and elimination in African countries. It seeks to understand the factors and enhancers that can promote mutual and innovative health development initiatives, and those that are necessary in generating reliable and quality data for evidence-based contextual policy, priorities and programs.We examined the China-Africa health cooperation in supporting global health agenda on infectious diseases such as malaria, schistosomiasis, Ebola, TB, HIV/AIDS, neglected tropical diseases (NTDs) prevention, control and elimination spanning a period of 10 years. We reviewed referenced publications, global support data, and extensive sources related to and other emerging epidemics and infectious diseases of poverty, programs and interventions, health systems development issues, challenges, opportunities and investments. Published literature in PubMed, Scopus, Google Scholar, Books and web-based peer-reviewed journal articles, government annual reports were assessed from the first Forum on China-Africa Cooperation (FOCAC) in November 2006 to December 2015 Third Ministerial conferences.Our findings highlight current shared public health challenges and emphasize the need to nurture, develop and establish effective, functional and sustainable health systems capacity to detect and respond to all public health threats and epidemic burdens, evidence-based programs and quality care outcomes. China's significant health diplomacy emphasizes the importance of health financing in establishing health development commitment and investment in improving the gains and opportunities, importantly efficiency and value health priorities and planning.Strengthening China-Africa health development agenda towards collective commitment and investment in quality care delivery, effective programs coverage and efficiency, preparedness and emergency response is needed in transforming African health information systems, and local health governance structures and management in emerging epidemics. Furthermore, innovative evidence of operational joint solutions and strategies are critical in advancing healthcare delivery, and further enhancing Universal Health Care, and Sustainable Development Goals to attain global health improvements and economic prosperity. Abstract HIGHLIGHTS NTZ amplifies RNA sensor and type I interferon activities and induces GADD34 expression NTZ inhibits infectious Ebola virus (EBOV) via RIG-I and PKR, but not GADD34 NTZ inhibits a second negative-strand RNA virus, VSV, via RIG-I and GADD34, but not PKR NTZ holds promise as an oral therapy against EBOV Jasenosky et al., iScience 19,Here, we show that the US Food and Drug Administration-approved oral drug nitazoxanide (NTZ) broadly amplifies the host innate immune response to viruses and inhibits Ebola virus (EBOV) replication. We find that NTZ enhances retinoic-acid-inducible protein I (RIG-I)-like-receptor, mitochondrial antiviral signaling protein, interferon regulatory factor 3, and interferon activities and induces transcription of the antiviral phosphatase GADD34. NTZ significantly inhibits EBOV replication in human cells through its effects on RIG-I and protein kinase R (PKR), suggesting that it counteracts EBOV VP35 protein's ability to block RIG-I and PKR sensing of EBOV. NTZ also inhibits a second negative-strand RNA virus, vesicular stomatitis virus (VSV), through RIG-I and GADD34, but not PKR, consistent with VSV's distinct host innate immune evasion mechanisms. Thus, NTZ counteracts varied virus-specific immune evasion strategies by generally enhancing the RNA sensing and interferon axis that is triggered by foreign cytoplasmic RNA exposure, and holds promise as an oral therapy against EBOV. Abstract In this review, we report that the receptor of mouse hepatitis virus (MHV), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), is an important determinant of mouse susceptibility to MHV infection. This finding was revealed by using mouse strains with two different allelic forms of the MHV receptor, Ceacam1a and Ceacam1b. Although previous studies indicated that susceptibility is determined by a single gene, Ceacam1, our recent work in gene-replaced mice with chimeric Ceacam1 pointed toward the involvement of other host factors (genes) in the susceptibility. Studies on mouse susceptibility to MHV, as well as the factors involved in their susceptibility, are overviewed. Abstract The protective efficacy of DNA plasmids encoding avian infectious bronchitis virus (IBV) S1, N, or M protein was investigated in chickens. Chickens were inoculated monovalently (with plasmid pVAX1-16S1, pVAX1-16M, or pVAX1-16N alone) or multivalently (combination of the three different plasmids, pVAX1-16S1/M/N). A prime-boost immunization protocol against IBV was developed. Chickens were immunized with the multivalent DNA vaccine twice and then boosted with an inactivated vaccine once. Antibody titers of the chickens immunized with pVAX1-16S1/M/N were much higher than those of the monovalent groups (p ＜ 0.01). A protective rate up to 90% was observed in the pVAX1-16S1/M/N group. The serum antibody titers in the prime-boost birds were significantly higher than those of the multivalent DNA vaccine group (p ＜ 0.01) but not significantly different compared to the inactivated vaccine group at 49 days of age. Additionally, the prime-boost group also showed the highest level of IBV-specific cellular proliferation compared to the monovalent groups (p ＜ 0.01) but no significant difference was found compared to the multivalent DNA vaccine group, and the prime-boost group completely protected from followed viral challenge. Abstract Artificial blood is an innovative concept of transfusion medicine where specifically designed compounds perform the task of transport and delivery of oxygen in the body to replace this function of allogenic human blood transfusion. Several molecules have been developed in the past few decades to achieve this objective and continous refinements are being continuously made in the quest of the ideal blood substitute. Currently, available technology manufactures artificial blood from haemoglobin obtained from outdated human/bovine blood (Haemoglobin Based Oxygen Carriers) or utilizing Perfluorocarbons. These synthetic blood substitutes are advantageous in that they do not require compatibility testing, are free from blood borne infections, have prolonged shelf life and do not require refrigeration. Artificial blood is projected to have a significant impact on the development of medical care in the future. It can complement the current blood products for transfusion and create a stable supply of safe and effective products. It is likely to reduce the requirements of blood transfusions drastically especially in settings of trauma and surgery thereby reducing the reliance on banked donated blood. Abstract We report a case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection in a 61-year-old businessman returning from Kuwait. The patient arrived there on August 16, 2018, developed watery diarrhea on August 28 (day 0), and came back to Korea on September 7 (day 10) as his condition worsened. Upon arrival, he complained of diarrhea and weakness, but denied any respiratory symptoms, and he directly went to visit an emergency room. Chest radiography revealed interstitial infiltrates in the lungs, and he was immediately transferred to an isolation unit. Quantitative real-time PCR analysis of sputum samples taken on day 11 returned positive for MERS-CoV. No secondary MERS-CoV infection was identified among people who had close contact with him. This case underscores the importance of a high index of suspicion of MERS-CoV infection in any febrile patients who present after a trip to the Middle East. Abstract Adrenal insufficiency is a rare, potentially life-threatening condition whose diagnosis requires a high index of suspicion. Adrenal insufficiency may be primary, secondary, or tertiary with varied etiologies. Primary insufficiency may be part of a cluster of autoimmune diseases, referred to as autoimmune polyglandular syndrome(s) (APS). We describe a case of a 15-year-old male who presents to a local emergency department complaining of fatigue, fever, abdominal pain, nausea, and vomiting for a few days with a preceding viral illness. The patient was hyponatremic and hyperkalemic with skin hyperpigmentation, raising concern for adrenal insufficiency. Laboratory workup confirmed autoimmune primary adrenal insufficiency, with subsequent laboratory studies revealing autoimmune thyroiditis and celiac disease. Concomitant Addison's and Hashimoto's diseases led to a diagnosis of APS type 2. The patient was started on steroid replacement with rapid clinical improvement. Abstract COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sarl GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP-and GTP-restricted forms of Sarl and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and Abstract In several EU member states, bovine spongiform encephalopathy (BSE) cases have been identified in cattle born after the reinforced ban (BARB cases), for reasons that are not entirely clear. Epidemiological investigation of these cases has proved challenging. The European Food Safety Authority recently recommended the collection of a predefined set of epidemiological data from BSE suspects and confirmed BSE cases to aid future investigations. In this study, we present an epidemiological framework and BSE investigation questionnaire to aid the investigation of suspect BSE cases, and illustrate its application during the investigation of a BSE case in Ireland in 2015. It is recommended that the framework and questionnaire are used concurrently: the framework provides structure and focus, whereas the questionnaire (with 135 questions) aids data collection. The framework focuses on confirmation and discrimination, estimating the date and location of exposure, and determining the method/ source of exposure. The BSE case in Ireland in 2015 was a BARB case born in 2010. It was identified with classical BSE at an authorised knackery as part of Ireland's targeted active surveillance programme for BSE. No definitive source of infection with the BSE agent could be attributed in this case. Abstract Since mid-December of 2019, coronavirus disease 2019 (COVID-19) has been spreading from Wuhan, China. The confirmed COVID-19 patients in South Korea are those who came from or visited China. As secondary transmissions have occurred and the speed of transmission is accelerating, there are rising concerns about community infections. The 54-year old male is the third patient diagnosed with COVID-19 in Korea. He is a worker for a clothing business and had mild respiratory symptoms and intermittent fever in the beginning of hospitalization, and pneumonia symptoms on chest computerized tomography scan on day 6 of admission. This patient caused one case of secondary transmission and three cases of tertiary transmission. Hereby, we report the clinical findings of the index patient who was the first to cause tertiary transmission outside China. Interestingly, after lopinavir/ritonavir (Kaletra, AbbVie) was administered, β-coronavirus viral loads significantly decreased and no or little coronavirus titers were observed. Abstract Purpose: Human adenovirus infection mimics Kawasaki disease (KD) but can be detected in KD patients. The aim of this study was to determine the clinical differences between KD with adenovirus infection and only adenoviral infection and to identify biomarkers for prediction of adenovirus-positive KD from isolated adenoviral infection. Methods: A total of 147 patients with isolated adenovirus were identified by quantitative polymerase chain reaction. In addition, 11 patients having KD with adenovirus, who were treated with intravenous immunoglobulin therapy during the acute phase of KD were also evaluated. Results: Compared with the adenoviral infection group, the KD with adenovirus group was significantly associated with frequent lip and tongue changes, skin rash and changes in the extremities. In the laboratory parameters, higher C-reactive protein (CRP) level and presence of hypoalbuminemia and sterile pyuria were significantly associated with the KD group. In the multivariate analysis, lip and tongue changes (odds ratio [OR], 1.416; 95% confidence interval [CI], 1.151-1.741; P=0.001), high CRP level (OR, 1.039; 95% CI 1.743-1.454; P= 0.021) and sterile pyuria (OR 1.052; 95% CI 0.861-1.286; P=0.041) were the significant predictive factors of KD. In addition, the cutoff CRP level related to KD with adenoviral detection was 56 mg/L, with a sensitivity of 81.8% and a specificity of 75.9%. Conclusion: Lip and tongue changes, higher serum CRP level and sterile pyuria were significantly correlated with adenovirus-positive KD. Abstract The large virus family Paramyxoviridae includes some of the most significant human and livestock viruses, such as measles-, distemper-, mumps-, parainfluenza-, newcastle disease-, respiratory syncytial virus and metapneumoviruses. Here we identify an estimated 66 new paramyxoviruses in a worldwide sample of 119 bat and rodent species (9,278 individuals). major discoveries include evidence of an origin of Hendra-and nipah virus in Africa, identification of a bat virus conspecific with the human mumps virus, detection of close relatives of respiratory syncytial virus, mouse pneumonia-and canine distemper virus in bats, as well as direct evidence of sendai virus in rodents. Phylogenetic reconstruction of host associations suggests a predominance of host switches from bats to other mammals and birds. Hypothesis tests in a maximum likelihood framework permit the phylogenetic placement of bats as tentative hosts at ancestral nodes to both the major Paramyxoviridae subfamilies (Paramyxovirinae and Pneumovirinae). Future attempts to predict the emergence of novel paramyxoviruses in humans and livestock will have to rely fundamentally on these data. Abstract To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RAbV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RAbV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RAbV virus titer. When cells pre-challenged with RAbV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RAbV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RAbV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RAbV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RAbV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RAbV multiplication, inhibited RAbV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies. Abstract Ascitic feline coronavirus (FCoV) RNA was examined in 854 cats with suspected feline infectious peritonitis (FIP) by RT-PCR. The positivity was significantly higher in purebreds (62.2%) than in crossbreds (34.8%) (P<0.0001). Among purebreds, the positivities in the Norwegian forest cat (92.3%) and Scottish fold (77.6%) were significantly higher than the average of purebreds (P=0.0274 and 0.0251, respectively). The positivity was significantly higher in males (51.5%) than in females (35.7%) (P<0.0001), whereas no gender difference has generally been noted in FCoV antibody prevalence, indicating that FIP more frequently develops in males among FCoV-infected cats. Genotyping was performed for 377 gene-positive specimens. Type I (83.3%) was far more predominantly detected than type II (10.6%) (P<0.0001), similar to previous serological and genetic surveys. Abstract Nosocomial transmission is an important characteristic of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection. Risk factors for transmission of MERS-CoV in healthcare settings are not well defined. During the Korean outbreak in 2015, 186 patients had laboratory-confirmed MERS-CoV infection. Those suspected as a source of viral transmission were categorized into the spreader groups (super-spreader [n = 5] and usualspreader [n = 10]) and compared to the non-spreader group (n = 171). Body temperature of ≥ 38.5°C (adjusted odds ratio [aOR], 5.54; 95% confidence interval [CI], 1.38-22.30; P = 0.016), pulmonary infiltration of ≥ 3 lung zones (aOR, 7.33; 95% CI, 1.93-27.79; P = 0.003), and a more nonisolated in-hospital days (aOR, 1.32 per 1 day; 95% CI, 1.09-1.60; P = 0.004) were significant risk factors in the spreader group. There was no different clinical factor between super-spreaders and usual-spreaders. Nonisolated in-hospital days was the only factor which tended to be higher in super-spreaders than usual-spreaders (Mean, 6.6 vs. 2.9 days; P = 0.061). Early active quarantine might help reducing the size of an outbreak. Abstract Proteins p38 map kinase and ribosomal S6 kinase (S6K) as members of mitogen-activated protein kinases (MAPKs) play important roles against pathogens. In this study, Bmp38 and BmS6K were identified as differentially expressed proteins from iTRAQ database. Bmp38 and BmS6K were expressed, and recombinant proteins were purified. The bioinformatics analysis showed that both proteins have serine/threonine-protein kinases, catalytic domain (S_ TKc) with 360 and 753 amino acids, respectively. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that Bmp38 and BmS6K had high expression in the midgut and hemolymph. The comparative expression level of Bmp38 and BmS6K in BC9 was upregulated than in P50 in the midgut after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Western bolt results showed a positive correlation between RT-qPCR and iTRAQ data for Bmp38, but BmS6K data showed partial correlation with iTRAQ. Injection of anti-Bmp38 and anti-BmS6K serum suggested that Bmp38 may be involved against BmNPV infection, whereas BmS6K may require phosphorylation modification to inhibit BmNPV infection. Taken together, our results suggest that Bmp38 and BmS6k might play an important role in innate immunity of silkworm against BmNPV. Abstract RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1 s enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an 'open' to a 'closed'-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change. Abstract Old mice will have a subset of lesions as part of the progressive decline in organ function that defines aging. External and palpable lesions will be noted by the research, husbandry, or veterinary staff during testing, cage changing, or physical exams. While these readily observable lesions may cause alarm, not all cause undue distress or are life-threatening. In aging research, mice are maintained until near end of life that, depending on strain and genetic manipulation, can be upwards of 33 months. Aging research has unique welfare issues related to age-related decline, debilitation, fragility, and associated pain of chronic diseases. An effective aging research program includes the collaboration and education of the research, husbandry, and veterinary staff, and of the members of the institution animal care and use committee. This collaborative effort is critical to humanely maintaining older mice and preventing excessive censorship due to non-lethal diseases. Part of the educational process is becoming familiar with how old mice appear clinically, at necropsy and histopathologically. This baseline knowledge is important in making the determination of humane end points, defining health span, contributing causes of death and effects of interventions. The goal of this paper is to introduce investigators to age-associated diseases and lesion patterns in mice from clinical presentation to pathologic assessment. To do so, we present and illustrate the common clinical appearances, necropsy and histopathological lesions seen in subsets of the aging colonies maintained at the University of Washington. Abstract Autophagy is a homeostatic mechanism that discards not only invading pathogens but also damaged organelles and denatured proteins via lysosomal degradation. Increasing evidence suggests a role for autophagy in inflammatory diseases, including infectious diseases, Crohn's disease, cystic fibrosis, and pulmonary hypertension. These studies suggest that modulating autophagy could be a novel therapeutic option for inflammatory diseases. Eosinophils are a major type of inflammatory cell that aggravates airway inflammatory diseases, particularly corticosteroid-resistant inflammation. The eosinophil count is a useful tool for assessing which patients may benefit from inhaled corticosteroid therapy. Recent studies demonstrate that autophagy plays a role in eosinophilic airway inflammatory diseases by promoting airway remodeling and loss of function. Genetic variant in the autophagy gene ATG5 is associated with asthma pathogenesis, and autophagy regulates apoptotic pathways in epithelial cells in individuals with chronic obstructive pulmonary disease. Moreover, autophagy dysfunction leads to severe inflammation, especially eosinophilic inflammation, in chronic rhinosinusitis. However, the mechanism underlying autophagy-mediated regulation of eosinophilic airway inflammation remains unclear. The aim of this review is to provide a general overview of the role of autophagy in eosinophilic airway inflammation. We also suggest that autophagy may be a new therapeutic target for airway inflammation, including that mediated by eosinophils. Abstract We have characterized the structure, biogenesis, and localization of dipeptidyl aminopeptidase B (DPAP B), a membrane protein of the yeast vacuole. An antibody specific for DPAP B recognizes a 120-kD glycoprotein in yeast that behaves like an integral membrane protein in that it is not removed from membranes by high pH Na2COs treatment. Inspection of the deduced amino acid sequence of DPAP B reveals a hydrophobic domain near the NH2 terminus that could potentially span a lipid bilayer. The in vitro enzymatic activity and apparent molecular weight of DPAP B are unaffected by the allelic state of PEP4, a gene essential for the proteolytic activation of a number of soluble vacuolar hydrolases. DPAP B is synthesized as a glycosylated precursor that is converted to the mature 120-kD species by carbohydrate addition. The precur-J. H. Rothman's present address is Abstract These authors contributed equally to the work.In eukaryotes and viruses that infect them, the 5 0 end of mRNA molecules, and also many other functionally important RNAs, are modified to form a so-called cap structure that is important for interactions of these RNAs with many nuclear and cytoplasmic proteins. The RNA cap has multiple roles in gene expression, including enhancement of RNA stability, splicing, nucleocytoplasmic transport, and translation initiation. Apart from guanosine addition to the 5 0 end in the most typical cap structure common to transcripts produced by RNA polymerase II (in particular mRNA), essentially all cap modifications are due to methylation. The complexity of the cap structure and its formation can range from just a single methylation of the unprocessed 5 0 end of the primary transcript, as in mammalian U6 and 7SK, mouse B2, and plant U3 RNAs, to an elaborate m 7 Gpppm 6,6 AmpAmpCmpm 3 Um structure at the 5 0 end of processed RNA in trypanosomes, which are formed by as many as 8 methylation reactions. While all enzymes responsible for methylation of the cap structure characterized to date were found to belong to the same evolutionarily related and structurally similar Rossmann Fold Methyltransferase superfamily, that uses the same methyl group donor, S-adenosylmethionine; the enzymes also exhibit interesting differences that are responsible for their distinct functions. This review focuses on the evolutionary classification of enzymes responsible for cap methylation in RNA, with a focus on the sequence relationships and structural similarities and dissimilarities that provide the basis for understanding the mechanism of biosynthesis of different caps in cellular and viral RNAs. Particular attention is paid to the similarities and differences between methyltransferases from human cells and from human pathogens that may be helpful in the development of antiviral and antiparasitic drugs. Abstract The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The results indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully. Abstract RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNa/b production. In an attempt to identify novel tools that modulate IFNa/b production, we used SELEX technology to screen RNA aptamers that specifically target RIG-I protein.Most of the selected RIG-I aptamers contained polyU motifs in the second half regions that played critical roles in the activation of RIG-I-mediated IFNb production. Unlike other known ligands, RIG-I aptamer bound and activated RIG-I in a 5 0 -triphosphateindependent manner. The helicase and RD domain of RIG-I were used for aptamer binding, but intact RIG-I protein was required to exert aptamermediated signaling activation. Furthermore, replication of NDV, VSV and influenza virus in infected host cells was efficiently blocked by pre-or posttreatment with RIG-I aptamer. Based on these data, we propose that RIG-I aptamer has strong potential to be an antiviral agent that specifically boosts the RIG-I-dependent signaling cascade. Abstract Development of RNAi-based therapeutics has the potential to revolutionize treatment options for a range of human diseases. However, as with gene therapy, a major barrier to progress is the lack of methods to achieve and measure efficient delivery for systemic administration. We have developed a positive-readout pharmacodynamic transgenic reporter mouse model allowing noninvasive real-time assessment of siRNA activity. The model combines a luciferase reporter gene under the control of regulatory elements from the lac operon of Escherichia coli. Introduction of siRNA targeting lac repressor results in increased luciferase expression in cells where siRNA is biologically active. Five founder luciferase-expressing and three founder Lacexpressing lines were generated and characterized. Mating of ubiquitously expressing luciferase and lac lines generated progeny in which luciferase expression was significantly reduced compared with the parental line. Administration of isopropyl β-D-1-thiogalactopyranoside either in drinking water or given intraperitoneally increased luciferase expression in eight of the mice examined, which fell rapidly when withdrawn. Intraperitoneal administration of siRNA targeting lac in combination with Lipofectamine 2000 resulted in increased luciferase expression in the liver while control nontargeting siRNA had no effect. We believe a sensitive positive readout pharmacodynamics reporter model will be of use to the research community in RNAi-based vector development. Abstract Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT. Abstract Application of plant-based protein expression systems for bulk production of recombinant protein pharmaceuticals is building momentum. There are considerable regulatory challenges to consider in commercialization of plant-made pharmaceuticals (PMPs), some of which are inherent to plant-production systems and others that are common with other production systems, but are new to PMPs because of the youth of the industry. In this review, we discuss our recent and ongoing experience with bulk production of the HIV microbicide candidate, griffithsin (GRFT), utilizing plant-based transient protein expression, with specific focus on areas relevant to commercial manufacturing of bulk GRFT active pharmaceutical ingredient (API). Analytical programs have been developed for the qualification and monitoring of both the expression vector system and the API detailing our experience and plans for each. Monitoring postpurification protein modifications are discussed in relation to stability and safety programs. Expression, processing and analytics programs are associated with increased manufacturing costs in current good manufacturing practice (cGMP) production because of the required qualification testing. The impact of these costs on the overall cost of goods is particularly relevant to GRFT manufacturing because GRFT, as an HIV microbicide, is most needed in populations at high risk for HIV exposure in resource-poor countries. Consequently, GRFT for microbicide applications is a very cost-sensitive recombinant PMP. We have therefore emphasized maintaining a low cost of goods. We provide a review of the literature on the economics of PMPs with various expression systems and how they may impact production costs and complexity.1160 Abstract Today, accredited zoos are not just places for entertainment, they are actively involved in research for conservation and health. During recent decades in which the challenges for biodiversity conservation and public health have escalated, zoos have made significant changes to address these difficulties. Zoos increasingly have four key areas of focus: education, recreation, conservation, and research. These key areas are important in addressing an interrelated global conservation (i.e. habitat and wildlife loss) and public health crisis. Zoo and public health professionals working together within a One Health framework represent a powerful alliance to address current and future conservation and public health problems around the world. For researchers, practitioners, and students, the collaboration between zoos and public health institutions offers the opportunity to both teach and operationalize this transdisciplinary approach. Using examples from our programs, we give a template for moving forward with collaborative initiatives and sustainable solutions involving partners in both zoos and public health institutions. We provide examples of cooperative programs and suggest a model for consideration in the development of further activities in this area. Abstract In vitro cytotoxicity testing has become an integral aspect of drug discovery because it is a convenient, costeffective, and predictive means of characterizing the toxic potential of new chemical entities. The early and routine implementation of this testing is testament to its prognostic importance for humans. Although a plethora of assay chemistries and methods exist for 96-well formats, few are practical and sufficiently sensitive enough for application in high throughput screening (HTS). Here we briefly describe a handful of the currently most robust and validated HTS assays for accurate and efficient assessment of cytotoxic risk. We also provide guidance for successful HTS implementation and discuss unique merits and detractions inherent in each method. Lastly, we discuss the advantages of combining specific HTS compatible assays into multi-parametric, same-well formats. Abstract Regulations (IHR) aim to prevent, detect and respond to such threats, through increase in national public health core capacities, but whether IHR core capacity implementation is necessary and sufficient has been contested. With a longitudinal study we relate changes in national IHR core capacities to changes in cross-border infectious disease threat events (IDTE) between 2010 and 2016, collected through epidemic intelligence at the European Centre for Disease Prevention and Control (ECDC). By combining all IHR core capacities into one composite measure we found that a 10% increase in the mean of this composite IHR core capacity to be associated with a 19% decrease (p = 0.017) in the incidence of cross-border IDTE in the EU.With respect to specific IHR core capacities, an individual increase in national legislation, policy & financing; coordination and communication with relevant sectors; surveillance; response; preparedness; risk communication; human resource capacity; or laboratory capacity was associated with a significant decrease in cross-border IDTE incidence. In contrast, our analysis showed that IHR core capacities relating to point-of-entry, zoonotic events or food safety were not associated with IDTE in the EU. Due to high internal correlations between core capacities, we conducted a principal component analysis which confirmed a 20% decrease in risk of IDTE for every 10% increase in the core capacity score (95% CI: 0.73, 0.88). Globally (EU excluded), a 10% increase in the mean of all IHR core capacities combined was associated with a 14% decrease (p = 0.077) in cross-border IDTE incidence. We provide quantitative evidence that improvements in IHR core capacities at country-level are associated with fewer cross-border IDTE in the EU, which may also hold true for other parts of the world.epidemic, infectious diseases, International Health Regulations, outbreak, pandemic, threat events This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmerc ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Abstract A severe respiratory ongoing outbreak of pneumonia associated with 2019 novel coronavirus was recently emerged in China. Here, we reported the epidemiological, clinical, laboratory and radiological characteristics of 19 suspect cases. We compared J o u r n a l P r e -p r o o f Abstract Unlike those infected by H5N1, birds infected by the newly discovered H7N9 virus have no observable clinical symptoms. Public health workers in China do not know where the public health threat lies. In this study, we used a distributed focused crawler to analyze online knowledge of the live bird trade in first-wave provinces, namely, Jiangsu, Zhejiang, Anhui, and Shanghai, to track the new H7N9 virus and predict its spread. Of the 18 provinces proposed to be at high risk of infection, 10 reported human infections and one had poultry specimens that tested positive. Five provinces (Xinjiang, Yunnan, Guizhou, Shaanxi, and Tibet) as well as Hong Kong, Macao, and Taiwan were proposed to have no risk of H7N9 virus infection from the live bird trade. These data can help health authorities and the public to respond rapidly to reduce damage related to the spread of the virus. Abstract Site-directed mutagenesis in vitro was used to introduce stop codons in the genomic DNA of the a and/3 chains of the murine class II major histocompatibility complex antigen, I-A k. Mutated DNA was transfected into B lymphoma cells that were then selected by neomycin resistance and for their ability to express I-A k molecules on their plasma membrane. The translational diffusion coefficient (Dtat) of I-A k molecules composed of a wild-type/3 chain paired with an ot chain missing either 6 or 12 amino acids from the cytoplasmic domain is on the average threefold higher than the D,,, of wild-type I-A k molecules as measured by fluorescence photobleaching and recovery. The removal of 12 amino acids from the cytoplasmic domain of the/3 chain did not change the D~a~ value from that of wild-type I-A k if the truncated/3 chain was paired with a wild-type c~ chain. Removing all amino acids of the cytoplasmic domains of both the ot and/3 chains resulted in a 10-fold increase in the Dtat, the highest value for any of the truncated I-A k molecules tested. These data indicate that the carboxy-terminal six amino acids of the cytoplasmic domain of the et chain and the six plasma membrane-proximal amino acids of the/3 chain are important in constraining the translational diffusion of I-A k molecules in the plasma membrane. Abstract X-box binding protein 1 (XBP-1) is a key regulator required for cellular unfolded protein response (UPR) and plasma cell differentiation. In addition, involvement of XBP-1 in host cell-virus interaction and transcriptional regulation of viruses, such as human T-lymphotropic virus type 1 (HTLV-1), has been revealed recently. Two XBP-1 isoforms, XBP-1U and XBP-1S, which share an identical N-terminal domain, are present in cells. XBP-1S is a transcription activator while XBP-1U is the inactive isoform. Although the transactivation domain of XBP-1S has been identified within the XBP-1Sspecific C-terminus, molecular mechanism of the transcriptional activation by XBP-1S still remains unknown. Here we report the interaction between p300/CBP-associated factor (PCAF) and XBP-1S through the C-terminal domain of XBP-1S. No binding between XBP-1U and PCAF is detected. In a cell-based reporter assay, overexpression of PCAF further stimulates the XBP-1S-mediated cellular and HTLV-1 transcription while knockdown of PCAF exhibits the opposite effect. Expression of endogenous XBP-1S cellular target genes, such as BiP and CHOP, is significantly inhibited when PCAF is knocked down. Furthermore, PCAF is recruited to the promoters of XBP-1S target genes in vivo, in a XBP-1S-dependent manner. Collectively, our results demonstrate that PCAF mediates the XBP-1Sdependent transcription through the interaction with XBP-1S. Abstract Error-free replication and repair of DNA are pivotal to organisms for faithful transmission of their genetic information. Cells orchestrate complex signaling networks that sense and resolve DNA damage. Post-translational protein modifications by ubiquitin and ubiquitin-like proteins, including SUMO and NEDD8, are critically involved in DNA damage response (DDR) and DNA damage tolerance (DDT). The expression of interferon-stimulated gene 15 (ISG15), the first identified ubiquitin-like protein, has recently been shown to be induced under various DNA damage conditions, such as exposure to UV, camptothecin, and doxorubicin. Here we overview the recent findings on the role of ISG15 and its conjugation to target proteins (e.g., p53, Np63, and PCNA) in the control of cellular responses to genotoxic stress, such as the inhibition of cell growth and tumorigenesis. Abstract Copy number variations (CNVs) in the human genome are conventionally detected using highthroughput scanning technologies, such as comparative genomic hybridization and high-density single nucleotide polymorphism (SNP) microarrays, or relatively low-throughput techniques, such as quantitative polymerase chain reaction (PCR). All these approaches are limited in resolution and can at best distinguish a twofold (or 50%) difference in copy number. We have developed a new technology to study copy numbers using a platform known as the digital array, a nanofluidic biochip capable of accurately quantitating genes of interest in DNA samples. We have evaluated the digital array's performance using a model system, to show that this technology is exquisitely sensitive, capable of differentiating as little as a 15% difference in gene copy number (or between 6 and 7 copies of a target gene). We have also analyzed commercial DNA samples for their CYP2D6 copy numbers and confirmed that our results were consistent with those obtained independently using conventional techniques. In a screening experiment with breast cancer and normal DNA samples, the ERBB2 gene was found to be amplified in about 35% of breast cancer samples. The use of the digital array enables accurate measurement of gene copy numbers and is of significant value in CNV studies. Abstract The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamateenhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondriarelated pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases. Abstract The need for multidisciplinary research to address today's complex health and environmental challenges has never been greater. The One Health (OH) approach to research ensures that human, animal, and environmental health questions are evaluated in an integrated and holistic manner to provide a more comprehensive understanding of the problem and potential solutions than would be possible with siloed approaches. However, the OH approach is complex, and there is limited guidance available for investigators regarding the practical design and implementation of OH research. In this paper we provide a framework to guide researchers through conceptualizing and planning an OH study. We discuss key steps in designing an OH study, including conceptualization of hypotheses and study aims, identification of collaborators for a multi-disciplinary research team, study design options, data sources and collection methods, and analytical methods. We illustrate these concepts through the presentation of a case study of health impacts associated with land application of biosolids. Finally, we discuss opportunities for applying an OH approach to identify solutions to current global health issues, and the need for cross-disciplinary funding sources to foster an OH approach to research. Abstract The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in September 2012 in Saudi Arabia had attracted the attention of the global health community. In 2017 the Saudi Ministry of Health released a visual triage system with scoring to alert healthcare workers in emergency departments (EDs) and haemodialysis units for the possibility of occurrence of MERS-CoV infection. We performed a retrospective analysis of this visual score to determine its sensitivity and specificity. The study included all cases from 2014 to 2017 in a MERS-CoV referral centre in Riyadh, Saudi Arabia. During the study period there were a total of 2435 suspected MERS cases. Of these, 1823 (75%) tested negative and the remaining 25% tested positive for MERS-CoV by PCR assay. The application of the visual triage score found a similar percentage of MERS-CoV and non-MERS-CoV patients, with each score from 0 to 11. The percentage of patients with a cutoff score of 4 was 75% in patients with MERS-CoV infection and 85% in patients without MERS-CoV infection (p 0.0001). The sensitivity and specificity of this cutoff score for MERS-CoV infection were 74.1% and 18.6%, respectively. The sensitivity and specificity of the scoring system were low, and further refinement of the score is needed for better prediction of MERS-CoV infection. Abstract Here we introduce the Computational Recognition of Secondary Structure (CROSS) method to calculate the structural profile of an RNA sequence (singleor double-stranded state) at single-nucleotide resolution and without sequence length restrictions. We trained CROSS using data from high-throughput experiments such as Selective 2 -Hydroxyl Acylation analyzed by Primer Extension (SHAPE; Mouse and HIV transcriptomes) and Parallel Analysis of RNA Structure (PARS; Human and Yeast transcriptomes) as well as high-quality NMR/X-ray structures (PDB database). The algorithm uses primary structure information alone to predict experimental structural profiles with >80% accuracy, showing high performances on large RNAs such as Xist (17 900 nucleotides; Area Under the ROC Curve AUC of 0.75 on dimethyl sulfate (DMS) experiments). We integrated CROSS in thermodynamics-based methods to predict secondary structure and observed an increase in their predictive power by up to 30%.RNA molecules (29). We envisage that the combination of CROSS with thermodynamics-based approaches will be the key ingredient to improve predictions of RNA structure.CROSS is freely available at http://service.tartaglialab.com/ new submission/cross. Abstract Background: Nonantibiotic alternatives providing local gut immunity have been recommended for managing calf diarrhea.Animals: One hundred and two calves with diarrhea.Hypothesis: Oral supplementation with immunoglobulins in calves with diarrhea will reduce time to resolution of diarrhea, number of treatment events, and mortality rate.Methods: Randomized controlled trial. Calves were assigned into 1 of 3 groups. The treatment group was supplemented with 20 g of immunoglobulins in milk twice daily for 14 days. The placebo group was supplemented with 20 g of a product with similar nutritional value as the treatment group, but without immunoglobulins, in milk, twice daily for 14 days. The control group received no supplements. Medical treatments, time to resolution of diarrhea, and case fatality rates were compared.Results: There was no difference in the proportion of treatment events (treatment, 79% versus placebo, 77% versus control, 71%) among groups (P = .69). The median time to resolution of diarrhea was not different between the treatment (10.5 days; 95% confidence interval [CI], 7, 13) and control (8 days; 95% CI, 5, 10) groups (P = .08) or between the placebo (6.5 days; 95% CI, 3, 9) and control groups (P = .89).Median time to resolution was shorter (P = .008) in the placebo compared to the treatment group (6.5 versus 10.5 days). Case fatality rates among groups (treatment, 12% versus placebo, 3% versus control, 3%) were not different (P = .36).Conclusions and Clinical Importance: Expected benefits of conferring local gut immunity by immunoglobulin supplementation in calves with diarrhea were not evident.cattle, colostrum, fecal score, immunity, milk Abstract are useful and will be translated into actions and change in the system. Based on Taiwan's experiences, early stakeholder engagement and an experts' pre-JEE pilot visit would contribute to a successful JEE process. Abstract Surfactant proteins SP-A and SP-D are hydrophilic, collagen-containing calcium-dependent lectins, which appear to have a range of innate immune functions at pulmonary as well as extrapulmonary sites. These proteins bind to target ligands on pathogens, allergens, and apoptotic cells, via C-terminal homotrimeric carbohydrate recognition domains, while the collagen region brings about the effector functions via its interaction with cell surface receptors. SP-A and SP-D deal with various pathogens, using a range of innate immune mechanisms such as agglutination/aggregation, enhancement of phagocytosis, and killing mechanisms by phagocytic cells and direct growth inhibition. SP-A and SP-D have also been shown to be involved in the control of pulmonary inflammation including allergy and asthma. Emerging evidence suggest that SP-A and SP-D are capable of linking innate immunity with adaptive immunity that includes modulation of dendritic cell function and helper T cell polarization.This review enumerates immunological properties of SP-A and SP-D inside and outside lungs and discusses their importance in human health and disease. Abstract In 2010, Yemen started the surveillance for severe acute respiratory infections (SARIs) by establishing 2 sentinel sites in Sana'a and Aden city. This study aims to determine the proportions of influenza and noninfluenza viruses among SARI patients and to determine the severity of SARI and its associated factors. The data of SARI patients who were admitted to SARI surveillance sites at Al Johory hospital in Sana'a and Al Wahdah hospital in Aden city during the period 2011-2016 were analyzed. The proportions of positive influenza viruses (type A, B) and noninfluenza viruses (respiratory syncytial, adenovirus, human parainfluenza, and human metapneumovirus), intensive care unit (ICU) admission rate, and fatality rate among SARI patients were calculated. A total of 1811 of SARI patients were admitted during 2011-2016. Of those, 78% were <15 years old. A total of 89 (5%) patients had influenza viruses and 655 (36%) had noninfluenza viruses. The overall ICU admission rate was 40% and the case-fatality rate was 8%. Infection by influenza type (A, B) and mixed (adenovirus, human parainfluenza) was significantly associated with lower ICU admission. Age <15 years old, infection with influenza B, pre-existence of chronic diseases, and admission to Aden site were significantly associated with higher fatality rate among patients. In conclusion; SARI patients in Yemen had a high ICU admission and case-fatality rates. Influenza type B, chronic diseases, and admission to Aden site are associated with higher fatality rate. Expanding surveillance sites and panel of laboratory tests to involve other pathogens will help to provide accurate diagnosis for SARI etiology and give more comprehensive picture. Training staff for SARI case management will help to reduce severe outcomes. Abstract One sentence summary: We consider various approaches to develop the toxins of the type II family as possible candidates to drug discovery; druggability of toxins-antitoxins could be possible as antivirals. As antibacterials, they might be considered as druggable but delivery and formulation may not be simple so far.Editor: Grzegorz WegrzynType II (proteic) toxin-antitoxin (TA) operons are widely spread in bacteria and archaea. They are organized as operons in which, usually, the antitoxin gene precedes the cognate toxin gene. The antitoxin generally acts as a transcriptional self-repressor, whereas the toxin acts as a co-repressor, both proteins constituting a harmless complex. When bacteria encounter a stressful environment, TAs are triggered. The antitoxin protein is unstable and will be degraded by host proteases, releasing the free toxin to halt essential processes. The result is a cessation of cell growth or even death. Because of their ubiquity and the essential processes targeted, TAs have been proposed as good candidates for development of novel antimicrobials. We discuss here the possible druggability of TAs as antivirals and antibacterials, with focus on the potentials and the challenges that their use may find in the 'real' world. We present strategies to develop TAs as antibacterials in view of novel technologies, such as the use of very small molecules (fragments) as inhibitors of protein-protein interactions. Appropriate fragments could disrupt the T:A interfaces leading to the release of the targeted TA pair. Possible ways of delivery and formulation of Tas are also discussed. Abstract The Middle East respiratory syndrome coronavirus (MERS-CoV) presents an ideal example for developing One Health concepts. Dromedary camels are the principal reservoir for the virus. Infected camels shed the virus in body secretions, particularly nasal discharges. MERS-CoV has the potential to remain active in the environment for some time under optimum conditions of temperature and humidity. This shedding sustains the virus in endemic communities and thus contact with camels is considered a major risk factor for human infection. Reducing virus shedding from camels will have a great positive impact on reducing the human risk of infection. Our main objective is to highlight the potential aspects of reducing virus shedding from camels to the environment, thereby reducing the possibility of human infection. We will focus on the potential roles of camel markets, camel shows, importation, transportation and grazing in the amplification and shedding of the virus, providing some novel concepts for the control approaches for the MERS-CoV. Abstract Background: New approaches are urgently needed to fight influenza viral infection. Previous research has shown that zirconia nanoparticles can be used as anticancer materials, but their antiviral activity has not been reported. Here, we investigated the antiviral effect of zirconia (ZrO 2 ) nanoparticles (NPs) against a highly pathogenic avian influenza virus. Materials and Methods: In this study, the antiviral effects of ZrO 2 on H5N1 virus were assessed in vivo, and the molecular mechanism responsible for this protection was investigated. Results: Mice treated with 200 nm positively-charged NPs at a dose of 100 mg/kg showed higher survival rates and smaller reductions in weight. 200 nm ZrO 2 activated mature dendritic cells and initially promoted the expression of cytokines associated with the antiviral response and innate immunity. In the lungs of H5N1-infected mice, ZrO 2 treatment led to less pathological lung injury, significant reduction in influenza A virus replication, and overexpression of pro-inflammatory cytokines. Conclusion: This antiviral study using zirconia NPs shows protection of mice against highly pathogenic avian influenza virus and suggests strong application potential for this method, introducing a new tool against a wide range of microbial infections. Abstract Journal homepage: http://www.kcdcphrp.orgObjectives: This research aimed to understand the popularity of topics in the field of women's health through analysis of online news articles which were chronologically classified and examined to determine how women's health and diseases had changed over time. Methods: Women's health and disease news articles were collated from a popular news website between 1993 to 2015 and preprocessed using gynecological medical terminology, Korean words and nouns (excluding general nouns not related to women's healthcare topics). The resultant articles (N = 7,710) were analyzed using the Latent Dirichlet Allocation algorithm and major topics were extracted. Topic trends were analyzed by year and period for women's health. Results: It was observed that most of the women's health articles were focused on "Healthcare", and 9 other topics were identified that represented a relatively small proportion in Abstract Since May 2013, outbreaks of porcine epidemic diarrhea have devastated the U.S. swine industry, causing immense economic losses. Two different swine enteric coronaviruses (porcine epidemic diarrhea virus and Delta coronavirus) have been isolated from the affected swine population. The disease has been reported from at least 32 states of the United States and other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, Ecuador, and Ukraine, with repeated outbreaks in previously infected herds. Here we report the isolation and characterization of a novel mammalian orthoreovirus 3 (MRV3) from diarrheic feces of piglets from these outbreaks in three states and ring-dried swine blood meal from multiple sources. MRV3 could not be isolated from healthy or pigs that had recovered from epidemic diarrhea from four states. Several MRV3 isolates were obtained from chloroform-extracted pig feces or blood meal in cell cultures or developing chicken embryos. Biological characterization of two representative isolates revealed trypsin resistance and thermostability at 90°C. NextGen sequencing of ultrapurified viruses indicated a strong homology of the S1 segment to mammalian and bat MRV3. Neonatal piglets experimentally infected with these viruses or a chloroform extract of swine blood meal developed severe diarrhea and acute gastroenteritis with 100% mortality within 3 days postinfection. Therefore, the novel porcine MRV3 may contribute to enteric disease along with other swine enteric viruses. The role of MRV3 in the current outbreaks of porcine epidemic diarrhea in the United States remains to be determined, but the pathogenic nature of the virus warrants further investigations on its epidemiology and prevalence.We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea. X-J, Elankumaran S. 2015. A novel pathogenic mammalian orthoreovirus from diarrheic pigs and swine blood meal in the United States. mBio 6(3):e00593-15. Abstract Objective: The aim of this study was to determine the etiology and prevalence of pediatric CAP in Beijing using a real-time polymerase chain reaction (PCR) technique. Methods: Between February 15, 2011 and January 18, 2012, 371 pediatric patients with CAP were enrolled at Beijing Children's Hospital. Sixteen respiratory viruses and two bacteria were detected from tracheal aspirate specimens using commercially available multiplex real-time reverse transcription PCR (RT-PCR) kits. Results: A single viral pathogen was detected in 35.3% of enrolled patients, multiple viruses in 11.6%, and virus/bacteria coinfection in 17.8%. In contrast, only 6.5% of patients had a single bacterial pathogen and 2.2% were infected with multiple bacteria. The etiological agent was unknown for 26.7% of patients. The most common viruses were respiratory syncytial virus (RSV) (43.9%), rhinovirus (14.8%), parainfluenza virus (9.4%), and adenovirus (8.6%). In patients under three years of age, RSV (44.6%), rhinovirus (12.8%), and Streptococcus pneumoniae (9.9%) were the most frequent pathogens. In children aged 3e7 years, S. pneumoniae (38.9%), RSV (30.6%), Haemophilus influenzae (19.4%), and adenovirus (19.4%) were most prevalent. Finally in children over seven years, RSV (47.3%), S. pneumoniae (41.9%), and rhinovirus (21.5%) infections were most frequent. Conclusions: Viral pathogens, specifically RSV, were responsible for the majority of CAP in pediatric patients. However, both S. pneumoniae and H. influenzae contributed as major causes of disease. Commercially available multiplexing real-time PCR allowed for rapid detection of the etiological agent. Abstract Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1 st or the 3 rd position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3 rd position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV-and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation. Abstract In this study the toxicity of antimalarial drug chloroquine (CQ) on certain enzymological (GOT, GPT and LDH) and histopathological alterations (Gill, liver and kidney) of a freshwater fish Cyprinus carpio was studied after acute (96 h) and sublethal (35 days) exposure. The median lethal concentration (96 h) of CQ was 31.62 mg/ml. During acute treatment (CQ at 31.62 mg/ml) the treated fish groups showed a significant increase in GOT and GPT activities in blood plasma; whereas LDH activity was decreased when compare to control groups. To analyse the effects of drug at the lowest concentration, the fish were exposed to 3.16 mg/ml (1/10th of 96 h LC50 value) for 96 h. In sublethal treatment (3.16 mg/ml) GOT activity increased up to 14th day and decreased during the rest of the exposure period (21, 28 and 35th day). A biphasic response in GPT activity was observed. LDH activity was found to be increased throughout the study period (35 days) compare to control groups. The alterations in enzyme activities in blood plasma were found to be significant at p < 0.05 (DMRT). Many histopathological changes in vital organs such as gill, liver and kidney of fish were observed in CQ treated group (acute and sub-lethal) compare to normal group. The alterations in the enzymological and histopathological study in the present investigation indicate that the drug CQ has toxic effects on non-target organisms. We conclude that the alterations in enzymological parameters and histopathological changes can be used as biomarker to assess the health of the aquatic organism/environment. Further data on molecular studies are needed to define the mode of action and toxicity of these emerging pollutants. Abstract During the 2015 outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Korea, 186 persons were infected, resulting in 38 fatalities. We isolated MERS-CoV from the oropharyngeal sample obtained from a patient of the outbreak. Cytopathic effects showing detachment and rounding of cells were observed in Vero cell cultures 3 days after inoculation of the sample. Spherical virus particles were observed by transmission electron microscopy. Full-length genome sequence of the virus isolate was obtained and phylogenetic analyses showed that it clustered with clade B of MERS-CoV. Abstract This study examined links among unrealistic optimism, sex, and risk perception of type 2 diabetes onset in college students. Participants included 660 college students who consented to complete a questionnaire. The results showed significant differences between students who perceived that they were at risk for type 2 diabetes onset and those who thought their peers were the ones at risk. A higher prevalence of participants thought their peers were the ones at risk for type 2 diabetes. Women were more likely than men to report a higher risk perception, indicating that their peers were at lower risk for diabetes onset. Abstract Infection of Lewis rats with the murine coronavirus JHM leads to a persistent infection in the central nervous system (CNS) and a disease process that shares some features with experimental allergic encephalomyelitis (EAE) (1-4). After an incubation period of weeks, a subacute demyelinating encephalomyelitis (SDE) develops that is characterized by paralysis (2). Histologically, the main lesion consists of a primary demyelination associated with perivascular cuffings and a cellular infiltration of macrophages as well as helper and cytotoxic T lymphocytes (1, 2) . SDE animals reveal a cell-mediated immune (CMI) response to myelin basic protein (MBP) as documented in adoptive transfer experiments (1). In addition, genetic experiments demonstrate that Brown-Norway (BN) rats are resistant to SDE, similar to what has been observed in EAE (4) . These findings indicate an immunopathological reaction in coronavirus-induced SDE in Lewis rats that is lacking in BN rats.A CMI response is genetically controlled at at least two levels : (a) the ability of the animal to generate an encephalolytogenic T cell response to antigen within the brain (foreign or autoantigen) from its genetically determined T cell repertoire and (b) the degree to which the antigen is presented to these T cells in the context of class I and II (Ia) histocompatibility molecules within a particular tissue . Class II-restricted T cells initiate an immune response to infection and both class I-and II-restricted T cells provide cytolysis of infected cells (5). Since la molecules are normally lacking on cells of the brain (6), the induction of la within the brain is crucial for the initial local presentation of viral antigen during infection . It may therefore be important that astrocytes of Lewis rat brain can be induced to express la directly by JHM virus (7, 8) as well as by other viruses. IFN-y subsequently released by antigen-activated T cells can further induce the antigen-presenting function of astrocytes (9-11) .The direct induction of la on astrocytes by JHM virus is also thought to play a crucial role in eliciting the excessive T cell response in susceptible rats . Supporting this notion are recent observations by us (12) showing that astrocytes of rat and mouse strains susceptible to EAE express much higher levels of la than astrocytes of resistant strains after treatment with IFN-y. We now extend these studies and analyze JHM virus induction of la on astrocytes of rats susceptible or resistant to SDE and show that, unlike astrocytes of susceptible Lewis rats, induction of astrocyte la by JHM virus is not detectable in the resistant BN rat. J. Exp. MED. Abstract Case summary A 2-year-old male domestic shorthair cat presented to the University of Liverpool Small Animal Teaching Hospital with a 2 week history of altered mentation, blindness and focal epileptic seizures. MRI examination revealed generalised cerebral and cerebellar atrophy, diffuse T2-weighted hyperintensity of the white matter and meningeal thickening. Neuronal ceroid lipofuscinosis was confirmed on post-mortem examination. Relevance and novel information This is the first report of the MRI findings of neuronal ceroid lipofuscinosis in a cat. Abstract The twentieth century was marked by extraordinary advances in our understanding of microbes and infectious disease, but pandemics remain, food and waterborne illnesses are frequent, multidrug-resistant microbes are on the rise, and the needed drugs and vaccines have not been developed. The scientific approaches of the past-including the intense focus on individual genes and proteins typical of molecular biology-have not been sufficient to address these challenges. The first decade of the twenty-first century has seen remarkable innovations in technology and computational methods. These new tools provide nearly comprehensive views of complex biological systems and can provide a correspondingly deeper understanding of pathogen-host interactions. To take full advantage of these innovations, the National Institute of Allergy and Infectious Diseases recently initiated the Systems Biology Program for Infectious Disease Research. As participants of the Systems Biology Program, we think that the time is at hand to redefine the pathogen-host research paradigm. Abstract Rationale: HSV is one of the most widespread human viral pathogens. HSV-1 infects a large portion of the human population and causes severe diseases. The current clinical treatment for HSV-1 is based on nucleoside analogues, the use of which is limited due to drug resistance, side effects and poor bioavailability. AMPs have been identified as potential antiviral agents that may overcome these limitations. Therefore, we screened anti-HSV-1 peptides from a scorpion-derived AMP library and engineered one candidate into a histidine-rich peptide with significantly improved antiviral activity and development potential. Methods: A venomous gland cDNA library was constructed from the scorpion Euscorpiops validus in the Yunnan Province of China. Six putative AMPs were characterized from this cDNA library, and the synthesized peptides were screened via plaque-forming assays to determine their virucidal potential. Time of addition experiments according to the infection progress of HSV-1 were used to identify the modes of action for peptides of interest. The histidine-rich modification was designed based on structural analysis of peptides by a helical wheel model and CD spectroscopy. Peptide cellular uptake and distribution were measured by flow cytometry and confocal microscopy, respectively. Results: The peptide Eval418 was found to have high clearance activity in an HSV-1 plaque reduction assay. Eval418 exhibited dose-dependent and time-dependent inactivation of HSV-1 and dose-dependent inhibition of HSV-1 attachment to host cells. However, Eval418 scarcely suppressed an established HSV-1 infection due to poor cellular uptake. We further designed and modified Eval418 into four histidine-rich derivative peptides with enhanced antiviral activities and lower cytotoxicities. All of the derivative peptides suppressed established HSV-1 infections. One of these peptides, Eval418-FH5, not only had strong viral inactivation activity and enhanced attachment inhibitory activity but also had high inhibitory activity against intracellular HSV-1, which was consistent with its improved intracellular uptake and distribution as confirmed by confocal microscopy and flow cytometry. Conclusion: We successfully identified an anti-HSV-1 peptide, Eval418, from a scorpion venom peptide library and designed a histidine-rich Eval418 derivative with significantly improved potential for further development as an anti-HSV-1 drug. This successful modification can provide a design strategy to improve the bioavailability, cellular distribution and antiviral activity of peptide agents. Abstract Objective To assess virus-specific hospitalisation rates, risk factors for illness severity and seasonal trends in children hospitalised with acute respiratory infections (ARI). Design Prospective cohort study. setting A government hospital serving low-income and middle-income population in Amman, Jordan. Participants Children under 2 years of age hospitalised with fever and/or respiratory symptoms (n=3168) from 16 March 2010 to 31 March 2013. Children with chemotherapy-associated neutropenia and newborns who had never been discharged after birth were excluded from the study. Outcome measures Hospitalisation rates and markers of illness severity: admission to intensive care unit (ICU), mechanical ventilation (MV), oxygen therapy, length of stay (LOS) and death. results Of the 3168 subjects, 2581 (82%) had at least one respiratory virus detected, with respiratory syncytial virus (RSV) being the most predominant pathogen isolated. During admission, 1013 (32%) received oxygen therapy, 284 (9%) were admitted to ICU, 111 (4%) were placed on MV and 31 (1%) children died. Oxygen therapy was higher in RSV-only subjects compared with human rhinovirus-only (42%vs29%, p<0.001), adenovirus-only (42%vs21%, p<0.001) and human parainfluenza virusonly (42%vs23%, p<0.001) subjects. The presence of an underlying medical condition was associated with oxygen therapy (adjusted OR (aOR) 1.95, 95% CI 1.49 to 2.56), ICU admission (aOR 2.51, 95% CI 1.71 to 3.68), MV (aOR 1.91, 95% CI 1.11 to 3.28) and longer LOS (aOR1.71, 95% CI 1.37 to 2.13). Similarly, younger age was associated with oxygen therapy (0.23, 95% CI 0.17 to 0.31), ICU admission (aOR 0.47, 95% CI 0.30 to 0.74), MV (0.28, 95% CI 0.15 to 0.53) and longer LOS (aOR 0.47, 95% CI 0.38 to 0.59). Pneumonia was strongly associated with longer LOS (aOR 2.07, 95% CI 1.65 to 2.60), oxygen therapy (aOR 2.94, 95% CI 2.22 to 3.89), ICU admission (aOR 3.12, 95% CI 2.16 to 4.50) and MV (aOR 3.33, 95% CI 1.85 to 6.00). Virus-specific hospitalisation rates ranged from 0.5 to 10.5 per 1000 children. Conclusion Respiratory viruses are associated with severe illness in Jordanian children hospitalised with ARI. Prevention strategies such as extended breast feeding, increased access to palivizumab and RSV vaccine development could help decrease hospitalisation rates and illness severity, particularly in young children with underlying medical conditions. Abstract The phylogenetic and taxonomic relationships among the Old World leaf-nosed bats (Hipposideridae) and the closely related horseshoe bats (Rhinolophidae) remain unresolved. In this study, we generated a novel approximately 10-kb molecular data set of 19 nuclear exon and intron gene fragments for 40 bat species to elucidate the phylogenetic relationships within the families Rhinolophidae and Hipposideridae. We estimated divergence times and explored potential reasons for any incongruent phylogenetic signal. We demonstrated the effects of outlier taxa and genes on phylogenetic reconstructions and compared the relative performance of intron and exon data to resolve phylogenetic relationships. Phylogenetic analyses produced a well-resolved phylogeny, supporting the familial status of Hipposideridae and demonstrated the paraphyly of the largest genus, Hipposideros. A fossil-calibrated timetree and biogeographical analyses estimated that Rhinolophidae and Hipposideridae diverged in Africa during the Eocene approximately 42 Ma. The phylogram, the timetree, and a unique retrotransposon insertion supported the elevation of the subtribe Rhinonycterina to family level and which is diagnosed herein. Comparative analysis of diversification rates showed that the speciose genera Rhinolophus and Hipposideros underwent diversification during the Mid-Miocene Climatic Optimum. The intron versus exon analyses demonstrated the improved nodal support provided by introns for our optimal tree, an important finding for large-scale phylogenomic studies, which typically rely on exon data alone. With the recent outbreak of Middle East respiratory syndrome, caused by a novel coronavirus, the study of these species is urgent as they are considered the natural reservoir for emergent severe acute respiratory syndrome (SARS)-like coronaviruses. It has been shown that host phylogeny is the primary factor that determines a virus's persistence, replicative ability, and can act as a predictor of new emerging disease. Therefore, this newly resolved phylogeny can be used to direct future assessments of viral diversity and to elucidate the origin and development of SARS-like coronaviruses in mammals. Abstract Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12-18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element. Abstract Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC.The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. Abstract Rhinovirus infection is typically associated with the common cold and has rarely been reported as a cause of severe pneumonia in immunocompetent adults. A 55-year-old previous healthy woman, who consumed half a bottle of alcohol daily, presented with respiratory failure after one week of upper respiratory infection symptoms. Radiography revealed bilateral, diffuse ground glass opacity with patchy consolidation in the whole lung field; bronchoalveolar lavage fluid analysis indicated that rhinovirus was the causative organism. After five days of conservative support, the symptoms and radiographic findings began to improve. We report this rare case of rhinovirus pneumonia in an otherwise healthy host along with a review of references.causative and commensal pathogens in specimen obtained by nasopharyngeal aspiration. Data on viral pneumonia are increasing; however, severe rhinovirus pneumonia resulting respiratory failure in an immunocompetent host is still regarded as extremely unusual.A 55-year-old woman visited our emergency department in September with two-day history of progressive dyspnea. The patient had never smoked and had no other previous medical history, but consumed half a bottle of alcohol daily. The patient had got a common cold one week prior, but her symptoms aggravated despite empirical treatment. After 5 days, she began to experience shortness of breath. Exertional dyspnea progressed to resting dyspnea the following day and at the emergency room, her initial peripheral oxygen saturation was 70%. Wet crackles and expiratory wheezing sounds were present in the whole lung field area. Evaluation of the patient' s vital signs revealed a blood pressure of 140/80 mm Hg, body temperature of 36.6 o C, pulse rate of 108 beats per minute, and respiration rate of 30 breaths per minute. Simple chest radiography showed the predominance of bilateral consolidation with ground glass opacity (GGO) in both lower lobes ( Figure 1A ). Chest computed tomography indicated diffuse GGO Abstract A febrile respiratory infectious disease unit (FRIDU) with a negative pressure ventilation system was constructed outside the emergency department (ED) of the Samsung Medical Center in 2015, to screen for patients with contagious diseases requiring isolation. We evaluated the utility of the FRIDU during 1 year of operation. We analyzed 1,562 patients who were hospitalized after FRIDU screening between August 2015 and July 2016. The level of isolation recommended during their screening at the FRIDU was compared with the level deemed appropriate given their final diagnosis. Of the 1,562 patients screened at the FRIDU, 198 (13%) were isolated, 194 (12%) were reverse isolated, and 1,170 (75%) were not isolated. While hospitalized, 97 patients (6%) were confirmed to have a contagious disease requiring isolation, such as tuberculosis; 207 patients (13%) were confirmed to be immunocompromised and to require reverse isolation, mainly due to neutropenia; and the remaining 1,258 patients (81%) did not require isolation. The correlation coefficient for isolation consistency was 0.565 (P < 0.001). The sensitivity and negative predictive value of FRIDU screening for diagnosing contagious disease requiring isolation are 76% and 98%, respectively. No serious nosocomial outbreaks of contagious diseases occurred. During FRIDU screening, 114 patients were admitted to the resuscitation zone due to clinical instability, and three of these patients died. The initial isolation levels resulting from FRIDU screening were moderately well correlated with the isolation levels required by the final diagnosis, demonstrating the utility of pre-hospitalization screening units. However, the risks of deterioration during the screening process remain challenges. Abstract We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ($3700 18-mers or $2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus. Abstract How to cite: Bhuiyan ZA, Ali MZ, Moula MM, Bary MA, Arefin N, Giasuddin M, et al. Seroprevalence of major avian respiratory diseases in broiler and sonali chicken in selected areas of Bangladesh. J Adv Vet Anim Res 2019; 6(4):Objective: This study was conducted to investigate different respiratory diseases in broiler and sonali birds in some selected districts of Bangladesh. Materials and Methods: We were collected a total of 460 blood samples from 46 farms with 36 broiler farms and 10 sonali farms (cross-breed) from 2015 to 2017. All the collected serum samples were tested for determining specific antibodies of avian rhinotracheitis (ART) virus, infectious laryngotracheitis (ILT) virus, infectious bronchitis (IBV) virus, and Ornithobacterium rhinotracheale (ORT) infection using commercially available enzyme-linked immunosorbent assay kits. Results: The overall seropositivity was highest in ORT (45.9%), followed by IBV (37.6%), ART (2.6%), and ILT (0.4%). Out of 360 broiler samples, highest seropositivity was recorded in ORT (43.3%) and lowest in IBV (31.4%). Surprisingly, no broiler samples were found positive for ART and ILT. In case of sonali, the seropositivity was highest in IBV (60%) and lowest in ILT (2%). With respect to types of birds and age groups, the seropositive percentage of all four pathogens was found higher in sonali than broiler. Between two age groups of sonali, the seropositive percentage of ART (12%), ORT (55%), ILT (2%), and IBV (60%) was highest at 21-60 weeks of age compared to 5-20 weeks of age. However, based on location, the seropositive of ORT and IBV was highest in Jamalpur (63.3%) and Fulbariya and Trishal (50%) and lowest in Sreepur (16.7%) and Jamalpur (3.3%). Conclusion: The four pathogens are ubiquitous in nature for the sonali chickens, and the prevalence of ORT and IBV was the most prevalent viruses in the study areas. This study indicates a need for improved surveillance and characterization of ORT and ART circulating in all types of poultry in Bangladesh.ARTICLE HISTORY Abstract Within the last decade new technologies have been developed and implemented which employ light, often in the presence of a photosensitizer, to inactivate pathogens that reside in human blood products for the purpose of transfusion. These pathogen reduction technologies attempt to find the proper balance between pathogen kill and cell quality. Each system utilizes various chemistries that not only impact which pathogens they can inactivate and how, but also how the treatments affect the plasma and cellular proteins and to what degree. This paper aims to present the various chemical mechanisms for pathogen reduction in transfusion medicine that are currently practiced or in development. Abstract Citation Fouchier RAM. 2015. Reply to "Comments on Fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory." mBio 6(2):e00407-15. Abstract In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10-to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10 9 CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID 50 of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 · 10 9 CFU of p3D-NT56/S. cho were protected in 9 days.foot-and-mouth disease virus / RNA interference / Salmonella choleraesuis / conserved sequence / swine Abstract HIV-1 uses a programmed -1 ribosomal frameshift to synthesize the precursor of its enzymes, Gag-Pol. The frameshift efficiency that is critical for the virus replication, is controlled by an interaction between the ribosome and a specific structure on the viral mRNA, the frameshift stimulatory signal. The rate of cap-dependent translation initiation is known to be altered by the TAR RNA structure, present at the 5' and 3' end of all HIV-1 mRNAs. Depending upon its concentration, TAR activates or inhibits the double-stranded RNA-dependent protein kinase (PKR). We investigated here whether changes in translation initiation caused by TAR affect HIV-1 frameshift efficiency. CD4+ T cells and 293T cells were transfected with a dual-luciferase construct where the firefly luciferase expression depends upon the HIV-1 frameshift. Translation initiation was altered by adding TAR in cis or trans of the reporter mRNA. We show that HIV-1 frameshift efficiency correlates negatively with changes in the rate of translation initiation caused by TAR and mediated by PKR. A model is presented where changes in the rate of initiation affect the probability of frameshifting by altering the distance between elongating ribosomes on the mRNA, which influences the frequency of encounter between these ribosomes and the frameshift stimulatory signal. Abstract Purpose: This study evaluated the specificity of different avian secondary antibodies used in Western blot and dot-blot ELISA to detect avian bornavirus antibodies in bird plasma. Methods: Plasma samples were collected from: two Blue and gold macaws, one positive and one negative for avian bornavirus by RT-PCR; a Cockatiel and a Monk parakeet prior to and following experimental infection; and, two Mallards, one positive and one negative for avian bornavirus by RT-PCR Samples were analyzed by Western blot and dot-blot ELISA that incorporated recombinant avian bornavirus nucleoprotein as the target analyte. Four species-specific anti-IgY secondary antibodies were used in the assays: goat anti-macaw IgY, goat anti-bird IgY, goat anti-duck IgY, and rabbit anti-chicken IgY. Results: In the Western blot, anti-macaw IgY secondary antibody produced strong signals with Blue and gold macaw and Cockatiel positive plasma, but no signal with Mallard positive plasma. Anti-bird IgY secondary antibody produced strong signals with Blue and gold macaw, Cockatiel, and Mallard positive plasma. Anti-duck and anti-chicken IgY secondary antibody produced a strong and moderate signal, respectively, only with Mallard positive plasma. In the dot-blot ELISA, there was a distinct and significant difference (P<0.05) in the signal intensity between the different secondary antibodies within a bird species. Anti-macaw IgY secondary antibody produced significantly (P<0.05) stronger signals than the other secondary antibodies in Blue and gold macaw, Cockatiel, and Monk parakeet positive plasma, while anti-duck IgY secondary antibody produced significantly (P<0.05) stronger signals than the other secondary antibodies in Mallard positive plasma. Conclusion: In testing psittacines with immunoassays, and especially in assays that incorporate short incubation reaction times such as a dot-blot ELISA, species-specific anti-IgY secondary antibodies provided more accurate results. Abstract Background: During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits. (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively.Results: The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition.The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls. Abstract Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to vi.rus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon.A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of.VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-~-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-~-Nacetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal. Abstract Hajj is a unique gathering with Mecca and Kaaba being spiritually important to many faiths across the globe, especially Muslims. This is because of the proclamation of the prophet's father, Ibrahaam, when he called all mankind to perform Hajj. That is why all Muslims on Earth feel that they have to visit Mecca and Kaaba on a specific date and time, and that is the reason this small location hosts one of the largest human gatherings in the world. Hajj is one of the five pillars of Islam that every financially and physically able Muslim must perform once in his/her lifetime. For 14 centuries countless millions of Muslim men and women from the four corners of the earth have undertaken pilgrimage to Mecca.In conclusion this review article confirm that Hajj is oldest and largest mass gathering in all mankind and there is some issues influence the health response such as size of gathering. diversity of population, climate and health facilities around hajj site, also we discuss the infectious and non infectious related illness in hajj and their prevention methods. Abstract In April 2009, a new influenza A (H1N1 2009) virus emerged that rapidly spread around the world. While current variants of this virus have caused widespread disease, particularly in vulnerable Abstract Background: The accurate and rapid identification of the causative viruses is important for the timely diagnosis and management of respiratory infections. Multiplex molecular diagnostic techniques have been widely adopted to detect respiratory viruses. We compared the results of a newly upgraded, multiplex, molecular bead-based respiratory viral panel (RVP) assay with the results of Anyplex II RV16 detection kit and AdvanSure RV real-time RT-PCR assay.Methods: We tested 254 respiratory specimens and cultured viral strains using the Luminex xTAG RVP Fast v2 assay (Luminex Molecular Diagnostics, Canada) and Anyplex II RV16 detection kit and compared the results. Specimens showing discordant results between the two assays were tested with a AdvanSure RV real-time RT-PCR assay.Results: Of the 254 respiratory specimens, there was total agreement in the results between the xTAG RVP Fast v2 assay and the other real-time PCR assay in 94.1-100% of the specimens. The agreement levels were relatively low (94.1-97.6%) for specimens of adenovirus, coronavirus NL63, and parainfluenza type 3. In comparison to the other assay, the xTAG RVP Fast v2 assay detected a higher number of parainfluenza type 3 (4 cases) and metapneumovirus (9 cases).The xTAG RVP Fast v2 assay showed comparable capabilities compared with the other assays; it will be useful for identifying respiratory viral infections in patients with respiratory symptoms. Clinicians should be aware of the characteristics of the assays they use, since different assays show different detectability for each virus. Abstract ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi. To identify the targeting signals that mediate this recycling, N-glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to in-creased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER-ERGIC-cis- Abstract The sudden outbreak of severe acute respiratory syndrome (SARS) in 2002 prompted the establishment of a global scientific network subsuming most of the traditional rivalries in the competitive field of virology. Within months of the SARS outbreak, collaborative work revealed the identity of the disastrous pathogen as SARS-associated coronavirus (SARS-CoV). However, although the rapid identification of the agent represented an important breakthrough, our understanding of the deadly virus remains limited. Detailed biological knowledge is crucial for the development of effective countermeasures, diagnostic tests, vaccines and antiviral drugs against the SARS-CoV. This article reviews the present state of molecular knowledge about SARS-CoV, from the aspects of comparative genomics, molecular biology of viral genes, evolution, and epidemiology, and describes the diagnostic tests and the anti-viral drugs derived so far based on the available molecular information. Abstract Clinical application of siRNA-based therapeutics outside of the liver has been hindered by the inefficient delivery of siRNA effector molecules into extra-hepatic organs and cells of interest. To understand the parameters that enable RNAi activity in vivo, it is necessary to develop a systematic approach to identify which cells within a tissue are permissive to oligonucleotide internalization and activity. In the present study, we evaluate the distribution and activity within the lung of chemically stabilized siRNA to characterize cell-type tropism and structure-activity relationship. We demonstrate intratracheal delivery of fully modified siRNA for RNAi-mediated target knockdown in lung CD11c + cells (dendritic cells, alveolar macrophages) and alveolar epithelial cells. Finally, we use an allergen-induced model of lung inflammation to demonstrate the capacity of inhaled siRNA to induce target knockdown in dendritic cells and ameliorate lung pathology. Abstract The biochemical properties of mouse LSECtin, a glycanbinding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcβ1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcβ1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor. Abstract Inhibition of host-encoded targets, such as the cyclophilins, provides an opportunity to generate potent, high barrier to resistance antivirals for the treatment of a broad range of viral diseases. However, many host-targeted agents are natural products which can be difficult to optimize using synthetic chemistry alone. We describe the orthogonal combination of bioengineering and semisynthetic chemistry to optimize the drug-like properties of sanglifehrin A, a known cyclophilin inhibitor of mixed non-ribosomal peptide/polyketide origin in order to generate the drug candidate NVP018 (formerly BC556). NVP018 is a potent inhibitor of HBV, HCV and HIV-1 replication, shows minimal inhibition of major drug transporters and has a high barrier to generation of both HCV and HIV-1 resistance. Abstract Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway that modulates cellular redox homeostasis via the regeneration of NADPH. G6PD-deficient cells have a reduced ability to induce the innate immune response, thus increasing host susceptibility to pathogen infections. An important part of the immune response is the activation of the inflammasome. G6PD-deficient peripheral blood mononuclear cells (PBMCs) from patients and human monocytic (THP-1) cells were used as models to investigate whether G6PD modulates inflammasome activation. A decreased expression of IL-1β was observed in both G6PD-deficient PBMCs and PMA-primed G6PD-knockdown (G6PD-kd) THP-1 cells upon lipopolysaccharide (LPS)/adenosine triphosphate (ATP) or LPS/nigericin stimulation. The pro-IL-1β expression of THP-1 cells was decreased by G6PD knockdown at the transcriptional and translational levels in an investigation of the expression of the inflammasome subunits. The phosphorylation of p38 MAPK and downstream c-Fos expression were decreased upon G6PD knockdown, accompanied by decreased AP-1 translocation into the nucleus. Impaired inflammasome activation in G6PD-kd THP-1 cells was mediated by a decrease in the production of reactive oxygen species (ROS) by NOX signaling, while treatment with hydrogen peroxide (H 2 O 2 ) enhanced inflammasome activation in G6PD-kd THP-1 cells. G6PD knockdown decreased Staphylococcus aureus and Escherichia coli clearance in G6PD-kd THP-1 cells and G6PD-deficient PBMCs following inflammasome activation. These findings support the notion that enhanced pathogen susceptibility in G6PD deficiency is, in part, due to an altered redox signaling, which adversely affects inflammasome activation and the bactericidal response.Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; NOX, NADPH oxidase; NO, nitric oxide; NOS, nitric oxide synthase; UP-LPS, ultrapure lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; ATP, adenosine triphosphate; MAPK, mitogenactivated protein kinases; AP-1, activator protein 1; ROS, reactive oxygen species; PBMC, peripheral blood mononuclear cells; H 2 O 2 , hydrogen peroxide; THP-1, human monocytic cells; G6PD-kd, G6PD knockdown Abstract Objective: To assess the clinical process of gonorrhoea diagnosis and report in China, and to determine the difference of sex ratio between reported incidence based on reporting data and true diagnosis rate based on reference tests of gonorrhoea.Setting: A total of 26 dermatology and sexually transmitted disease (STD) departments, 34 obstetricsgynaecology clinics and 28 urology outpatient clinics selected from 34 hospitals of Shenzhen regarded as our study sites.Participants: A total of 2754 participants were recruited in this study, and 2534 participants completed the questionnaire survey and provided genital tract secretion specimens. There were 1106 male and 1428 female participants. Eligible participants were patients who presented for outpatient STD care at the selected clinics for the first time in October 2012 were at least 18 years old, and were able to give informed consent.Outcome measures: Untested rate, true-positive rate, false-negative rate and unreported rate of gonorrhoea, as well as reported gonorrhoea incidence sex ratio and true diagnosis sex ratio were calculated and used to describe the results. Abstract We have evaluated the fate of misfolded protein domains in the Saccharomyces cerevisiae secretory pathway by fusing mutant forms of the NH2-terminal domain of h repressor protein to the secreted protein invertase. The hybrid protein carrying the wild-type repressor domain is mostly secreted to the cell surface, whereas hybrid proteins with amino acid substitutions that cause the repressor domain to be thermodynamically unstable are retained intracellularly. Surprisingly, the retained hybrids are found in the vacuole, where the repressor moiety is degraded by vacuolar proteases. The following observations indicate that receptor-mediated recognition of the mutant repressor domain in the Golgi lumen targets these hybrid fusions to the vacuole. (a) The invertase-repressor fusions, like wild-type invertase, behave as soluble proteins in the ER lumen. (b) Targeting to the vacuole is saturable since overexpression of the hybrids carrying mutant repressor increases the fraction of fusion protein that appears at the cell surface. (c) Finally, deletion of the VPSIO gene, which encodes the transmembrane Golgi receptor responsible for targeting carboxypeptidase Y to the vacuole, causes the mutant hybrids to be diverted to the cell surface. Together these findings suggest that yeast have a salvage pathway for degradation of nonnative luminal proteins by receptor-mediated transport to the vacuole. Abstract Objectives: Actively recruit and intensively follow pregnant women receiving a dose of acellular pertussis vaccine for 4 weeks after vaccination. Design and settings: A prospective observational study conducted in 2 New Zealand regions. Participants: Women in their 28th-38th week of pregnancy, recruited from primary care and antenatal clinics at the time of Tdap administration. Telephone interviews were conducted at 48 h and 4 weeks postvaccination.Main outcomes measures: Outcomes were injection site reactions, systemic symptoms and serious adverse events (SAEs). Where available, data have been classified and reported according to Brighton Collaboration definitions. Abstract While general medical practitioners (GPs) and veterinarians are often the first line responders in the face of a disease outbreak, pathways to improving the One Health efficacy of these clinicians remain unclear. A two-phase modified Delphi survey of professionals with known expertise in One Health ('expert panel') was used to 1) identify key knowledge, attitudes and practices (KAPs) of GPs and veterinarians that would be consistent with a One Health approach to zoonoses; and 2) determine priorities for future surveys with Australian GPs and veterinarians to identify important gaps that impede effective diagnosis and management of zoonoses. A list of 13 topics/sub-topics, as well as a list of 25 specific zoonotic diseases/agents emerged from the first phase of the survey. In the second phase the expert panel identified general knowledge of the clinical aspects and epidemiological aspects of zoonoses, as well as risk management practices, as the most important KAPs and research priorities for both GPs and veterinarians. In terms of diseases, the expert panel regarded knowledge of Hendra virus, Q fever, Australian bat lyssavirus (ABLV), anthrax and Brucella suis most important for veterinarians, whilst for GPs, Q fever, gastrointestinal/foodborne diseases, influenza, ABLV and local vector-borne diseases were found to be most important by the expert panel. Some differences were noted in terms of prioritization of topics/sub-topics and diseases/agents according to expert background (veterinary and non-veterinary). The Delphi survey technique enabled efficient collection of data from a diverse range of One Health 'experts'/specialists and provided clear priorities for proposed future research, and potentially for educational interventions to improve One Health efficacy of clinicians. Abstract SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5 0 -overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the singlestranded 5 0 -tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5 0 -overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5 0 -overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5 0 -overhang account for the observed enhanced processivity of DNA unwinding. Abstract Mesenchymal stem cells (MSCs) have the capacity to proliferate and differentiate into multiple connective tissue lineages, which include cartilage, bone, and fat. Cartilage differentiation and chondrocyte maturation are required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. This study was designed to identify novel genes that might help clarify the molecular mechanisms of chondrogenesis. Chondrogenesis was induced by culturing human bone marrow (BM) derived MSCs in micromass pellets in the presence of defined medium for 3, 7, 14 or 21 days. Several genes regulated during chondrogenesis were then identified by reverse transcriptase-polymerase chain reaction (RT-PCR). Using an ABI microarray system, we determined the differential gene expression profiles of differentiated chondrocytes and BM-MSCs. Normalization of this data resulted in the identification of 1,486 differentially expressed genes. To verify gene expression profiles determined by microarray analysis, the expression levels of 10 genes with high fold changes were confirmed by RT-PCR. Gene expression patterns of 9 genes (Hrad6B, annexinA2, BMP-7, contactin-1, peroxiredoxin-1, heat shock transcription factor-2, synaptotagmin IV, serotonin receptor-7, Axl) in RT-PCR were similar to the microarray gene expression patterns. These findings provide novel information concerning genes involved in the chondrogenesis of human BM-MSCs. Abstract Bioinformatics and genome science (BGS) are gradually gaining roots in Africa, contributing to studies that are leading to improved understanding of health, disease, agriculture and food security. While a few African countries have established foundations for research and training in these areas, BGS appear to be limited to only a few institutions in specific African countries. However, improving the disciplines in Africa will require pragmatic efforts to expand training and research partnerships to scientists in yet-unreached institutions. Here, we discuss the need to expand BGS programmes in Africa, and propose mechanisms to do so. Abstract Proteolytic cleavage regulates numerous processes in health and disease. One key player is the ubiquitously expressed serine protease furin, which cleaves a plethora of proteins at polybasic recognition motifs. Mammalian substrates of furin include cytokines, hormones, growth factors and receptors. Thus, it is not surprising that aberrant furin activity is associated with a variety of disorders including cancer. Furthermore, the enzymatic activity of furin is exploited by numerous viral and bacterial pathogens, thereby enhancing their virulence and spread. In this review, we describe the physiological and pathophysiological substrates of furin and discuss how dysregulation of a simple proteolytic cleavage event may promote infectious diseases and cancer. One major focus is the role of furin in viral glycoprotein maturation and pathogenicity. We also outline cellular mechanisms regulating the expression and activation of furin and summarise current approaches that target this protease for therapeutic intervention. Abstract Oncolytic viruses (OVs) are engineered and/or evolved to propagate selectively in cancerous tissues. They have a dual mechanism of action; direct killing of infected cancer cells crossprimes anticancer immunity to boost the killing of uninfected cancer cells. The goal of the field is to develop OVs that are easily manufactured, efficiently delivered to disseminated sites of cancer growth, undergo rapid intratumoral spread, selectively kill tumor cells, cause no collateral damage and pose no risk of transmission in the population. Here we discuss the many virus engineering strategies that are being pursued to optimize delivery, intratumoral spread and safety of OVs derived from different virus families. With continued progress, OVs have the potential to transform the paradigm of cancer care. Abstract Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China. Abstract In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development. Abstract Graphical Abstract Highlights d Several unique conformational states of an elongating RdRp exist d Only one conformation incorporates nucleotide analogs with therapeutic potential d An analog thought to be a chain terminator actually promotes RdRp backtracking d Distinctive behavior of backtrack-inducing analog on virus variants in cell culture SUMMARYRNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotidesubstrate-binding and catalytic sites. Conventional, quantitative, kinetic analysis of antiviral ribonucleotides monitors one or a few incorporation events.Here, we use a high-throughput magnetic tweezers platform to monitor the elongation dynamics of a prototypical RdRp over thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors. We observe multiple RdRp-RNA elongation complexes; only a subset of which are competent for analog utilization. Incorporation of a pyrazine-carboxamide nucleotide analog, T-1106, leads to RdRp backtracking. This analysis reveals a mechanism of action for this antiviral ribonucleotide that is corroborated by cellular studies. We propose that induced backtracking represents a distinct mechanistic class of antiviral ribonucleotides. Abstract The Epstein-Barr virus (EBV) genome encodes several hundred transcripts. We have used ribosome profiling to characterize viral translation in infected cells and map new translation initiation sites. We show here that EBV transcripts are translated with highly variable efficiency, owing to variable transcription and translation rates, variable ribosome recruitment to the leader region and coverage by monosomes versus polysomes. Some transcripts were hardly translated, others mainly carried monosomes, showed ribosome accumulation in leader regions and most likely represent non-coding RNAs. A similar process was visible for a subset of lytic genes including the key transactivators BZLF1 and BRLF1 in cells infected with weakly replicating EBV strains. This suggests that ribosome trapping, particularly in the leader region, represents a new checkpoint for the repression of lytic replication. We could identify 25 upstream open reading frames (uORFs) located upstream of coding transcripts that displayed 5 leader ribosome trapping, six of which were located in the leader region shared by many latent transcripts. These uORFs repressed viral translation and are likely to play an important role in the regulation of EBV translation. Abstract Translational science for new biotechnologies (e.g. drugs, vaccines, devices, or diagnostics) depend on the development of a robust 'business case'. This is driven by complex scientific, technical, logistical, financial and operational elements to determine the feasibility and probability of traversing the "valleys of death" leading to licensure. The potential results in terms of profitability and financial realization, called 'product value proposition' play a crucial role in establishing incentives for investment during and after development. With this review, our goal is to summarize the challenges in taking vaccines against neglected tropical diseases (NTDs) from development through licensure and provide a perspective that these vaccines can have measurable public health and economic profitability and market success. Understanding these processes and its challenges would open the opportunity to accelerate and advance these essential NTD vaccines through the last mile towards licensure and for the delivery to afflicted populations in low-and middle-income countries.ARTICLE HISTORY Abstract Acute respiratory distress syndrome (ARDS) is a life-threatening noncardiogenic circulatory disorder of the lungs associated with critical illnesses such as sepsis, trauma, and immune and collagen vascular disease. Its mortality rate is marginally improved with the best supportive care. The demise occurs due to progressive pulmonary hypoxia and multi-organ dysfunction syndrome (MODS) with severe inflammation. Complement activation is a part of immune response against pathogen or insult in which membrane attack complex (MAC) is formed and eliminates microbes. If complement regulatory protein such as endothelial CD59 is underexpressed, MAC may also cause pulmonary vascular injury to the innocent bystander endothelial cell of host and provokes endotheliopathy that causes inflammation and pulmonary vascular microthrombosis, leading to ARDS. Abstract One of the daunting challenges facing modern medicine lies in the understanding and treatment of tumor heterogeneity. Most tumors show intra-tumor heterogeneity at both genomic and proteomic levels, with marked impacts on the responses of therapeutic targets. Therapeutic target-related gene expression pathways are affected by hypoxia and cellular stress. However, the finding that targets such as eukaryotic initiation factor (eIF) 4E (and its phosphorylated form, p-eIF4E) are generally homogenously expressed throughout tumors, regardless of the presence of hypoxia or other cellular stress conditions, opens the exciting possibility that malignancies could be treated with therapies that combine targeting of eIF4E phosphorylation with immune checkpoint inhibitors or chemotherapy. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. rVSVDG-ZEBOV-GP (also designated V920) recombinant vesicular stomatitis virus pseudotyped with Ebola Zaire Glycoprotein: Standardized template with key considerations for a risk/benefit assessment a b s t r a c tThe Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and characteristics of live, recombinant viral vector vaccines. A recent publication by the V3SWG described live, attenuated, recombinant vesicular stomatitis virus (rVSV) as a chimeric virus vaccine for HIV-1 (Clarke et al., 2016). The rVSV vector system is being explored as a platform for development of multiple vaccines. This paper reviews the molecular and biological features of the rVSV vector system, followed by a template with details on the safety and characteristics of a rVSV vaccine against Zaire ebolavirus (ZEBOV). The rVSV-ZEBOV vaccine is a live, replication competent vector in which the VSV glycoprotein (G) gene is replaced with the glycoprotein (GP) gene of ZEBOV. Multiple copies of GP are expressed and assembled into the viral envelope responsible for inducing protective immunity. The vaccine (designated V920) was originally constructed by the National Microbiology Laboratory, Public Health Agency of Canada, further developed by NewLink Genetics Corp. and Merck & Co., and is now in final stages of registration by Merck. The vaccine is attenuated by deletion of the principal virulence factor of VSV (the G protein), which also removes the primary target for anti-vector immunity. The V920 vaccine caused no toxicities after intramuscular (IM) or intracranial injection of nonhuman primates and no reproductive or developmental toxicity in a rat model. In multiple studies, cynomolgus macaques immunized IM with a wide range of virus doses rapidly developed ZEBOV-specific antibodies measured in IgG ELISA and neutralization assays and were fully protected against lethal challenge with ZEBOV virus. Over 20,000 people have received the vaccine in clinical trials; the vaccine has proven to be safe and well tolerated. During the first few days after vaccination, many vaccinees experience a mild acute-phase reaction with fever, headache, myalgia, and arthralgia of short duration; this period is associated with a low-level viremia, activation of anti-viral genes, and increased levels of chemokines and cytokines. Oligoarthritis and rash appearing in the second week occur at a low incidence, and are typically mild-moderate in severity and self-limited. V920 vaccine was used in a Phase III efficacy trial during the West African Ebola epidemic in 2015, showing 100% protection against Ebola Virus Disease, and it has subsequently been deployed for emergency control of Ebola outbreaks in central Africa. The template Abstract Objective: To investigate the long-term efficacy of a combination treatment of alendronate, extracorporeal shock and hyperbaric oxygen for osteonecrosis of the femoral head (ONFH) of post-severe acute respiratory syndrome (SARS) patients. Patients and methods: The retrospective study was performed including a total of 37 post-SARS ONFH patients (66 hip joints) in the Department of Orthopedics of the General Hospital of Tianjin Medical University between November 2003 and November 2015, consisting of 6 males (11 hip joints) and 31 females (55 hip joints), with age between 19 and 47 years (average 29.9 years). Visual analog scale (VAS) score, Harris score and Association Research Circulation Osseous (ARCO) stage of imaging examination were compared among those before treatment, and at 1, 3, 6, 9 and 12 years after treatment. Paired t-test was used for statistical analysis of VAS and Harris score before and after treatment. Difference of effective rate on all stages was analyzed with Chi-square test. Results: With 12-year follow-up, significant improvements on VAS (6.81 of pre-treatment vs 3.94 of 12-year post-treatment) and Harris score (74.54 of pre-treatment vs 80.14 of 12-year post-treatment) were observed (all p,0.05). Effective rate showed statistical significance among three stages of ARCO (p,0.05). The combined treatment showed different efficacies on different ARCO stages; the best was on ARCO Phase I. Conclusion: The combined treatment may delay or discontinue the development of ONFH in post-SARS patients. Abstract Background. Few studies have prospectively assessed viral etiologies of acute respiratory infections in communitybased elderly individuals. We assessed viral respiratory pathogens in individuals ≥65 years with influenza-like illness (ILI).Methods. Multiplex reverse-transcriptase polymerase chain reaction identified viral pathogens in nasal/throat swabs from 556 episodes of moderate-to-severe ILI, defined as ILI with pneumonia, hospitalization, or maximum daily influenza symptom severity score (ISS) >2. Cases were selected from a randomized trial of an adjuvanted vs nonadjuvanted influenza vaccine conducted in elderly adults from 15 countries.Results. Respiratory syncytial virus (RSV) was detected in 7.4% (41/556) moderate-to-severe ILI episodes in elderly adults. Most (39/41) were single infections. There was a significant association between country and RSV detection (P = .004). RSV prevalence was 7.1% (2/28) in ILI with pneumonia, 12.5% (8/64) in ILI with hospitalization, and 6.7% (32/480) in ILI with maximum ISS > 2. Any virus was detected in 320/556 (57.6%) ILI episodes: influenza A (104/556, 18.7%), rhinovirus/enterovirus (82/556, 14.7%), coronavirus and human metapneumovirus (each 32/556, 5.6%).Conclusions. This first global study providing data on RSV disease in ≥65 year-olds confirms that RSV is an important respiratory pathogen in the elderly. Preventative measures such as vaccination could decrease severe respiratory illnesses and complications in the elderly. a Members of the core writing team. b Members of the Influence-65 publication steering committee. c Authors are presented in alphabetical order. Abstract Naturally occurring plant compounds including tannins, saponins and essential oils are extensively assessed as natural alternatives to in-feed antibiotics. Tannins are a group of polyphenolic compounds that are widely present in plant region and possess various biological activities including antimicrobial, anti-parasitic, anti-viral, antioxidant, anti-inflammatory, immunomodulation, etc. Therefore, tannins are the major research subject in developing natural alternative to in-feed antibiotics. Strong protein affinity is the well-recognized property of plant tannins, which has successfully been applied to ruminant nutrition to decrease protein degradation in the rumen, and thereby improve protein utilization and animal production efficiency. Incorporations of tannin-containing forage in ruminant diets to control animal pasture bloat, intestinal parasite and pathogenic bacteria load are another 3 important applications of tannins in ruminant animals. Tannins have traditionally been regarded as "anti-nutritional factor" for monogastric animals and poultry, but recent researches have revealed some of them, when applied in appropriate manner, improved intestinal microbial ecosystem, enhanced gut health and hence increased productive performance. The applicability of plant tannins as an alternative to in-feed antibiotics depends on many factors that contribute to the great variability in their observed efficacies. Abstract Genome sequence analysis of Middle East respiratory syndrome coronavirus (MERS-CoV) variants from patient specimens has revealed the evolutionary dynamics and mechanisms of pathogenesis of the virus. However, most studies have analyzed the consensus sequences of MERS-CoVs, precluding an investigation of intrapatient heterogeneity. Here, we analyzed non-consensus sequences to characterize intrapatient heterogeneity in cases associated with the 2015 outbreak of MERS in South Korea. Deepsequencing analysis of MERS-CoV genomes performed on specimens from eight patients revealed significant intrapatient variation; therefore, sequence heterogeneity was further analyzed using targeted deep sequencing. A total of 35 specimens from 24 patients (including a super-spreader) were sequenced to detect and analyze variants displaying intrapatient heterogeneity. Based on the analysis of non-consensus sequences, we demonstrated the intrapatient heterogeneity of MERS-CoVs, with the highest level in the super-spreader specimen. The heterogeneity could be transmitted in a close association with variation in the consensus sequences, suggesting the occurrence of multiple MERS-CoV infections. Analysis of intrapatient heterogeneity revealed a relationship between D510G and I529T mutations in the receptor-binding domain (RBD) of the viral spike glycoprotein. These two mutations have been reported to reduce the affinity of the RBD for human CD26. Notably, although the frequency of both D510G and I529T varied greatly among specimens, the combined frequency of the single mutants was consistently high (87.7% ± 1.9% on average). Concurrently, the frequency of occurrence of the wild type at the two positions was only 6.5% ± 1.7% on average, supporting the hypothesis that selection pressure exerted by the host immune response played a critical role in shaping genetic variants and their interaction in human MERS-CoVs during the outbreak. Abstract In the recently published paper by Zhang et al 1 in Drug Des Develop Ther, the authors have evaluated the role of signal transducer and activator of transcription 3 (STAT3) in the antifibrotic activity of paclitaxel in vitro and in mice. They have reported that the treatment of paclitaxel at 2-4 µM reduced the level of phosphorylated STAT3 at Tyr705 in a dose-and time-dependent manner, and downregulated the expression of fibronectin, α-smooth muscle actin (α-SMA), and collagen I in cultured rat renal interstitial fibroblast NRK-49F cells derived from normal kidney. Treatment of the cells with the selective STAT3 inhibitor S3I-201 at 50 mM suppressed the expression of fibronectin, α-SMA, and collagen I in NRK-49F cells. However, S3I-201 treatment increased the expression of phosphorylated STAT1 but did not affect that of phosphorylated STAT5M. The immunoprecipitation assay has revealed that paclitaxel inhibited the STAT3 activity by disrupting the binding of STAT3 with tubulin independently of the effect on STAT3 phosphorylation and by inhibiting STAT3 nucleus translocation. 1 Furthermore, paclitaxel treatment by intraperitoneal injection at 0.3 mg/kg twice a week ameliorated renal interstitial fibrosis by inhibiting the expression of fibronectin, α-SMA, and collagen I in a male C57 mouse model of unilateral ureteral obstruction. Paclitaxel administration also suppressed the infiltration of macrophages and neutrophils and production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, transforming growth factor (TGF)-β, and intercellular adhesion molecule 1 (ICAM-1) by inhibition of STAT3 activity in mouse obstructive nephropathy. 1 These findings indicate that paclitaxel suppresses renal interstitial fibrosis via inhibition of STAT3-mediated pathway and production of proinflammatory cytokines. The findings from this study indicate that in addition to being a clinically used anticancer agent, paclitaxel may represent a new agent that manages renal fibrosis.Through indication discovery or therapeutic switching, drugs that have been approved for clinical use may be used for new indications, and this process is called drug repositioning or drug repurposing. 2-7 Drug repositioning is different from drug coincidence or "serendipity", which arises from unintentional mishaps in the drug discovery process as exemplified by drugs such as sildenafil and thalidomide. Apart from the staggering manufacturing cost and time reduction, drug repositioning facilitates drug discovery that will overcome bottlenecks in the therapeutic development process and prolong patent life, thereby obtaining largest investment return throughout the development process coupled with a significantly higher rate of success and reduced development risk. The benefits of drug repositioning for patients are evident in that newly arising diseases such as severe acute respiratory syndrome Abstract Liver diseases are one of the fatal syndromes due to the vital role of the liver. Most of the effective treatment of liver conditions are of natural origin. Silymarin (SI) is the standard drug used for treatment of impaired liver functions. Two natural compounds possessing promising liver protection and with different chemical structures namely; the bioflavonoid hinokiflavone (HF) isolated from Junipers phoenicea family Cupressaceae and the sweet saponin Glycyrrhizin (GL) present in Glycyrrhiza glabra (liquorice) were selected for the current study. Since the two compounds are of different nature, they may act by different mechanisms and express synergistic effect. Combination of the two compounds using to dose levels were challenged with single doses of HF, GL and SI as well. The comparison was monitored via measuring serum biochemical parameters including, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltranspeptidase (GGT), alkaline phosphatase (ALP) and total bilirubin, tissue parameters such as MDA, NP-SH and TP, histopathological study using light and electron microscope. Protective effect on kidney was also monitored histopathologically and biochemically through observing the levels of LDH, creatinine, creatinine-kinase, urea and uric acid. The combinations of HF and GL showed protective effect more than the used single doses of HF and GL alone. However, SI was superior to the used combination in the two used doses in all the measured parameters. The liver and kidney cells appearance under normal and electron microscope showed that SI treated groups showed almost normal cells with slight toxic signs. Cells from group treated with the higher doses of the combination of HF and GL showed slight signs of intoxication under light and electron microscope indicating good level of protection. Although the combination of HF and GL expressed good protection in the higher dose, however, the combination did not exceed the protective effect of SI. Abstract Repeat-associated disorders caused by expansions of short sequences have been classified as coding and noncoding and are thought to be caused by protein gain-of-function and RNA gain-of-function mechanisms, respectively. The boundary between such classifications has recently been blurred by the discovery of repeat-associated non-AUG (RAN) translation reported in spinocerebellar ataxia type 8, myotonic dystrophy type 1, fragile X tremor/ataxia syndrome and C9ORF72 amyotrophic lateral sclerosis and frontotemporal dementia. This noncanonical translation requires no AUG start codon and can initiate in multiple frames of CAG, CGG and GGGGCC repeats of the sense and antisense strands of diseaserelevant transcripts. RNA structures formed by the repeats have been suggested as possible triggers; however, the precise mechanism of the translation initiation remains elusive. Templates containing expansions of microsatellites have also been shown to challenge translation elongation, as frameshifting has been recognized across CAG repeats in spinocerebellar ataxia type 3 and Huntington's disease. Determining the critical requirements for RAN translation and frameshifting is essential to decipher the mechanisms that govern these processes. The contribution of unusual translation products to pathogenesis needs to be better understood. In this review, we present current knowledge regarding RAN translation and frameshifting and discuss the pro-posed mechanisms of translational challenges imposed by simple repeat expansions. Abstract In recent years, many studies have reported potential associations between cytokine gene polymorphisms and the development, course, and outcome of sepsis, often with apparently conflicting results. The objective of this study was to investigate single nucleotide polymorphism (SNP) in the interleukin (IL)-1β -31 T/C, IL-6 -174 G/C, tumor necrosis factor α (TNF-α) -308 G/A, and interferon γ (IFN-γ) +874 A/T genes for their possible association with susceptibility to early onset sepsis (EOS) in Saudi newborn infants. A total of 205 newborn infants aged 1-2 days were consecutively enrolled onto the study having met the inclusion criteria (as per the research protocol). DNA was extracted from filter papers using the Chelex-100 method. The cytokines SNP were genotyping using Taqman 5' nuclease allelic discrimination. For cytokine measurements we used the commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kit. Our results show that the circulating IL-1β, IL-6, TNF-α, and IFN-γ were significantly (p < 0.001) elevated in EOS patients compared to suspected and sepsis-free control groups; and IL-1β -31C, IL-6 -174G, TNF-α -308G, and IFN-γ +874A alleles were associated with EOS in Saudi infants. In conclusion, analysis of cytokines concentrations and SNP for the four tested genes can be used as a predictor of sepsis outcome in newborns. Abstract To investigate whether kokuvirus is present in Japanese dogs, we examined the fecal samples obtained from 94 diarrheal household dogs and 50 clinically healthy kenneled dogs by RT-PCR. The gene was detected in 37.2% and 48.0% in the former and the latter, respectively, suggesting that canine kobuvirus (CaKoV) is circulating among Japanese dogs. From the result of the latter, however, CaKoV may not be a primary pathogen. Furthermore, all gene-positive dogs were purebreds aged four months or younger. This finding suggests that CaKoV endemic is confined in multi-dog environments, and the dogs have a strong age-dependent resistance to CaKoV. Abstract Genome-wide transcriptional profiling provides a global view of cellular state and how this state changes under different treatments (e.g. drugs) or conditions (e.g. healthy and diseased). Here, we present ProTINA (Protein Target Inference by Network Analysis), a network perturbation analysis method for inferring protein targets of compounds from gene transcriptional profiles. ProTINA uses a dynamic model of the cell-type specific protein-gene transcriptional regulation to infer network perturbations from steady state and time-series differential gene expression profiles. A candidate protein target is scored based on the gene network's dysregulation, including enhancement and attenuation of transcriptional regulatory activity of the protein on its downstream genes, caused by drug treatments. For benchmark datasets from three drug treatment studies, ProTINA was able to provide highly accurate protein target predictions and to reveal the mechanism of action of compounds with high sensitivity and specificity. Further, an application of ProTINA to gene expression profiles of influenza A viral infection led to new insights of the early events in the infection. Figure 1. Protein target prediction by ProTINA. (A) The protein-gene network describes direct and indirect regulations of gene expression by transcription factors (TF) and their protein partners (P), respectively. A drug interaction with a protein is expected to cause differential expression of the downstream genes in the PGN. (B)Based on a kinetic model of gene transcriptional process, ProTINA infers the weights of the protein-gene regulatory edges, denoted by a kj , using gene expression data. The variable a kj describes the regulation of protein j on gene k, where the magnitude and sign of a kj indicate the strength and mode (+a kj : activation, -a kj : repression) of the regulatory interaction, respectively. (C) A candidate protein target is scored based on the deviations in the expression of downstream genes from the PGN model prediction (P j : log 2 FC expression of protein j, G k : log 2 FC expression of gene k). The colored dots in the plots illustrate the log 2 FC data of a particular drug treatment, while the lines show the predicted expression of gene k by the (linear) PGN model. The variable z k denotes the z-score of the deviation of the expression of gene k from the PGN model prediction. A drug-induced enhancement of protein-gene regulatory interactions is indicated by a positive (negative) z k in the expression of genes that are activated (repressed) by the protein (i.e. a kj z k > 0). Vice versa, a drug-induced attenuation is indicated by a negative (positive) z k in the expression of genes that are activated (repressed) by the protein (i.e. a kj z k < 0). (D) The score of a candidate protein target is determined by combining the z-scores of the set of regulatory edges associated with the protein in the PGN. A positive (negative) score indicates a drug-induced enhancement (attenuation). The larger the magnitude of the score, the more consistent is the drug induced perturbations (enhancement/attenuation) on the protein-gene regulatory edges. Abstract After Ͼ 8,000 infections and Ͼ 700 deaths worldwide, the pathogenesis of the new infectious disease, severe acute respiratory syndrome (SARS), remains poorly understood. We investigated 18 autopsies of patients who had suspected SARS; 8 cases were confirmed as SARS. We evaluated white blood cells from 22 confirmed SARS patients at various stages of the disease. T lymphocyte counts in 65 confirmed and 35 misdiagnosed SARS cases also were analyzed retrospectively. SARS viral particles and genomic sequence were detected in a large number of circulating lymphocytes, monocytes, and lymphoid tissues, as well as in the epithelial cells of the respiratory tract, the mucosa of the intestine, the epithelium of the renal distal tubules, the neurons of the brain, and macrophages in different organs. SARS virus seemed to be capable of infecting multiple cell types in several organs; immune cells and pulmonary epithelium were identified as the main sites of injury. A comprehensive theory of pathogenesis is proposed for SARS with immune and lung damage as key features. Abstract Nanomedicines often involve the use of nanocarriers as a delivery system for drugs or genes for maximizing the therapeutic effect and/or minimizing the adverse effect. From drug administration to therapeutic activity, nanocarriers must evade the host's immune system, specifically and efficiently target and enter the cell, and release their payload into the cell cytoplasm by endosomal escape. These processes constitute the early infection stage of viruses. Viruses are a powerful natural nanomaterial for the efficient delivery of genetic information by sophisticated mechanisms. Over the past two decades, many virus-inspired nanocarriers have been generated to permit successful drug and gene delivery. In this review, we summarize the early infection machineries of viruses, of which the part has so far been utilized for delivery systems. Furthermore, we describe basics and applications of the bio-nanocapsule, which is a hepatitis B virus-mimicking nanoparticle harboring nearly all activities involved in the early infection machineries (i.e., stealth activity, targeting activity, cell entry activity, endosomal escaping activity). Abstract We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL) recognition in vitro compared to wild-type HLA-A*02:01. This mutant contains a single amino acid substitution from histidine to leucine at position 74 (H74L) that is located in the peptide-binding groove. To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT) in which three components involving a peptide, β2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers. We generated recombinant adenovirus expressing SCT comprised influenza A matrix protein (FMP)-derived peptide, β2 microglobulin and the H74L heavy chain. HLA-A*02:01 transgenic mice were immunized with the adenovirus, and the induction of peptide-specific CTLs and antitumor immunity was investigated. It was clearly shown that the H74L mutation enabled the HLA-A*02:01 SCT molecule to dramatically enhance both in vivo priming of FMP-specific CTLs and protection against a lethal challenge of tumor cells expressing FMP. These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.A single-chain HLA mutant enhances CTL priming M Matsui et al. Abstract This article is a non-systematic review of selected recent publications in community-acquired pneumonia, including a comparison of various guidelines. Risk stratification of patients has recently been advanced by the addition of several useful biomarkers. The issue of single versus dual antibiotic treatment remains controversial and awaits a conclusive randomized controlled trial. However, in the meantime, there is a working consensus that more severe patients should receive dual therapy. Abstract The sudden outbreak of severe acute respiratory syndrome (SARS) in 2002 prompted the establishment of a global scientific network subsuming most of the traditional rivalries in the competitive field of virology. Within months of the SARS outbreak, collaborative work revealed the identity of the disastrous pathogen as SARS-associated coronavirus (SARS-CoV). However, although the rapid identification of the agent represented an important breakthrough, our understanding of the deadly virus remains limited. Detailed biological knowledge is crucial for the development of effective countermeasures, diagnostic tests, vaccines and antiviral drugs against the SARS-CoV. This article reviews the present state of molecular knowledge about SARS-CoV, from the aspects of comparative genomics, molecular biology of viral genes, evolution, and epidemiology, and describes the diagnostic tests and the anti-viral drugs derived so far based on the available molecular information. Abstract Salmonella Typhimurium is one of the most important zoonotic pathogens worldwide and a major cause of economic losses in the pig production chain. The emergence of multi-drug resistant strains over the past years has led to considerations about an enhanced surveillance of bacterial food contamination. Currently, ELISA is the method of choice for high throughput identification of S. Typhimurium. The sensitivity and specificity of this assay might be improved by application of new diagnostic antibodies. We focused on plant-based expression of candidate diagnostic TM43-E10 antibodies discovered using as antigen the S. Typhimurium OmpD protein. The scFv-TM43-E10 and scFv-Fc-TM43-E10 antibody derivatives have been successfully produced in N. benthamiana using a deconstructed movementdeficient PVX vector supplemented with the gb silencing suppressor from Poa semilatent virus. The plant-made antibodies showed the same antigen-binding specificity as that of the microbial/mammalian cell-produced counterparts and could recognize the OmpD antigen in S. Typhimurium infected plant samples. Abstract Influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. In this review, we describe the current medical need resulting from respiratory infections caused by RNA viruses, which justifies drug discovery efforts to identify new therapeutic agents. The RNA polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of RNA chain synthesis. Here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of RNA respiratory viruses: Orthomyxoviridae, Pneumoviridae/Paramyxoviridae, Coronaviridae, and Picornaviridae. We summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. This includes molecules with very broad antiviral spectrum such as ribavirin and T-705 (favipiravir), and others targeting more specifically one or a few virus families. Recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs. Abstract BACKGROUND: Cases of H1N1 and other pulmonary infections evolve to acute respiratory failure and death when co-infections or lung injury predominate over the immune response, thus requiring early diagnosis to improve treatment.To perform a detailed histopathological analysis of the open lung biopsy specimens from five patients with ARDS with confirmed H1N1.Lung specimens underwent microbiologic analysis, and examination by optical and electron microscopy. Immunophenotyping was used to characterize macrophages, natural killer, T and B cells, and expression of cytokines and iNOS.The pathological features observed were necrotizing bronchiolitis, diffuse alveolar damage, alveolar hemorrhage and abnormal immune response. Ultrastructural analysis showed viral-like particles in all cases.CONCLUSIONS: Viral-like particles can be successfully demonstrated in lung tissue by ultrastructural examination, without confirmation of the virus by RT-PCR on nasopharyngeal aspirates. Bronchioles and epithelium, rather than endothelium, are probably the primary target of infection, and diffuse alveolar damage the consequence of the effect of airways obliteration and dysfunction on innate immunity, suggesting that treatment should be focused on epithelial repair. MIS. Pathological and ultrastructural analysis of surgical lung biopsies in patients with swine-origin influenza type A/H1N1 and acute respiratory failure. Abstract A method is described for the detection of certain nucleotide modifications adjacent to the 5' 7-methyl guanosine cap of mRNAs from individual genes. The method quantitatively measures the relative abundance of 2'-O-methyl and N 6 ,2'-O-dimethyladenosine, two of the most common modifications. In order to identify and quantitatify the amounts of N 6 ,2'-O-dimethyladenosine, a novel method for the synthesis of modified adenosine phosphoramidites was developed. This method is a one step synthesis and the product can directly be used for the production of N 6 ,2'-O-dimethyladenosine containing RNA oligonucleotides. The nature of the cap-adjacent nucleotides were shown to be characteristic for mRNAs from individual genes transcribed in liver and testis. Abstract The World Health Organization lists a constellation of 17 tropical diseases that afflict approximately one in six individuals on the planet and, until recently, few resources have been devoted to the treatment and eradication of those diseases. They are often referred to as the diseases of the "bottom billion," because they are most prevalent among the poorest individuals in impoverished tropical nations. However, the few studies that have been performed reveal an extraordinary world of molecular and cellular adaptations that facilitate the pathogens' survival in hosts ranging from insects to humans. A compelling case can be made that even a modest investment toward understanding the basic molecular and cell biology of these neglected pathogens has a high probability of yielding exciting new cellular mechanisms and insights into novel ways of combating these diseases. Abstract Systems vaccinology has proven a fascinating development in the last decade. Where traditionally vaccine development has been dominated by trial and error, systems vaccinology is a tool that provides novel and comprehensive understanding if properly used. Data sets retrieved from systemsbased studies endorse rational design and effective development of safe and efficacious vaccines. In this review we first describe different omicstechniques that form the pillars of systems vaccinology. In the second part, the application of systems vaccinology in the different stages of vaccine development is described. Overall, this review shows that systems vaccinology has become an important tool anywhere in the vaccine development chain. Abstract research; C.D. contributed new reagents/analytic tools; G.D., A.R., and M.P. analyzed data; and L.L.M.P., S.P., and M.P. wrote the paper. Reviewers: S.S.M., Columbia University; and V.J.M., National Institutes of Health. The authors declare no conflict of interest. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). Abstract Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin I showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from MansGIcNAc to MansGIcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus. preparation of this manuscript and to Ms. H. Snitkin for her help in the tissue culture experiments. We also thank Ms. H. Plesken, Ms. J. Culkin, and Mr. B. Zeitlow for preparing the illustrations and Ms. M. Cort and Ms. T. Singfield for typing the manuscript. Abstract tRNA-derived RNA fragments (tRFs) are an emerging class of non-coding RNAs (ncRNAs). A growing number of reports have shown that tRFs are not random degradation products but are functional ncRNAs made of specific tRNA cleavage. They play regulatory roles in several biological contexts such as cancer, innate immunity, stress responses, and neurological disorders. In this review, we summarize the biogenesis and functions of tRFs. Abstract 766 | E. Ahat et al.MBoC | ARTICLE GRASP depletion-mediated Golgi destruction decreases cell adhesion and migration via the reduction of α5β1 integrin ABSTRACT The Golgi apparatus is a membrane-bound organelle that serves as the center for trafficking and processing of proteins and lipids. To perform these functions, the Golgi forms a multilayer stacked structure held by GRASP55 and GRASP65 trans-oligomers and perhaps their binding partners. Depletion of GRASP proteins disrupts Golgi stack formation and impairs critical functions of the Golgi, such as accurate protein glycosylation and sorting. However, how Golgi destruction affects other cellular activities is so far unknown. Here, we report that depletion of GRASP proteins reduces cell attachment and migration. Interestingly, GRASP depletion reduces the protein level of α5β1 integrin, the major cell adhesion molecule at the surface of HeLa and MDA-MB-231 cells, due to decreased integrin protein synthesis. GRASP depletion also increases cell growth and total protein synthesis. These new findings enrich our understanding on the role of the Golgi in cell physiology and provide a potential target for treating protein-trafficking disorders. Abstract Pneumococcal infections are the leading cause of community-acquired pneumonia. Although the type 1 interferon-α (IFN-α) is a well-known antiviral cytokine, the role of IFN-α in antipneumococcal host defense and its therapeutic potential remain poorly understood. We have investigated these issues by using a murine transgene expression model. We found that in control animals, Streptococcus pneumoniae infection caused severe weight loss and excessive lung inflammation, associated with rapid bacterial outgrowth. In contrast, the animals that received a single dose of an adenoviral vector expressing IFN-α prior to pneumococcal infection demonstrated rapid and effective control of bacterial replication and lung inflammation and improved clinical outcome. Enhanced protection by IFN-α was due to increased activation of neutrophils and macrophages with increased release of reactive oxygen and nitrogen species and bacterial killing. Furthermore, we found that raised levels of IFN-α in the lung remained immune protective even when the gene transfer vector was given at a time postpneumococcal infection. Our study thus shows that the classically antiviral type 1 IFN can be exploited for enhancing immunity against pneumococcal infection via its activating effects on innate immune cells. Our findings hold implications for the therapeutic use of IFN-α gene transfer strategies to combat pneumococcal infections. Abstract Microglia are specialized parenchymal-resident phagocytes of the central nervous system (CNS) that actively support, defend and modulate the neural environment. Dysfunctional microglial responses are thought to worsen CNS diseases; nevertheless, their impact during neuroinflammatory processes remains largely obscure. Here, using a combination of single-cell RNA sequencing and multicolour flow cytometry, we comprehensively profile microglia in the brain of lipopolysaccharide (LPS)-injected mice. By excluding the contribution of other immune CNS-resident and peripheral cells, we show that microglia isolated from LPS-injected mice display a global downregulation of their homeostatic signature together with an upregulation of inflammatory genes. Notably, we identify distinct microglial activated profiles under inflammatory conditions, which greatly differ from neurodegenerative disease-associated profiles. These results provide insights into microglial heterogeneity and establish a resource for the identification of specific phenotypes in CNS disorders, such as neuroinflammatory and neurodegenerative diseases. Abstract This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 790-794 h t t p : / / w w w . b j m i c r o b i o l . c o m . b r / Distemper Parvovirus Diarrhea Dog Co-infection a b s t r a c t Although the use of vaccines has controlled enteric diseases in dogs in many developed countries, vaccine coverage is still under optimal situation in Brazil. There is a large population of nonimmunized dogs and few studies about the identification of the viruses associated with diarrhea. To address this situation, stool samples from 325 dogs were analyzed by polymerase chain reaction for the detection of common enteric viruses such as Canine adenovirus (CAdV), Canine coronavirus (CCoV), Canine distemper virus (CDV), Canine rotavirus (CRV)and Carnivorous protoparvovirus 1 (canine parvovirus 2; CPV-2). At least one of these species was detected in 56.6% (184/325) of the samples. The viruses detected most frequently in either diarrheic or nondiarrheic dog feces were CPV-2 (54.3% of the positive samples), CDV (45.1%) and CCoV (30.4%), followed by CRV (8.2%) and CAdV (4.9%). Only one agent was detected in the majority of the positive samples (63%), but co-infections were present in 37% of the positive samples and mainly included CDV and CPV-2. The data presented herein can improve the clinical knowledge in regions with low vaccine coverage and highlight the need to improve the methods used to control these infectious diseases in domestic dogs. Abstract Case summary A 10-month-old neutered male domestic shorthair cat presented with a 4 month history of polyuria and polydipsia. After a thorough diagnostic work-up the only abnormal findings were hyposthenuria and an elevated random plasma osmolality level. Trial therapy with the oral and ophthalmic forms of desmopressin failed to concentrate urine. A modified water deprivation test confirmed the ability to concentrate urine above a urine specific gravity (USG) of 1.035. After transitioning the cat to a higher sodium diet and instituting several enrichment changes to the cat's environment, average water consumption and urine output levels decreased to almost normal levels and USG increased from 1.006 to 1.022. These findings provide strong evidence that primary polydipsia was the underlying etiology of the cat's condition. Relevance and novel information This case report exemplifies the challenges faced when a cat presents for polyuria and polydipsia without an obvious cause identified on routine diagnostics. To our knowledge, this is the first report of primary polydipsia in a cat. Abstract Objective: This study was conducted to assess the correlation between central venous pressure (CVP) and venous blood gas (VBG) analysis parameters, to facilitate management of severe sepsis and septic shock in emergency department. Material and methods: This diagnostic study was conducted from January 2014 until June 2015 in three major educational medical centers, Tehran, Iran. For patients selected with diagnosis of septic shock, peripheral blood sample was taken for testing the VBG parameters and the anion gap (AG) was calculated. All the mentioned parameters were measured again after infusion of 500 cc of normal saline 0.9% in about 1 h. Results: Totally, 93 patients with septic shock were enrolled, 63 male and 30 female. The mean age was 72.53 ± 13.03 and the mean Shock Index (SI) before fluid therapy was 0.79 ± 0.30. AG and pH showed significant negative correlations with CVP, While HCO3 showed a significant positive correlation with CVP. These relations can be affected by the treatment modalities used in shock management such as fluid therapy, mechanical ventilation and vasopressor treatment. Conclusion: It is likely that there is a significant statistical correlation between VBG parameters and AG with CVP, but further research is needed before implementation of the results of this study. Abstract Purpose: This study represents the current epidemiological status of Middle East respiratory syndrome coronavirus (MERS-CoV) worldwide in the first three months of 2019. Patients and methods: Full details of the MERS-CoV cases available and published in the disease outbreak news on the WHO website were retrieved. Related details of laboratoryconfirmed MERS-CoV were extracted and analyzed by standard statistical methods. Results: A total of 107 cases of MERS-CoV, including 18 deaths (overall case fatality rate (CFR), 16.8%; male-specific CFR was 17.5% [14/80] and female-specific CFR was 14.8% [4/27]) were reported to WHO from the National International Health Regulation Focal Points of Saudi Arabia and Oman. The overall mean age was 50±17 years and 80 patients (74.8%) were male. The average time from the onset of the symptoms to the first hospitalization was 3±3.3 days; from the first hospitalization to laboratory confirmation was 3.6±6.5 days; from the onset of symptom to death was 17.5±11.7 days; and the mean length of hospitalization for patients with MERS-CoV was 3.5±3.9 days. Males in comparison to females had a 1.5-fold increased chance (adjusted OR =1.5 [95% CI: 1.3-1.8]) of death related to MERS-CoV infection; 1.05 [95% CI: 1.1-3.3], 1.05 [95% CI: 1.2-2.8] and 1.06 [95% CI: 1.2-2.0] for those who had exposure to camels, camel milk consumption, and close contact with MERS-CoV cases, respectively. Health care workers had 2.4 fold [95% CI: 1.2-3.1] greater odds of death compared to other people. Conclusion:The knowledge obtained from this study can contribute to the development of a prevention program and early system warning against MERS-CoV infection. Abstract The Allplex Respiratory Panel 1/2/3 (All16) is a multiplex PCR assay for detecting 16 respiratory viruses with influenza A virus (FluA) subtyping, and the first clinical assay based on multiple detection temperatures. We compared the results between All16 and Anyplex II RV16 (Any16) in 426 clinical samples. Samples showing discrepancies between the two tests were further tested using monoplex PCR. FluA subtyping based on the hemagglutinin type results of All16, which yielded H1, H3, and non-H1/H3, was compared with the results of the BioFire FilmArray respiratory panel. The positive and negative percent agreements and kappa value for each virus between All16 and Any16 ranged from 54.5-100.0%, 84.7-100.0%, and 0.57-1.00, respectively. FluA subtype results from All16 for 26 samples were consistent with those from FilmArray. Good agreement was observed between the two methods, except when analyzing human enterovirus (kappa value 0.70), and the All16 showed reliable FluA subtyping results. For parainfluenza virus 3, the All16 was more sensitive than Any16. When testing 28 samples simultaneously, the mean test time and hands-on time were 4.3 and 0.5 hours, respectively in All16. In conclusion, All16 showed reliable performance, but further studies are needed regarding human enterovirus analysis. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The severe acute respiratory syndrome (SARS) epidemic started in late 2002 and swiftly spread across 5 continents with a mortality rate of around 10%. Although the epidemic was eventually controlled through the implementation of strict quarantine measures, there continues a need to investigate the SARS coronavirus (SARS-CoV) and develop interventions should it re-emerge. Numerous studies have shown that neutralizing antibodies against the virus can be found in patients infected with SARS-CoV within days upon the onset of illness and lasting up to several months. In contrast, there is little data on the kinetics of T cell responses during SARS-CoV infection and little is known about their role in the recovery process. However, recent studies in mice suggest the importance of T cells in viral clearance during SARS-CoV infection. Moreover, a growing number of studies have investigated the memory T cell responses in recovered SARS patients. This review covers the available literature on the emerging importance of T cell responses in SARS-CoV infection, particularly on the mapping of cytotoxic T lymphocyte (CTL) epitopes, longevity, polyfunctionality and human leukocyte antigen (HLA) association as well as their potential implications on treatment and vaccine development. Abstract Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), account for severe economic losses in the poultry industry. The continuous emergence of a multitude of IBV variants poses many challenges for its diagnosis and control, and live attenuated vaccines, despite their routine use, still plays a significant role in driving IBV evolution, further complicating the epidemiological scenario. Unfortunately, the impact of different vaccination strategies on IB control, epidemiology, and diagnosis has rarely been investigated.This work presents the results of a large-scale diagnostic survey performed in Poland to study IBV molecular epidemiology and how vaccination may affect the viral circulation in the field. To this purpose, 589 samples were collected between May 2017 and January 2019, tested by reverse transcription-PCR for IBV and sequenced. Vaccine and field strains were discriminated based on genetic and anamnestic information.The most commonly detected lineages were 793B (79%) and variant 2 (17.4%), with sporadic detections of QX, Mass, and D274-like strains. Most of the detected strains had a vaccine origin: 46.3% matched one of the applied vaccines, while 36.5% were genetically related to vaccines not implemented in the respective protocol. Besides their practical value for the proper planning of vaccination protocols in Poland, these results suggest that only a fraction (17.2%) of the circulating strains are field ones, imposing a careful assessment of the actual IBV field menaces. Moreover, phenomena like vaccine spreading and persistence seem to occur commonly, stressing the need to further study the epidemiological consequences of the extensive use of live vaccines. Abstract Platform technologies for the changing need of diagnostics are one of the main challenges in medical device technology. From one point-of-view the demand for new and more versatile diagnostic is increasing due to a deeper knowledge of biomarkers and their combination with diseases. From another point-of-view a decentralization of diagnostics will occur since decisions can be made faster resulting in higher success of therapy. Hence, new types of technologies have to be established which enables a multiparameter analysis at the point-of-care. Within this review-like article a system called Fraunhofer ivD-platform is introduced. It consists of a credit-card sized cartridge with integrated reagents, sensors and pumps and a read-out/processing unit. Within the cartridge the assay runs fully automated within 15-20 minutes. Due to the open design of the platform different analyses such as antibody, serological or DNA-assays can be performed. Specific examples of these three different assay types are given to show the broad applicability of the system. Abstract A major advantage of DNA vaccination is the ability to induce both humoral and cellular immune responses. DNA vaccines are currently used in veterinary medicine, but have not achieved widespread acceptance for use in humans due to their low immunogenicity in early clinical studies. However, recent clinical data have re-established the value of DNA vaccines, particularly in priming high-level antigenspecific antibody responses. Several approaches have been investigated for improving DNA vaccine efficacy, including advancements in DNA vaccine vector design, the inclusion of genetically engineered cytokine adjuvants, and novel non-mechanical delivery methods. These strategies have shown promise, resulting in augmented adaptive immune responses in not only mice, but also in large animal models. Here, we review advancements in each of these areas that show promise for increasing the immunogenicity of DNA vaccines. Abstract Linear motifs are short, evolutionarily plastic components of regulatory proteins and provide low-affinity interaction interfaces. These compact modules play central roles in mediating every aspect of the regulatory functionality of the cell. They are particularly prominent in mediating cell signaling, controlling protein turnover and directing protein localization. Given their importance, our understanding of motifs is surprisingly limited, largely as a result of the difficulty of discovery, both experimentally and computationally. The Eukaryotic Linear Motif (ELM) resource at http:// elm.eu.org provides the biological community with a comprehensive database of known experimentally validated motifs, and an exploratory tool to discover putative linear motifs in user-submitted protein sequences. The current update of the ELM database comprises 1800 annotated motif instances representing 170 distinct functional classes, including approximately 500 novel instances and 24 novel classes. Several older motif class entries have been also revisited, improving annotation and adding novel instances. Furthermore, addition of full-text search capabilities, an enhanced interface and simplified batch download has improved the overall accessibility of the ELM data. The motif discovery portion of the ELM resource has added conservation, and structural attributes have been incorporated to aid users to discriminate biologically relevant motifs from stochastically occurring non-functional instances. Figure 5 . Schema of the ELM resource and data life cycle. Annotated ELM classes, and instances thereof, can be searched by database query. Via sequence search by the motif detection pipeline, annotated ELM classes yield putative instances in query sequences. By adding experimental evidence and references, these putative instances become candidate instances for annotation, and, with further curation, ultimately become fully annotated instances. Abstract Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots and frameshifting efficiency has previously been found; however, the physical mechanism behind frameshifting still remains to be fully understood. In this study, we utilized synthetic sequences predicted to form mRNA pseudoknotlike structures. Surprisingly, the structures predicted to be strongest lead only to limited frameshifting. Two-dimensional gel electrophoresis of pulse labelled proteins revealed that a significant fraction of the ribosomes were frameshifted but unable to pass the pseudoknot-like structures. Hence, pseudoknots can act as ribosomal roadblocks, prohibiting a significant fraction of the frameshifted ribosomes from reaching the downstream stop codon. The stronger the pseudoknot the larger the frameshifting efficiency and the larger its roadblocking effect. The maximal amount of full-length frameshifted product is produced from a structure where those two effects are balanced. Taking ribosomal roadblocking into account is a prerequisite for formulating correct frameshifting hypotheses. Abstract Contact monitoring is an essential component of the public health response to a Middle East respiratory syndrome coronavirus outbreak, and is required for an effective quarantine to contain the epidemic. The timeliness of a quarantine is associated with its effectiveness. This paper provides a conceptual framework to describe the process of contact monitoring, and proposes a new measure called the "timely quarantined proportion" as a tool to assess the adequacy of a public health response. Abstract To impart principles of antimicrobial stewardship (AS) and infection prevention and control (IPC), we developed a curriculum tailored to the diverse aptitudes of learners at our medical center.Methods. We integrated case-based modules, group learning activities, smartphone applications (apps), decision support tools, and prescription audit and feedback into curricula of the medical school, medicine residency program, infectious diseases (ID) fellowship program, and hospital medicine program operations. Interventions were implemented in 2012-2016 using a quasi-experimental before-and-after study design, and this was assessed using pre-and postintervention surveys or audit of antibiotic prescriptions.Results. Over 180 medical students participated in the AS and IPC seminars. After smartphone app introduction, 69% reported using the app as their preferred source of antibiotic information. Approximately 70% of students felt comfortable prescribing antibiotics for a known infection compared with 40% at baseline (P = .02), and approximately 83% were able to identify the appropriate personal protective equipment for specific scenarios. Approximately 99% agreed that they have a role in promoting patient safety and preventing healthcare-associated infections as medical students. At 20 months, appropriateness of trainee antibiotic prescriptions increased by 20% (P < .01). Almost all ID fellows indicated that the AS and IPC seminar was a vital training supplement. Uptake of internist antibiotic recommendations using AS decision support tools was approximately 70%.Conclusions. All 5 interventions addressed learning objectives and knowledge gaps and are applicable across a range of environments. Evaluating long-term impact of our curriculum is the focus of future study. Abstract Consensus Statements of the American College of Veterinary Internal Medicine (ACVIM) provide the veterinary community with up-to-date information on the pathophysiology, diagnosis, and treatment of clinically important animal diseases. The ACVIM Board of Regents oversees selection of relevant topics, identification of panel members with the expertise to draft the statements, and other aspects of assuring the integrity of the process. The statements are derived from evidence-based medicine whenever possible and the panel offers interpretive comments when such evidence is inadequate or contradictory. A draft is prepared by the panel, followed by solicitation of input by the ACVIM membership which may be incorporated into the statement. It is then submitted to the Journal of Veterinary Internal Medicine, where it is edited before publication. The authors are solely responsible for the content of the statements. Abstract The discussion of H5N1 influenza virus gain-of-function research has focused chiefly on its risk-to-benefit ratio. Another key component of risk is the level of containment employed. Work is more expensive and less efficient when pursued at biosafety level 4 (BSL-4) than at BSL-3 or at BSL-3 as modified for work with agricultural pathogens (BSL-3-Ag). However, here too a risk-to-benefit ratio analysis is applicable. BSL-4 procedures mandate daily inspection of facilities and equipment, monitoring of personnel for signs and symptoms of disease, and logs of dates and times that personnel, equipment, supplies, and samples enter and exit containment. These measures are not required at BSL-3 or BSL-3-Ag. Given the implications of inadvertent or deliberate release of high-threat pathogens with pandemic potential, it is imperative that the World Health Organization establish strict criteria for biocontainment that can be fairly applied in the developing world, as well as in more economically developed countries.Citation Lipkin WI. 2012. Biocontainment in gain-of-function infectious disease research. mBio 3(5):e00290-12. Abstract The relationship between knowledge, risk perceptions, health belief towards seasonal influenza and vaccination and the vaccination behaviours of nurses was explored. Qualified nurses attending continuing professional education courses at a large London university between 18 April and 18 October 2010 were surveyed (522/672; response rate 77 . 7 %). Of these, 82 . 6% worked in hospitals ; 37 . 0 % reported receiving seasonal influenza vaccination in the previous season and 44 . 9% reported never being vaccinated during the last 5 years. All respondents were categorized using two-step cluster analyses into never, occasionally, and continuously vaccinated groups. Nurses vaccinated the season before had higher scores of knowledge and risk perception compared to the unvaccinated (P<0 . 001). Nurses never vaccinated had the lowest scores of knowledge and risk perception compared to other groups (P<0 . 001). Nurses' seasonal influenza vaccination behaviours are complex. Knowledge and risk perception predict uptake of vaccination in nurses. Abstract Individual patients with life-threatening or severely debilitating diseases can petition the U.S. Food and Drug Administration (FDA) through their physicians to have expanded access (EA) to drugs that are in clinical trials but have not reached full FDA approval (the "single-patient" investigational new drug [IND] application). Additionally, recent state and federal laws-so-called "right to try legislation"-allow patients to approach drug companies directly for access prior to FDA approval. While these pathways provide potential access for individual patients to investigational drugs, different EA pathways permit entire groups of certain patients to access investigational drugs prior to FDA approval. This review focuses on special categories of EA INDs intended for multiple patients-the intermediate-group IND and the widespreadtreatment IND-as well as emergency authorization for use of investigational drugs and biological products (e.g., Abstract The 3rd Workshop on Paediatric Virology, which took place on October 7th, 2017 in Athens, Greece, highlighted the role of breast feeding in the prevention of viral infections during the first years of life. Moreover, it focused on the longterm outcomes of respiratory syncytial virus and rhinovirus infections in prematurely born infants and emphasised the necessity for the development of relevant preventative strategies. Other topics that were covered included the vaccination policy in relation to the migration crisis, mother-to-child transmission of hepatitis B and c viruses, vaccination against human papilloma viruses in boys and advances on intranasal live-attenuated vaccination against influenza. Emphasis was also given to the role of probiotics in the management of viral infections in childhood, the potential association between viral infections and the pathogenesis of asthma, fetal and neonatal brain imaging and the paediatric intensive care of children with central nervous system viral infections. Moreover, an interesting overview of the viral causes of perinatal mortality in ancient Greece was given, where recent archaeological findings from the Athenian Agora's bone well were presented. Finally, different continuing medical educational options in Paediatric Virology were analysed and evaluated. The present review provides an update of the key topics discussed during the workshop. Abstract In addition to the impact of a disease itself, public reaction could be considered another outbreak to be controlled during an epidemic. Taiwan's experience with SARS in 2003 highlighted the critical role played by the media during crisis communication. After the SARS outbreak, Taiwan's Centers for Disease Control (Taiwan CDC) followed the WHO outbreak communication guidelines on trust, early announcements, transparency, informing the public, and planning, in order to reform its risk communication systems. This article describes the risk communication framework in Taiwan, which has been used to respond to the 2009-2016 influenza epidemics, Ebola in West Africa (2014-16), and MERS-CoV in South Korea (2015) during the post-SARS era. Many communication strategies, ranging from traditional media to social and new media, have been implemented to improve transparency in public communication and promote civic engagement. Taiwan CDC will continue to maintain the strengths of its risk communication systems and resolve challenges as they emerge through active evaluation and monitoring of public opinion to advance Taiwan's capacity in outbreak communication and control. Moreover, Taiwan CDC will continue to implement the IHR (2005) and to promote a global community working together to fight shared risks and to reach the goal of ''One World, One Health.'' Abstract The evolution of new and reemerging historic virulent strains of respiratory viruses from animal reservoirs is a significant threat to human health. Inefficient human-to-human transmission of zoonotic strains may initially limit the spread of transmission, but an infection may be contracted by touching contaminated surfaces. Enveloped viruses are often susceptible to environmental stresses, but the human coronaviruses responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have recently caused increasing concern of contact transmission during outbreaks. We report here that pathogenic human coronavirus 229E remained infectious in a human lung cell culture model following at least 5 days of persistence on a range of common nonbiocidal surface materials, including polytetrafluoroethylene (Teflon; PTFE), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. We have shown previously that noroviruses are destroyed on copper alloy surfaces. In this new study, human coronavirus 229E was rapidly inactivated on a range of copper alloys (within a few minutes for simulated fingertip contamination) and Cu/Zn brasses were very effective at lower copper concentration. Exposure to copper destroyed the viral genomes and irreversibly affected virus morphology, including disintegration of envelope and dispersal of surface spikes. Cu(I) and Cu(II) moieties were responsible for the inactivation, which was enhanced by reactive oxygen species generation on alloy surfaces, resulting in even faster inactivation than was seen with nonenveloped viruses on copper. Consequently, copper alloy surfaces could be employed in communal areas and at any mass gatherings to help reduce transmission of respiratory viruses from contaminated surfaces and protect the public health.that this is a significant infection risk to anyone touching a contaminated surface. However, rapid inactivation, irreversible destruction of viral RNA, and massive structural damage were observed in coronavirus exposed to copper and copper alloy surfaces. Incorporation of copper alloy surfaces in conjunction with effective cleaning regimens and good clinical practice could help to control transmission of respiratory coronaviruses, including MERS and SARS.Citation Warnes SL, Little ZR, Keevil CW. 2015. Human coronavirus 229E remains infectious on common touch surface materials. mBio 6(6):e01697-15. Abstract Graphical Abstract Highlights d ER stress pathways can mediate immune recognition of zoonotic viruses d Glycosylation status of viral proteins regulates activation of ER stress d Acute lung injury from pandemic influenza viruses is dependent on this activation d Adaptation through glycan addition mediates immune escape of seasonal IAV Abstract General practitioners stand at the front line of healthcare provision and have a pivotal role in the fight against increasing antibiotic resistance. In this respect, targeted antibiotic prescribing by general practitioners would help reduce the unnecessary use of antibiotics, leading to reduced treatment failures, fewer side-effects for patients and a reduction in the (global) spread of antibiotic resistances. Current 'gold standard' antibiotic resistance detection strategies tend to be slow, taking up to 48 h to obtain a result, although the implementation of point-of-care testing by general practitioners could help achieve the goal of targeted antibiotic prescribing practices. However, deciding on which antibiotic resistances to include in a point-of-care diagnostic is not a trivial task, as outlined in this publication. Abstract Human rhinoviruses (HRVs) are the major cause of the common cold. HRVs were recently reclassified into the Enterovirus genus (HEV) in the Picornaviridae family. HRVs and other members of the HEV genus share many common features, including sense RNA genomes and partial nucleotide sequence identity. The aim of this study was to evaluate different HRV detection strategies. Samples from adults with acute respiratory infection (n = 291) who were treated in Sao Paulo Hospital (2001Hospital ( -2003 were tested using three assays. The first assay detected picornaviruses by RT-PCR and hybridization, the second detected rhinoviruses using RT-PCR/sequencing, and the third differentiated HRV from HEV using duplex seminested-RT-PCR. Analysis of the results obtained from the first two strategies revealed 83% concordance.Discordant samples were then evaluated by the third protocol, and 82% were negative. The picornavirus detection protocol was more sensitive but less specific than the rhinovirus detection protocols. The seminested protocol utilized in the present study was less sensitive and was not useful in differentiating HRV from HEV. Sequencing assays examining different genes would address the best strategy of confirming rhinovirus and enterovirus infections. Abstract Antibodies to Encephalitozoon cuniculi (E. cuniculi) were examined by enzyme-linked immunosorbent assay using E. cuniculi PTP2 recombinant protein from serum samples that had been collected from a total of 295 cats in Japan. Of these samples, 6.1% (18/295) had antibodies against E. cuniculi, which included 6.3% (6/96) of the male cats and 6.0% (12/199) of the female cats. The incidence was slightly higher in feral cats (8.3%, 11/132) compared to domesticated cats (4.3%, 7/163). This suggests the possibility that the cats of our country have become a reservoir of E. cuniculi. This study is the first to demonstrate the prevalence of E. cuniculi infection in cats in Japan. Abstract Hand hygiene and isolation are basic, but very effective, means of preventing the spread of pathogens in healthcare. Although the principle may be straightforward, this review highlights some of the controversies regarding the implementation and efficacy of these interventions.Hand hygiene compliance is an accepted measure of quality and safety in many countries. The evidence for the efficacy of hand hygiene in directly reducing rates of hospital-acquired infections has strengthened in recent years, particularly in terms of reduced rates of staphylococcal sepsis. Defining the key components of effective implementation strategies and the ideal method(s) of assessing hand hygiene compliance are dependent on a range of factors associated with the healthcare system. Although patient isolation continues to be an important strategy, particularly in outbreaks, it also has some limitations and can be associated with negative effects. Recent detailed molecular epidemiology studies of key healthcareacquired pathogens have questioned the true efficacy of isolation, alone as an effective method for the routine prevention of disease transmission.Hand hygiene and isolation are key components of basic infection control. Recent insights into the benefits, limitations and even adverse effects of these interventions are important for their optimal implementation. Abstract The 4a and 4b proteins of the Middle East respiratory syndrome coronavirus (MERS-CoV) have been described for their antagonism on host innate immunity. However, unlike clustering patterns of the complete gene sequences of human and camel MERS-CoVs, the 4a and 4b protein coding regions did not constitute species-specific phylogenetic groups. Moreover, given the estimated evolutionary rates of the complete, 4a, and 4b gene sequences, the 4a and 4b proteins might be less affected by species-specific innate immune pressures. These results suggest that the 4a and 4b proteins of MERS-CoV may function against host innate immunity in a manner independent of host species and/or evolutionary clustering patterns.Human infection with the Middle East respiratory syndrome coronavirus (MERS-CoV), a positive-sense single-stranded RNA virus of the family Coronaviridae, was first reported in 2012 [1]. Since then, more than 2,200 laboratory-confirmed cases have been described with an estimated 35.5% case-fatality rate [2] . Middle East countries appeared to be mainly affected by the life-threatening pathogenicity of the virus, but some exported cases were also reported in Asia, Europe, and the United States of America [1,3]. Given zoonotic transmission of the virus from dromedary camels to humans and its lethality in humans [1], vaccines and/or antivirals should be prepared, but no approved medical countermeasures are available, yet.In line with the pathogenic mechanisms reported with MERS-CoV [4], such as acute respiratory symptoms and local immune responses, viral antagonism against host innate immune responses may be of great significance in terms of the pandemic potential and host range restriction of the virus. Viral antagonism had been already described for , and, of the MERS-CoV proteins, protein 4a was suggested responsible for its role against RNA-dependent protein kinase-mediated antiviral immunity and activation of retinoic acidinducible gene I and Melanoma Differentiation-Associated protein 5 of host cells [6, 7] . The antagonistic effects of MERS-CoV protein 4b were also demonstrated against nuclear factor-κB-dependent immunity and RNase L activation of host cells [8, 9] . As shown for influenza and Zika viruses [10, 11] , the species-specific effects of viral antagonism of MERS-CoV on J Vet Sci. Abstract Animal Model Exp Med. 2018;1:247-249.wileyonlinelibrary.com/journal/ame2 | 247 mimic the pathological process or symptoms of human diseases, at least partially, should be acknowledged as ideal models. As a result, they believe that comparative medicine is an interdisciplinary subject of Laboratory Animal Science and Medicine, focusing on the similarities and differences in medical problems in humans and animals of different species. By establishing animal models of various human diseases, they have studied the occurrence, development, and outcome of diseases caused by the same pathogen in different species, and systematically compared and studied the molecular, organizational, and overall mechanisms that cause these differences in order to understand the nature of human diseases, aging, and life span. They have applied this theory in infectious diseases that have troubled human beings throughout history. In the past 30 years, many new infectious diseases, including AIDS, severe acute respiratory syndrome (SARS), Lyme disease, Hantavirus pneumonia, Nile virus infection, Ebola virus hemorrhagic fever, avian influenza, have emerged, and some diseases are still spreading around the world. Animal models of infectious diseases are indispensable for the study of pathogen-host interactions, immune regulation, and evaluation of vaccines and therapeutic drugs. There are a few key points that need to be considered in the development of infectious disease animal models. Which animals are sensitive to new pathogens? Will the animal-specific diagnostic reagents be developed in time since the clinical ones cannot be applied to animal models? Is there any animal model for simulating the whole process of infectious diseases to support the research? According to these concerns, Prof. Qin Chuan and her team have analyzed the receptors of pathogens and immune systems of different species and strains of animals by comparative medicine methods. The resource bank of susceptible animals with different pathogens has been established to solve the problem of timeliness in the establishment of animal models. By comparing and analyzing the genome, immune factors of humans and different species of animals, and eliminating the background interference of animals, 19 kinds of pathogen monitoring reagents for animal models have been developed. This has solved the problem of clinical reagents being unsuitable for animal detection due to species differences and the short time scale for establishing models. Aimed at the same pathogens, the animal model system (AMS) of different animal species and genetically modified animals has been established, so that factors such as genome, receptor, immunity, pathology, and transmission have been clinically compared and analyzed. The AMS is able to reflect the whole process of various diseases, which has solved the problem of a single animal model being unable to simulate diseases comprehensively. Under this guideline, in 2004, Prof. Qin and her team developed the first nonhuman primate model for SARS infection, which reflects the pathogenesis process of clinical SARS infection. 1 In 2009, they developed the H1N1 transmission model to predict the risk of spread and the carrying of pathogens in animals in close contact with humans. 2 In 2010, the Abstract Background. Beginning in December 2013, an epidemic of chikungunya virus (CHIKV) infection spread across the Caribbean and into virtually all countries in the Western hemisphere, with >2.4 million cases reported through the end of 2017.Methods. We monitored a cohort of school children in rural Haiti from May 2014, through February 2015, for occurrence of acute undifferentiated febrile illness, with clinical and laboratory data available for 252 illness episodes.Results. Our findings document passage of the major CHIKV epidemic between May and July 2014, with 82 laboratory-confirmed cases. Subsequent peaks of febrile illness were found to incorporate smaller outbreaks of dengue virus serotypes 1 and 4 and Zika virus, with identification of additional infections with Mayaro virus, enterovirus D68, and coronavirus NL63. CHIKV and dengue virus serotype 1 infections were more common in older children, with a complaint of arthralgia serving as a significant predictor for infection with CHIKV (odds ratio, 16.2; 95% confidence interval, 8.0-34.4; positive predictive value, 66%; negative predictive value, 80%).Conclusions. Viral/arboviral infections were characterized by a pattern of recurrent outbreaks and case clusters, with the CHIKV epidemic representing just one of several arboviral agents moving through the population. Although clinical presentations of these agents are similar, arthralgias are highly suggestive of CHIKV infection. Abstract A draft genome was produced using reads from both the Illumina HiSeq and Roche 454 platforms using the following pipeline: first Illumina HiSeq reads were filtered and corrected using the standalone read correction tool ErrorCorrectReads.pl available with ALLPATHS-LG (1). Resulting reads were filtered for possible cross-contamination by aligning reads against human, rhesus macaque monkey and mycoplasma reference genomes using the tool Bowtie2 (2) and removed from subsequent processing steps. Due to their larger read length, Roche 454 single-end reads were filtered for contamination from the same sources using DECONSEQ (3). Reads aligned to the above contaminant genomes with 90% identity over at least 90% of their length were removed from further steps unless they also mapped to T. gondii. In addition, for paired-end reads, if either end of the read was aligned under default parameters with any of these possible contaminant genomes, both ends were removed from the data set. Single-end reads were then assembled using the short read assembler Ray (4), using a kmer value of 41 and default parameters. All contigs greater than 500 bp (N50=12,617) were subsequently split into pseudoreads of length 400 bp, with 200 bp of overlap remaining between adjacent pseudo-reads to maximize support for pre-assembled contig continuity in the next assembly step. Next, after filtering for adapter sequence contamination, 454 reads were combined with the filtered pairedend set of Illumina sequence data and assembled using Newbler v2.8 (454 Life Sciences, Roche Applied Science, Branford, CT). Subsequently, single-end Illumina reads assembled by Ray were added to the assembly project as pseudo-reads in a strategy that has previously been demonstrated in two diverse genome projects (5, 6). 75 contigs under 2000 bp in length were removed from the Newbler assembly as they aligned with the human genome using Bowtie2 with default settings. All 454 reads and assembled pseudo-reads were required to overlap by at least 90 bp with 98% identity. During assembly we ensured that all reads were used only once.Gene models were created using the tools Genemark-ES, Augustus, SNAP, exonerate, EVM, PASA,. Augustus parameters were obtained from those generally used to predict genes in the closest S. neurona relative available, T. gondii. Genemark-ES was trained automatically using the S. neurona scaffolds using default settings. SNAP was trained in MAKER on the genome of T. gondii by running Augustus, outputting predictions to zff files, and improving SNAP Hidden Markov model (HMM) parameters based on the new predictions. The subsequent HMM was then used to scan the S. neurona genome. Expressed sequence tags from S. neurona and T. gondii were downloaded from NCBI: redundant reads were removed using CD-HIT (15) using default parameters. Gene models from T. gondii, E. tenella, and N. caninum were downloaded from ToxoDB (v8.2) (16). All forms of physical evidence, as well as output from the tools Augustus and Genemark-ES were then combined using MAKER. SNAP was then adjusted using the novel gene models, and MAKER was run again. Repeats were masked on this second run using T. gondii's library of repeats (available from the Genetic Information Research Institute) as well as repeat families identified in the S. neurona genome after 4 rounds of the ab initio repeat family prediction tool, RepeatModeler (17). In a separate run, SNAP was iteratively trained completely independently of the T. gondii genome using Abstract Calf diarrhea is a commonly reported disease in young animals, and still a major cause of productivity and economic loss to cattle producers worldwide. In the report of the 2007 National Animal Health Monitoring System for U.S. dairy, half of the deaths among unweaned calves was attributed to diarrhea. Multiple pathogens are known or postulated to cause or contribute to calf diarrhea development. Other factors including both the environment and management practices influence disease severity or outcomes. The multifactorial nature of calf diarrhea makes this disease hard to control effectively in modern cow-calf operations. The purpose of this review is to provide a better understanding of a) the ecology and pathogenesis of well-known and potential bovine enteric pathogens implicated in calf diarrhea, b) describe diagnostic tests used to detect various enteric pathogens along with their pros and cons, and c) propose improved intervention strategies for treating calf diarrhea. Abstract The SNP rs12252-C allele alters the function of interferon-induced transmembrane protein-3 increasing the disease severity of influenza virus infection in Caucasians, but the allele is rare. However, rs12252-C is much more common in Han Chinese. Here we report that the CC genotype is found in 69% of Chinese patients with severe pandemic influenza A H1N1/09 virus infection compared with 25% in those with mild infection. Specifically, the CC genotype was estimated to confer a sixfold greater risk for severe infection than the CT and TT genotypes. More importantly, because the risk genotype occurs with such a high frequency, its effect translates to a large population-attributable risk of 54.3% for severe infection in the Chinese population studied compared with 5.4% in Northern Europeans. Interferon-induced transmembrane protein-3 genetic variants could, therefore, have a strong effect of the epidemiology of influenza in China and in people of Chinese descent. Abstract RNA viruses are the causative agents for AIDS, influenza, SARS, and other serious health threats. Development of rapid and broadly applicable methods for complete viral genome sequencing is highly desirable to fully understand all aspects of these infectious agents as well as for surveillance of viral pandemic threats and emerging pathogens. However, traditional viral detection methods rely on prior sequence or antigen knowledge. In this study, we describe sequenceindependent amplification for samples containing ultra-low amounts of viral RNA coupled with Illumina sequencing and de novo assembly optimized for viral genomes. With 5 million reads, we capture 96 to 100% of the viral protein coding region of HIV, respiratory syncytial and West Nile viral samples from as little as 100 copies of viral RNA. The methods presented here are scalable to large numbers of samples and capable of generating full or near full length viral genomes from clone and clinical samples with low amounts of viral RNA, without prior sequence information and in the presence of substantial host contamination. Abstract Most eukaryotic expression systems make use of host-cell nuclear transcriptional and posttranscriptional machineries. Here, we present the first generation of the chimeric cytoplasmic cappingprone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNAdependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems. Abstract In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development. Abstract Patients with acute respiratory failure secondary to tuberculous destroyed lung (TDL) have a poor prognosis. The aim of the present retrospective study was to develop a mortality prediction model for TDL patients who require mechanical ventilation. Methods: Data from consecutive TDL patients who had received mechanical ventilation at a single university-affiliated tertiary care hospital in Korea were reviewed. Binary logistic regression was used to identify factors predicting intensive care unit (ICU) mortality. A TDL on mechanical Ventilation (TDL-Vent) score was calculated by assigning points to variables according to β coefficient values. Results: Data from 125 patients were reviewed. A total of 36 patients (29%) died during ICU admission. On the basis of multivariate analysis, the following factors were included in the TDL-Vent score: age ≥65 years, vasopressor use, and arterial partial pressure of oxygen/fraction of inspired oxygen ratio <180. In a second regression model, a modified score was then calculated by adding brain natriuretic peptide. For TDL-Vent scores 0 to 3, the 60-day mortality rates were 11%, 27%, 30%, and 77%, respectively (p<0.001). For modified TDL-Vent scores 0 to ≥3, the 60-day mortality rates were 0%, 21%, 33%, and 57%, respectively (p=0.001). For both the TDL-Vent score and the modified TDL-Vent score, the areas under the receiver operating characteristic curve were larger than that of other illness severity scores.The TDL-Vent model identifies TDL patients on mechanical ventilation with a high risk of mortality. Prospective validation studies in larger cohorts are now warranted. Abstract The tick is a well-known vector for arthropod-borne pathogens, such as tick-borne encephalitis, Lyme disease, Japanese spotted fever and severe fever with thrombocytopenia syndrome. It is therefore important to know the tick population and distribution in our environment and wild animals in order to prevent tick-borne diseases. Here, we report the results of tick surveillance from May to September 2011 at 14 geographical points and in 5 wild boars in Kyoto City, Kyoto prefecture, Japan. We collected 3,198 ticks comprising 5 tick species, Haemaphysalis (H.) longicornis, H. flava, H. kitaokai, Amblyomma testudinarium and Dermacentor taiwanensis. Interestingly, the proportion of tick species varied according to geographical region within the city. The ticks collected in the city were reported as potential vectors of pathogens, such as rickettsiosis. We detected rickettsial DNA by PCR in 71.1% of 201 ticks investigated. The ticks that carried rickettsiae were distributed across the whole the city. The sequences of PCR-amplified DNA fragments were determined and showed similarities to spotted fever group rickettsiae. Although their pathogenicity for animals including humans is still unclear, it is important to stay alert and pay attention to tick-borne diseases in order to ensure the safety of the citizens of the city as well as that of visitors. Abstract Objective: Tumor-treating fields are currently used to successfully treat various cancers; however, the specific pathways associated with its efficacy remain unknown in the immune responses. Here, we evaluated tumor-treating fields-mediated initiation of the macrophage-specific immune response. Materials and Methods: We subjected RAW 264.7 mouse macrophages to clinically relevant levels of tumor-treating fields (0.9 V/cm, 150 kHz) and evaluated alterations in cytokine expression and release, as well as cell viability. Additionally, we investigated the status of immunomodulatory pathways to determine their roles in tumor-treating fields-mediated immune activation. Results and Discussion: Our results indicated that tumor-treating fields treatment at 0.9 V/cm decreased cell viability and increased cytokine messenger RNA/protein levels, as well as levels of nitric oxide and reactive oxygen species, relative to controls. The levels of tumor necrosis factor a, interleukin 1b, and interleukin 6 were markedly increased in tumor-treating fields-treated RAW 264.7 cells cocultured with 4T1 murine mammary carcinoma cells compared with those in 4T1 or RAW 264.7 cells with or without tumor-treating fields treatment. Moreover, the viability of 4T1 cells treated with the conditioned medium of tumor-treating fields-stimulated RAW 264.7 cells decreased, indicating that macrophage activation by tumor-treating fields effectively killed the tumor cells. Moreover, tumor-treating fields treatment activated the nuclear factor kB and mitogen-activated protein kinase pathways involved in immunomodulatory signaling. Conclusion: These results provide critical insights into the mechanisms through which tumor-treating fields affect macrophagespecific immune responses and the efficacy of this method for cancer treatment. Abstract Since 1997, several epizootic avian influenza viruses (AIVs) have been transmitted to humans, causing diseases and even deaths. The recent emergence of severe human infections with AIV (H7N9) in China has raised concerns about efficient interpersonal viral transmission, polygenic traits in viral pathogenicity and the management of newly emerging strains. The symptoms associated with viral infection are different in various AI strains: H5N1 and newly emerged H7N9 induce severe pneumonia and related complications in patients, while some H7 and H9 subtypes cause only conjunctivitis or mild respiratory symptoms. The virulence and tissue tropism of viruses as well as the host responses contribute to the pathogenesis of human AIV infection. Several preventive and therapeutic approaches have been proposed to combat AIV infection, including antiviral drugs such as M2 inhibitors, neuraminidase inhibitors, RNA polymerase inhibitors, attachment inhibitors and signal-transduction inhibitors etc. In this article, we summarize the recent progress in researches on the epidemiology, clinical features, pathogenicity determinants, and available or potential antivirals of AIV. Abstract Powassan virus (POWV) is an emerging tick-transmitted flavivirus that circulates in North America and Russia. Up to 5% of deer ticks now test positive for POWV in certain regions of the northern United States. Although POWV infections cause life-threatening encephalitis, there is no vaccine or counter-measure available for prevention or treatment. Here, we developed a lipid nanoparticle (LNP)-encapsulated modified mRNA vaccine encoding the POWV prM and E genes and demonstrated its immunogenicity and efficacy in mice following immunization with one or two doses. The POWV mRNA vaccine induced high titers of neutralizing antibody and sterilizing immunity against lethal challenge with different POWV strains. The mRNA vaccine also induced cross-neutralizing antibodies against multiple other tick-borne flaviviruses and protected mice against the distantly related Langat virus. These data demonstrate the utility of the LNP-mRNAThis is an open access article under the CC BY-NC-ND license. Abstract Graphical Abstract Highlights d We describe cooperative neutralization and in vivo protection d Cooperativity turns non-neutralizing ebolavirus antibodies into potent neutralizers d A hotspot for antibody cooperativity identified on Ebola virus glycoprotein Abstract The purpose of this prospective case-control study was to survey the detection rate of respiratory viruses in children with Kawasaki disease (KD) by using multiplex reverse transcriptasepolymerase chain reaction (RT-PCR), and to investigate the clinical implications of the prevalence of respiratory viruses during the acute phase of KD. Methods: RT-PCR assays were carried out to screen for the presence of respiratory syncytial virus A and B, adenovirus, rhinovirus, parainfluenza viruses 1 to 4, influenza virus A and B, metapneumovirus, bocavirus, coronavirus OC43/229E and NL63, and enterovirus in nasopharyngeal secretions of 55 KD patients and 78 control subjects. Results: Virus detection rates in KD patients and control subjects were 32.7% and 30.8%, respectively (P=0.811). However, there was no significant association between the presence of any of the 15 viruses and the incidence of KD. Comparisons between the 18 patients with positive RT-PCR results and the other 37 KD patients revealed no significant differences in terms of clinical findings (including the prevalence of incomplete presentation of the disease) and coronary artery diameter. Conclusion: A positive RT-PCR for currently epidemic respiratory viruses should not be used as an evidence against the diagnosis of KD. These viruses were not associated with the incomplete presentation of KD and coronary artery dilatation. Abstract Many emerging arboviruses are not transmitted by traditional mosquito vectors, but by lesser-studied arthropods such as ticks, midges, and sand flies. Small RNA (sRNA) silencing pathways are the main antiviral defence mechanism for arthropods, which lack adaptive immunity. Non-retroviral integrated RNA virus sequences (NIRVS) are one potential source of sRNAs which comprise these pathways. NIRVS are remnants of past germline RNA viral infections, where viral cDNA integrates into the host genome and is vertically transmitted. In Aedes mosquitoes, NIRVS are widespread and produce PIWI-interacting RNAs (piRNAs). These are hypothesised to target incoming viral transcripts to modulate viral titre, perhaps rendering the organism a more efficient arbovirus vector. To explore the NIRVS landscape in alternative arbovirus vectors, we validated the NIRVS landscape in Aedes spp. and then identified novel NIRVS in six medically relevant arthropods and also in Drosophila melanogaster. We identified novel NIRVS in Phlebotomus papatasi, Culicoides sonorensis, Rhipicephalus microplus, Anopheles gambiae, Culex quinquefasciatus, and Ixodes scapularis. Due to their unexpected abundance, we further characterised NIRVS in the blacklegged tick I. scapularis (n = 143). Interestingly, NIRVS are not enriched in R. microplus, another hard tick, suggesting this is an Ixodes-specific adaptation. I. scapularis NIRVS are enriched in bunya-and orthomyxo-like sequences, reflecting that ticks are a dominant host for these virus groups. Unlike in mosquitoes, I. scapularis NIRVS are more commonly derived from the nonstructural region (replicase) of negative-sense viruses, as opposed to structural regions (e.g. glycoprotein). Like other arthropods, I. scapularis NIRVS preferentially integrate into genomic piRNA clusters, and serve as a template for primary piRNA production in the commonly used embryonic I. scapularis ISE6 cell line. Interestingly, we identified a two-fold enrichment of nonlong terminal repeat (non-LTR) retrotransposons, in genomic proximity to NIRVS, contrasting with studeis in Ae. aegypti, where LTR retrotransposons are instead associated with NIRVS formation. We characterised NIRVS phylogeny and integration patterns in the important vector, I. scapularis, revealing they are distinct from those in Aedes spp. Future studies will explore the possible antiviral mechanism conferred by NIRVS to I. scapularis,which may help the transmission of pathogenic arboviruses. Finally, this study explored NIRVS as an untapped wealth of viral diversity in arthropods. Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.shown to inhibit the innate immune response by blocking the induction of interferon (IFN). In this study, we isolated two single-stranded RNA aptamers specific to NS1 with K d values of 1.62 ± 0.30 nM and 1.97 ± 0.27 nM, respectively, using a systematic evolution of ligand by exponential enrichment (SELEX) procedure. The selected aptamers were able to inhibit the interaction of NS1 with tripartite motif-containing protein 25 (TRIM25), and suppression of NS1 enabled retinoic acid inducible gene I (RIG-I) to be ubiquitinated regularly by TRIM25. Additional luciferase reporter assay and quantitative real-time PCR (RT-PCR) experiments demonstrated that suppression of NS1 by the selected aptamers induced IFN production. It is noted that viral replication was also inhibited through IFN induction in the presence of the selected aptamers. These results suggest that the isolated aptamers are strongly expected to be new therapeutic agents against influenza infection. Keywords: aptamer, influenza virus, non-structural 1, systematic evolution of ligands by exponential enrichment, ubiquiti nation 722 Mol. Cells 2019; 42(10): 721-728 RNA Aptamers Specific to NS1 Protein of Influenza Virus Hye-Min Woo et al. Abstract Background: Important knowledge gaps exist in our understanding of migration medicine practice and the impact of pathogens imported by Canadian travellers. We present here a comprehensive, Canada-specific surveillance summary of illness in a cohort of returned Canadian travellers and new immigrants.: We extracted and analyzed (using standard parametric and nonparametric techniques) data from the Canadian Travel Medicine Network (CanTravNet) database for ill returned Canadian travellers and new immigrants who Results: During the study period, 4365 travellers and immigrants presented to a CanTravNet site, 3943 (90.3%) of whom were assigned a travel-related diagnosis. Among the 3115 non-immigrant travellers with a definitive travel-related diagnosis, arthropod bite (n = 127 [4.1%]), giardiasis (n = 91 [2.9%]), malaria (n = 77 [2.5%]), latent tuberculosis (n = 73 [2.3%]), and strongyloidiasis (n = 66 [2.1%]) were the most common specific etiologic diagnoses. Among the 828 immigrants with definitive travel-related diagnoses, the most frequent etiologies were latent tuberculosis (n = 229 [27.7%]), chronic hepatitis B (n = 182 [22.0%]), active tuberculosis (n = 97 [11.7%]), chronic hepatitis C (n = 89 [10.7%]), and strongyloidiasis (n = 41 [5.0%]). Potentially serious infections, such as dengue fever (61 cases) and enteric fever due to Salmonella enterica serotype Typhi or Paratyphi (36 cases), were common. Individuals travelling for the purpose of visiting friends and relatives (n = 500 [11.6% of those with known reason for travel]) were over-represented among those diagnosed with malaria and enteric fever, compared with other illnesses (for malaria 34/94 [36.2%] v. 466/4221 [11.0%]; for enteric fever, 17/36 [47.2%] v. 483/4279 [11.3%]) (both p < 0.001). For cases of malaria, there was also overrepresentation (compared with other illnesses) from business travellers (22/94 [23.4%] v. 337/4221 [8.0%]) and males (62/94 [66.0%] v. 1964/4269 [46.0%]) (both p < 0.001).Malaria was more likely than other illnesses to be acquired in sub-Saharan Africa (p < 0.001), whereas dengue was more likely than other illnesses to be imported from the Caribbean and South East Asia (both p = 0.003) and enteric fever from South Central Asia (24/36 [66.7%]) (p < 0.001).Interpretation: This analysis of surveillance data on ill returned Canadian travellers has detailed the spectrum of imported illness within this cohort. It provides an epidemiologic framework for Canadian practitioners encountering ill returned travellers. We have confirmed that travel to visit friends and relatives confers particularly high risks, which underscores the need to improve pretravel intervention for a population that is unlikely to seek specific pretravel advice. Potentially serious and fatal illnesses such as malaria and enteric fever were common, as were illnesses of public health importance, such as tuberculosis and hepatitis B.Competing interests: From 2008 to 2009, Andrea Boggild served as a medical consultant to Shoreland Inc. In the past 2 years, Michael Libman has served on the advisory board of Sanofi Pasteur. In the past 5 years, Brian Ward has served as the medical officer for Medicago Inc., has held investigator-initiated grants shared with industrial partners (specifically GlaxoSmithKline and Medicago Inc.), has held contracts for clinical vaccine trials (with Pfizer, Sanofi Pasteur, GlaxoSmithKline, and Medicago Inc.), has received honoraria approximately 5 times per year for delivering talks (for which he had sole control over content), and has served as an expert witness for vaccine injury compensation programs in Quebec and the United States. Susan Abstract Oligonucleotide microarrays have been applied to microbial surveillance and discovery where highly multiplexed assays are required to address a wide range of genetic targets. Although printing density continues to increase, the design of comprehensive microbial probe sets remains a daunting challenge, particularly in virology where rapid sequence evolution and database expansion confound static solutions. Here, we present a strategy for probe design based on protein sequences that is responsive to the unique problems posed in virus detection and discovery. The method uses the Protein Families database (Pfam) and motif finding algorithms to identify oligonucleotide probes in conserved amino acid regions and untranslated sequences. In silico testing using an experimentally derived thermodynamic model indicated near complete coverage of the viral sequence database. Abstract Interest in the composition of the intestinal microbiota and possibilities of its therapeutic modifications has soared over the last decade and more detailed knowledge specific to the canine microbiota at different mucosal sites including the gut is available. Probiotics, prebiotics or their combination (synbiotics) are a way of modifying the intestinal microbiota and exert effects on the host immune response. Probiotics are proposed to exert their beneficial effects through various pathways, for example production of antimicrobial peptides, enhancing growth of favourable endogenous microorganisms, competition for epithelial colonisation sites and immunemodulatory functions. Despite widespread use of pro-, pre-and synbiotics, scientific evidence of their beneficial effects in different conditions of the dog is scarce. Specific effects of different strains, their combination or their potential side-effects have not been evaluated sufficiently. In some instances, in vitro results have been promising, but could not be transferred consistently into in vivo situations. Specific canine gastrointestinal (GI) diseases or conditions where probiotics would be beneficial, their most appropriate dosage and application have not been assessed extensively. This review summarises the current knowledge of the intestinal microbiome composition in the dog and evaluates the evidence for probiotic use in canine GI diseases to date. It wishes to provide veterinarians with evidence-based information on when and why these products could be useful in preventing or treating canine GI conditions. It also outlines knowledge about safety and approval of commercial probiotic products, and the potential use of faecal microbial transplantation, as they are related to the topic of probiotic usage. Abstract Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem-loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through 'kissing' loop-loop interactions. We also show that loop-loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop-loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis. Abstract We provide the first genetic sequence data for a Dipylidium species from a wild carnivore plus an analysis of the effects of ecological, demographic, physiological and behavioural factors on Dipylidium sp. infection prevalence in a social carnivore, the spotted hyaena (Crocuta crocuta), in the Serengeti National Park, Tanzania. Our sequence data from a mitochondrial gene fragment (1176 base pair long) had a similarity of between 99% and 89% to Dipylidium caninum. We determined infection prevalence in 146 faecal samples from 124 known animals in three social groups (termed clans) using molecular screening and Dipylidium proglottid presence. Our analysis revealed significantly higher infection prevalence in juveniles (55%) than adults (15.8%), indicating that predominantly juveniles maintained infection in clans. The likelihood of infection in juveniles significantly: (1) increased as the number of adults and older juveniles (>6 months) at communal dens increased, implying a positive relationship between this factor and the size of the intermediate host (probably a flea species) population at communal dens; (2) decreased as the number of younger juveniles (<6 months) increased, suggesting that the chance of susceptible juveniles ingesting infected fleas during self-grooming declined as the number of infected fleas per younger juvenile declined; and (3) decreased during periods of low prey abundance in clan territories when an increased reliance on long-distances foraging excursions reduces the number of clan members visiting communal dens, possibly resulting in a decline in flea populations at dens. Long-distance foraging also increases the intervals (in days) between nursing visits by lactating females to their offspring. Lengthy intervals between milk intake by infected juveniles may reduce adult Dipylidium fecundity and hence decrease infection prevalence in the den flea population. Our study provides useful insights into Dipylidium epidemiology in a social carnivore population subject to large fluctuations in prey abundance. Abstract Filoviruses cause severe and fatal viral hemorrhagic fever in humans. Filovirus research has been extensive since the 2014 Ebola outbreak. Due to their high pathogenicity and mortality, live filoviruses require Biosafety Level-4 (BSL-4) facilities, which have restricted the development of anti-filovirus vaccines and drugs. An HIV-based pseudovirus cell infection assay is widely used for viral entry studies in BSL-2 conditions. Here, we successfully constructed nine in vitro pseudo-filovirus models covering all filovirus genera and three in vivo pseudo-filovirus-infection mouse models using Ebola virus, Marburg virus, and Lloviu virus as representative viruses. The pseudo-filovirus-infected mice showed visualizing bioluminescence in a dose-dependent manner. A bioluminescence peak in mice was reached on day 5 post-infection for Ebola virus and Marburg virus and on day 4 post-infection for Lloviu virus. Two known filovirus entry inhibitors, clomiphene and toremiphene, were used to validate the model. Collectively, our study shows that all genera of filoviruses can be well-pseudotyped and are infectious in vitro. The pseudo-filovirus-infection mouse models can be used for in vivo activity evaluation of anti-filovirus drugs. This sequential in vitro and in vivo evaluation system of filovirus entry inhibitors provides a secure and efficient platform for screening and assessing anti-filovirus agents in BSL-2 facilities. Abstract Treatments targeting the Ebola virus may eventually be shown to work, but they will not have an impact on overall Ebola mortality in West Africa. Endothelial dysfunction is responsible for the fluid and electrolyte imbalances seen in Ebola patients. Because inexpensive generic statins and angiotensin receptor blockers restore endothelial barrier integrity, they can be used to treat the host response in these patients. In Sierra Leone, approximately 100 Ebola patients were treated with this combination, and reports indicate that survival was greatly improved.Citation Fedson DS, Jacobson JR, Rordam OM, Opal SM. 2015. Treating the host response to Ebola virus disease with generic statins and angiotensin receptor blockers. mBio 6(3):e00716-15. Abstract We attempted to prepare a cell line that produces maedi/visna virus (MVV) and is free of contamination by other viruses and mycoplasmas. Three cell lines, which originated from a sheep, goat and bat, were infected with MVV and passaged approximately every 5 days. The cultured cells were then subjected to polymerase chain reaction analysis for MVV provirus. As a result, a cell line persistently infected with MVV was established from ZZ-R cells, which originated from the fetal goat tongue. The 50-fold concentrated culture fluid formed a precipitation line against reference antiserum. Abstract In the analysis of differential peptide peak intensities (i.e. abundance measures), LC-MS analyses with poor quality peptide abundance data can bias downstream statistical analyses and hence the biological interpretation for an otherwise high-quality dataset. Although considerable effort has been placed on assuring the quality of the peptide identification with respect to spectral processing, to date quality assessment of the subsequent peptide abundance data matrix has been limited to a subjective visual inspection of run-by-run correlation or individual peptide components. Identifying statistical outliers is a critical step in the processing of proteomics data as many of the downstream statistical analyses [e.g. analysis of variance (ANOVA)] rely upon accurate estimates of sample variance, and their results are influenced by extreme values. Results: We describe a novel multivariate statistical strategy for the identification of LC-MS runs with extreme peptide abundance distributions. Comparison with current method (runby-run correlation) demonstrates a significantly better rate of identification of outlier runs by the multivariate strategy. Simulation studies also suggest that this strategy significantly outperforms correlation alone in the identification of statistically extreme liquid chromatography-mass spectrometry (LC-MS) runs. Abstract KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bidirectional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHVassociated diseases. Abstract Acute transverse myelitis is a rare spinal cord inflammatory disorder that manifests as sudden onset of motor, sensory, and autonomic dysfunctions. Here, we report a case of acute transverse myelitis in a 13year-old boy secondary to Mycoplasma pneumoniae infection. He presented with left facial palsy and contralateral upper extremity weakness without sensory or autonomic changes. The patient was diagnosed with transverse myelitis based on his magnetic resonance imaging findings, although his presentation was mainly motor dysfunction, which is more consistent with acute flaccid paralysis.A 13-year-old previously healthy boy presented to the emergency department (ED) in August 2018 with neck pain and left sided facial drooping which started on the day of presentation. He had history of nasal congestion, cough, and fever for five days. He was initially diagnosed with Bell's palsy and started on prednisone. The patient returned to the ED two days later with new onset right shoulder and arm weakness. He was tachypneic to 32 breaths/minute, tachycardic to 108 beats/minute, and afebrile with normal blood pressure. He was alert and oriented with Glasgow coma scale 15/15. Neurological examination was significant for left facial drooping and inability to fully close his left eye. He had neck weakness as well and was unable to hold his head unsupported. Pupils were equal and reactive to light. Muscle strength in the right upper extremity was 1/5 proximally and 4/5 distally. He had normal strength of left upper extremity and both lower extremities. Reflexes were absent in right biceps Abstract The objective was to investigate porcine epidemic diarrhea (PED) outbreak that occurred in 2014 in Japan and its effects on herdlevel productivity using a data recording system (PigINFO). The study herds were selected from farrow-to-finish herds (n=99) that entered in the PigINFO (PED epidemic), any herds with clinical signs of PED and feces positive for porcine epidemic diarrhea virus (PEDV) on polymerase chain reaction analysis and/or immunohistochemical staining were defined as PED-positive (n=38). They were further classified into those with long PED periods (L-PED-positive; n=28) and those with short PED periods (S-PED-positive; n=10). Herds with no clinical signs of PED were classified as PED-negative (n=61). Herdlevel production data, including preweaning mortality (%; PRWM), postweaning mortality (%; POWM), pigs weaned per litter (PWL), pigs born alive per litter, litters per mated female per year and pigs marketed per sow (MP), were calculated every 3 months during study period. During the PED epidemic, L-PED-positive herds had significantly higher PRWM and POWM than PED-negative herds, and L-PEDpositive and S-PED-positive herds had significantly lower PWL. During October-December 2014, L-PED-positive herds had significantly fewer MP than PED-negative herds. The PED outbreak increased mortality and consequently reduced the numbers of marketed pigs. The rapid control of an outbreak is important for reducing the financial losses arising from PED infections. Abstract Acute respiratory infections (ARIs) are the leading cause of infectious disease-related morbidity, hospitalization, and morbidity among children worldwide. This study aimed to assess the viral and bacterial causes of ARI morbidity and mortality in children under 5 years in Senegal. Nasopharyngeal samples were collected from children under 5 years who had ARI. Viruses and bacteria were identified using multiplex real-time reverse transcription-polymerase chain reaction and conventional biochemical techniques, respectively. Adenovirus was the most prevalent virus (50%; n = 81), followed by influenza virus (45.68%, n = 74), rhinovirus (40.12%; n = 65), enterovirus (25.31%; n = 41), and respiratory syncytial virus (16.05%; n = 26), whereas Streptococcus pneumoniae (17%; n = 29), Moraxella catarrhalis (15.43%; n = 25), and Haemophilus influenzae (8.02%; n = 13) were the most commonly isolated bacteria. Virus pathogens seem more likely to be more prevalent in our settings and were often associated with bacteria and S. pneumoniae (6%; 16) coinfection. Abstract In this study, we evaluated antibody and cell-mediated immune (CMI) responses in the mucosal and systemic compartments and protection against challenge with a nephropathogenic Brazilian (BR-I) strain of infectious bronchitis virus (IBV) in chickens submitted to a vaccination regime comprising a priming dose of heterologous live attenuated Massachusetts vaccine followed by a booster dose of an experimental homologous inactivated vaccine two weeks later. This immunization protocol elicited significant increases in serum and lachrymal levels of anti-IBV IgG antibodies and upregulated the expression of CMI response genes, such as those encoding CD8β chain and Granzyme homolog A in tracheal and kidney tissues at 3, 7, and 11 days post-infection in the vaccinated chickens. Additionally, vaccinated and challenged chickens showed reduced viral loads and microscopic lesion counts in tracheal and kidney tissues, and their antibody and CMI responses were negatively correlated with viral loads in the trachea and kidney. In conclusion, the combination of live attenuated vaccine containing the Massachusetts strain with a booster dose of an inactivated vaccine, containing a BR-I IBV strain, confers effective protection against infection with nephropathogenic homologous IBV strain because of the induction of consistent memory immune responses mediated by IgG antibodies and TCD8 cells in the mucosal and systemic compartments of chickens submitted to this vaccination regime. Abstract An unexplained increase in the incidence of parapneumonic empyema (PPE) in pneumonia cases has been reported in recent years. The present study investigated the genetic and biological specifications of new isolates of torque teno mini virus (TTMV) detected in pleural effusion samples from children hospitalised for severe pneumonia with PPE.A pathogen discovery protocol was applied in undiagnosed pleural effusion samples and led to the identification of three new isolates of TTMV (TTMV-LY). Isolated TTMV-LY genomes were transfected into A549 and human embryonic kidney 293T cells and viral replication was assessed by quantitative realtime PCR and full-length genome amplification. A549 cells were further infected with released TTMV-LY virions and the induced-innate immune response was measured by multiplex immunoassays.Genetic analyses of the three TTMV-LY genomes revealed a classic genomic organisation but a weak identity (,64%) with known sequences. We demonstrated the in vitro replication of TTMV-LY in alveolar epithelial cells and the effective release of infectious viral particles. We also showed a selective production of inflammatory mediators in response to TTMV infection.This study reports the description of replicative TTMV-LY isolated from parapneumonic effusions of children hospitalised with PPE, suggesting a potential role of the virus in the pathogenesis of pneumonia.@ERSpublications New TTMV isolates, identified in pleural effusions, may have a role in the pathogenesis of severe pneumonia in children Abstract To cite: Cresswell E, et al. A questionnaire-based survey on the uptake and use of cattle vaccines in the UK. Vet ABSTRACT Background: Vaccination is a widely used strategy for disease control in cattle in the UK and abroad. However, there has been limited research describing the uptake and use of cattle vaccines on UK farms. Aim: To describe the current uptake and usage of cattle vaccines in the UK.Design: A questionnaire, available in paper and online format, was distributed to cattle farmers by convenience sampling.Participants: All UK cattle farmers were eligible to participate in the study.Results: Eighty-six per cent of respondents (n=229/266) had vaccinated their cattle in the past year. Diseases most commonly vaccinated against were Bovine Viral Diarrhoea, Leptospirosis and Infectious Bovine Rhinotracheitis. Vaccination compliance was limited in certain areas, for example only 48 per cent of respondents stated that they administered the second dose in the primary course within the recommended timeframe, and 14 per cent of respondents stated that they vaccinated earlier than the youngest recommended age. Although outside the scope of this study, further work is needed to establish the extent of inadequate compliance and the effect this has on vaccine efficacy. The role of the veterinarian was highlighted as the main supplier of vaccines and preferred source of vaccination information. Respondents preferred to receive recommendations regarding vaccination by faceto-face communication with the veterinarian.The results provide a description of the current uptake and usage of cattle vaccines in the UK. Uptake is generally high but there are areas of usage of vaccines which could be improved upon. The veterinarian plays a key role as supplier of vaccines and a source of information for the majority of farmers. Although outside the scope of this study, further work is needed to establish the extent of inadequate compliance and the effect this has on vaccine efficacy. Although the respondents in this study represent a biased population of farmers, the findings indicate areas for future investigation in order to improve vaccination strategies in cattle in the UK.Cresswell E, et al. Vet Rec Open 2014;1:e000042. Abstract Purpose: The aim of this study was to evaluate whether infants with rhinovirus (RV) infection-induced wheezing and those with respiratory syncytial virus (RSV) infection-induced wheezing have different cytokine profiles in the acute stage. Methods: Of the infants with lower respiratory tract infection (LRTI) between September 2011 and May 2012, 88 were confirmed using reverse transcription polymerase chain reaction and hospitalized. Systemic interferon-gamma (IFN-γ), interleukin (IL)-2, IL-12, IL-4, IL-5, IL-13, and Treg-type cytokine (IL-10) responses were examined with multiplex assay using acute phase serum samples. Results: Of the 88 patients, 38 had an RV infection (RV group) and 50 had an RSV infection (RSV group). In the RV group, the IFN-γ and IL-10 concentrations were higher in the patients with than in the patients without wheezing (P=0.022 and P=0.007, respectively). In the RSV group, the differences in IFN-γ and IL-10 concentrations did not reach statistical significance between the patients with and the patients without wheezing (P=0.105 and P=0.965, respectively). The IFN-γ and IL-10 concentrations were not significantly different between the RV group with wheezing and the RSV group with wheezing (P=0.155 and P=0.801, respectively), in contrast to the significant difference between the RV group without wheezing and the RSV group without wheezing (P=0.019 and P=0.035, respectively). Conclusion: In comparison with RSV-induced LRTI, RV-induced LRTI combined with wheezing showed similar IFN-γ and IL-10 levels, which may have an important regulatory function. Abstract Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM 1 (GM 1 ) molecules, and the toxin-GM 1 complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM 1 by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476 -1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573-586, 2012), suggesting that multivalent binding to GM 1 is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM 1 binding sites from zero (nonbinding) to five (wild type). We found that a single GM 1 binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM 1 receptors is not essential for toxicity of CT.We tested this idea directly by using purified variants of CT with zero to five functional receptor-binding sites (BS). One BS enabled CT to intoxicate cells, supporting the conclusion that CT can enter cells by coopting an endogenous lipid-sorting pathway. Although multivalent receptor binding is not essential, it does increase CT toxicity. These findings suggest that achieving higher receptor binding avidity or affecting membrane dynamics by lipid clustering and membrane remodeling may be driving forces for evolution of AB 5 subunit toxins that can bind multivalently to cell membrane lipid receptors.Citation Jobling MG, Yang Z, Kam WR, Lencer WI, Holmes RK. 2012. A single native ganglioside GM 1 -binding site is sufficient for cholera toxin to bind to cells and complete the intoxication pathway. mBio 3(6):e00401-12. Abstract Asthmatics hospitalised because of influenza A infection are less likely to require intensive care or die compared with nonasthmatics. The reasons for this are unknown.We performed a retrospective analysis of data on 1520 patients admitted to 75 UK hospitals with confirmed influenza A/H1N1 2009 infection. A multivariable model was used to investigate reasons for the association between asthma and severe outcomes (intensive care unit support or death).Asthmatics were less likely than nonasthmatics to have severe outcome (11.2% versus 19.8%, unadjusted OR 0.51, 95% CI 0.36-0.72) despite a greater proportion requiring oxygen on admission (36.4% versus 26%, unadjusted OR 1.63) and similar rates of pneumonia (17.1% versus 16.6%, unadjusted OR 1.04). The results of multivariable logistic regression suggest the association of asthma with outcome (adjusted OR 0.62, 95% CI 0.36-1.05; p50.075) are explained by pre-admission inhaled corticosteroid use (adjusted OR 0.34, 95% CI 0.18-0.66) and earlier admission (f4 days from symptom onset) (adjusted OR 0.60, 95% CI 0.38-0.94). In asthmatics, systemic corticosteroids were associated with a decreased likelihood of severe outcomes (adjusted OR 0.36, 95% CI 0.18-0.72).Corticosteroid use and earlier hospital admission explained the association of asthma with less severe outcomes in hospitalised patients. Abstract Adenoviral vectors are widely employed against infectious diseases or cancers, as they can elicit specific antibody responses and T cell responses when they are armed with foreign genes as vaccine carriers, and induce apoptosis of the cancer cells when they are genetically modified for cancer therapy. In this review, we summarize the biological characteristics of adenovirus (Ad) and the latest development of Ad vectorbased strategies for the prevention and control of emerging infectious diseases or cancers. Strategies to circumvent the pre-existing neutralizing antibodies which dampen the immunogenicity of Ad-based vaccines are also discussed. Abstract The initial cluster of severe pneumonia cases that triggered the COVID-19 epidemic was identified in Wuhan, China in December 2019. While early cases of the disease were linked to a wet market, human-to-human transmission has driven the rapid spread of the virus throughout China. The Chinese government has implemented containment strategies of city-wide lockdowns, screening at airports and train stations, and isolation of suspected patients; however, the cumulative case count keeps growing every day. The ongoing outbreak presents a challenge for modelers, as limited data are available on the early growth trajectory, and the epidemiological characteristics of the novel coronavirus are yet to be fully elucidated. We use phenomenological models that have been validated during previous outbreaks to generate and assess short-term forecasts of the cumulative number of confirmed reported cases in Hubei province, the epicenter of the epidemic, and for the overall trajectory in China, excluding the province of Hubei. We collect daily reported cumulative confirmed cases for the 2019-nCoV outbreak for each Chinese province from the National Health Commission of China. Here, we provide 5, 10, and 15 day forecasts for five consecutive days, February 5th through February 9th, with quantified uncertainty based on a generalized logistic growth model, the Richards growth model, and a sub-epidemic wave model. Our most recent forecasts reported here, based on data up until February 9, 2020, largely agree across the three models presented and suggest an average range of 7409e7496 additional confirmed cases in Hubei and 1128e1929 additional cases in other provinces within the next five days. Models also predict an average total cumulative case count between 37,415 and 38,028 in Hubei and 11,588e13,499 in other provinces by February 24, 2020. Mean estimates and uncertainty bounds for both Hubei and other provinces have remained relatively stable in the last three reporting dates (February 7th e 9th). We also observe that each of the models predicts that the epidemic has reached saturation in both Hubei and other provinces. Our findings suggest that the containment strategies implemented in China are successfully reducing transmission and that the epidemic growth has slowed in recent days. Abstract Humanitarian catastrophes," conflicts and calamities generating both widespread human suffering and destructive events, require a wide range of emergency resources. This paper answers a number of questions that humanitarian catastrophes generate: Why and how do the most-developed countries-those with the resources, capabilities, and willingness to help-intervene in specific types of disasters? What ethical and legal guidelines shape our interventions? How well do we achieve our goals? It then suggests a number of changes to improve humanitarian responses, including better NGO-government cooperation, increased research on the best disaster response methods, clarification of the criteria and roles for humanitarian (military) interventions, and development of post-2015 Millennium Development Goals with more accurate progress measures. [West J Emerg Med. 2014;15(2):231-240.] Abstract Background. Few studies have evaluated the relative cross-protection conferred by infection with different groups of viruses through studies of sequential infections in humans. We investigated the presence of short-lived relative cross-protection conferred by specific prior viral infections against subsequent febrile respiratory illness (FRI).Methods. Men enlisted in basic military training between December 2009 and December 2014 were recruited, with the first FRI as the study entry point. ResPlex II assays and real-time polymerase chain reaction assays were used to detect viral pathogens in nasal wash samples, and survival analyses were performed to determine whether infection with particular viruses conferred short-lived relative cross-protection against FRI.Results. Prior infection with adenovirus (hazard ratio [HR], 0.24; 95% confidence interval [CI], .14-.44) or influenza virus (HR, 0.52; 95% CI, .38-.73) conferred relative protection against subsequent FRI episode. Results were statistically significant even after adjustment for the interval between enlistment and FRI (P < .001). Adenovirus-positive participants with FRI episodes tended to be protected against subsequent infection with adenovirus, coronavirus, enterovirus/rhinovirus, and influenza virus (P = .062-.093), while men with influenza virus-positive FRI episodes tended be protected against subsequent infection with adenovirus (P = .044) and influenza virus (P = .081).Conclusion. Prior adenovirus or influenza virus infection conferred cross-protection against subsequent FRI episodes relative to prior infection due to other circulating viruses. Abstract Viruses are the smallest known microbes, yet they cause the most significant losses in human health. Most of the time, the best-known cure for viruses is the innate immunological defense system of the host; otherwise, the initial prevention of viral infection is the only alternative. Therefore, diagnosis is the primary strategy toward the overarching goal of virus control and elimination. The introduction of a new class of nanoscale materials with multiple unique properties and functions has sparked a series of breakthrough applications. Gold nanoparticles (AuNPs) are widely reported to guide an impressive resurgence in biomedical and diagnostic applications. Here, we review the applications of AuNPs in virus testing and detection. The developed AuNP-based detection techniques are reported for various groups of clinically relevant viruses with a special focus on the applied types of bio-AuNP hybrid structures, virus detection targets, and assay modalities and formats. We pay particular attention to highlighting the functional role and activity of each core Au nanostructure and the resultant detection improvements in terms of sensitivity, detection range, and time. In addition, we provide a general summary of the contributions of AuNPs to the mainstream methods of virus detection, technical measures, and recommendations required in guidance toward commercial in-field applications. Abstract The lamin B receptor (LBR) is a polytopic 1. Abbreviations used/n th/spaper: LBR, lamin B receptor; NLS, nuclear localization signal. Abstract Background: We evaluated the analytical and clinical performances of the SD BIOLINE Rota/Adeno Rapid kit (SD Rota/Adeno Rapid; Standard Diagnostics, Inc., Korea), an immunochromatographic assay (ICA), for the simultaneous detection of rotaviruses and adenoviruses in human stool samples.We tested 400 clinical stool samples from patients with acute gastroenteritis and compared the ICA results with the results obtained by using ELISA, enzyme-linked fluorescent assays (ELFA), PCR, and multiplex reverse transcription-PCR (mRT-PCR). To assess the analytical performance of the SD BIOLINE Rota/Adeno Rapid kit, we determined its detection limit, reproducibility, cross-reactivity, and analytical reactivity for adenovirus subtypes, and performed interference studies.Results: The overall agreement rates among the tested methods were 91.5% for rotavirus and 85.5% for adenovirus. On the basis of mRT-PCR, the overall agreement, positive agreement, and negative agreement rates of the ICA were 95.6%, 100%, and 94.9% for rotavirus, and 94.0%, 71.4%, and 94.8% for adenovirus, respectively. Using the ICA, we detected all the subtypes of adenovirus tested, but the analytical reactivities for adenovirus subtypes were different between the 4 adenovirus detection methods. The high reproducibility was confirmed, and no cross-reactivity or interference was detected.Conclusions: The SD BIOLINE Rota/Adeno Rapid kit showed acceptable analytical and clinical performances. However, interpretation of adenovirus positive/negative result should be cautious because of different detectability for adenovirus subtypes among adenovirus detection methods. Abstract Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5 0 direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 AE 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome. Abstract Hepatitis E virus (HEV) is one of the leading causes of acute viral hepatitis. It also causes acute liver failure and acute-onchronic liver failure in many patients, such as those suffering from other infections/ liver injuries or organ transplant/chemotherapy recipients. Despite widespread sporadic and epidemic incidents, there is no specific treatment against HEV, justifying an urgent need for developing a potent antiviral against it. This review summarizes the known antiviral candidates and provides an overview of the potential targets for the development of specific antivirals against HEV. Citation of this article: Anang S, Kaushik N, Surjit M. Recent advances towards the development of a potent antiviral against the hepatitis E virus. Abstract Background. The epidemiology of pediatric febrile illness is shifting in sub-Saharan Africa, but malaria remains a major cause of childhood morbidity and mortality. The present study describes causes of febrile illness in hospitalized children in Ghana and aims to determine the burden of malaria coinfections and their association with parasite densities.Methods. In a prospective study, children (aged ≥30 days and ≤15 years) with fever ≥38.0°C were recruited after admission to the pediatric ward of a primary hospital in Ghana. Malaria parasitemia was determined and blood, stool, urine, respiratory, and cerebrospinal fluid specimens were screened for parasitic, bacterial, and viral pathogens. Associations of Plasmodium densities with other pathogens were calculated.Results. From November 2013 to April 2015, 1238 children were enrolled from 4169 admissions. A clinical/microbiological diagnosis could be made in 1109/1238 (90%) patients, with Plasmodium parasitemia (n = 728/1238 [59%]) being predominant. This was followed by lower respiratory tract infections/pneumonia (n = 411/1238 [34%]; among detected pathogens most frequently Streptococcus pneumoniae, n = 192/299 [64%]), urinary tract infections (n = 218/1238 [18%]; Escherichia coli, n = 21/32 [66%]), gastrointestinal infections (n = 210 [17%]; rotavirus, n = 32/97 [33%]), and invasive bloodstream infections (n = 62 [5%]; Salmonella species, n = 47 [76%]). In Plasmodium-infected children the frequency of lower respiratory tract, gastrointestinal, and bloodstream infections increased with decreasing parasite densities.Conclusions. In a hospital setting, the likelihood of comorbidity with a nonmalarial disease is inversely correlated with increasing blood levels of malaria parasites. Hence, parasite densities provide important information as an indicator for the probability of coinfection, in particular to guide antimicrobial medication. Abstract Background: Proenkephalin (PENK) has been suggested as a novel biomarker for kidney function. We investigated the diagnostic and prognostic utility of plasma PENK in comparison with neutrophil gelatinase-associated lipocalin (NGAL) and estimated glomerular filtration rates (eGFR) in septic patients.Methods: A total of 167 septic patients were enrolled: 99 with sepsis, 37 with septic shock, and 31 with suspected sepsis. PENK and NGAL concentrations were measured and GFR was estimated by using the isotope dilution mass spectrometry traceable-Modification of Diet in Renal Disease (MDRD) Study and three Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations: CKD-EPICr, CDK-EPICysC, and CKD-EPICr-CysC. The PENK, NGAL, and eGFR results were compared according to sepsis severity, presence or absence of acute kidney injury (AKI), and clinical outcomes.Results: The PENK, NGAL, and eGFR results were significantly associated with sepsis severity and differed significantly between patients with and without AKI only in the sepsis group (all P < 0.05). PENK was superior to NGAL in predicting AKI (P = 0.022) and renal replacement therapy (RRT) (P = 0.0085). Regardless of the variable GFR category by the different eGFR equations, PENK showed constant and significant associations with all eGFR equations. Unlike NGAL, PENK was not influenced by inflammation and predicted the 30day mortality.Conclusions: PENK is a highly sensitive and objective biomarker of AKI and RRT and is useful for prognosis prediction in septic patients. With its diagnostic robustness and predictive power for survival, PENK constitutes a promising biomarker in critical care settings including sepsis. Abstract Background. The aim of the study is to assess the long-term secondary effects of personal experience with the H1N1 pandemic of 2009/2010 and the perception of the institutional reaction to it on Italians' willingness to get vaccinated in case of a novel influenza pandemic.Design and Methods. We conducted 140 face-to-face interviews in the Registry Office of the Municipality of Milan, Italy, from October to December 2012.Results. Willingness to get vaccinated during a novel influenza pandemic was best predicted by having been vaccinated against the seasonal flu in the past (OR=5.18; 95%CI: 1.40 to 19.13) and fear of losing one's life in case of an infection with H1N1 (OR=4.09; 95%CI: 1.68 to 9.97). It was unaffected by the assessment of institutional performance.Conclusions. The findings of this study do not point to long-term secondary effects of the institutional handling of the H1N1 pandemic. The results highlight the fact that behavioural intention is not the same as behaviour, and that the former cannot simply be taken as an indicator of the latter. Abstract Preserved egg, a kind of alkaline-fermented food, is a traditional egg product in China. Here, we investigated the nutritional functions of preserved eggs by in vivo and in vitro experiments. The results of in vivo studies showed that the levels of triglycerides (TG), total cholesterol (TCHO) and low-density lipoprotein cholesterol/high density lipoprotein cholesterol (LDL-C/HDL-C) were significantly decreased (p<0.05) in the liver of rats treated with preserved eggs. Meanwhile, the levels of two important cancer markers, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were also significantly decreased (p<0.05) in treated rats. In vitro studies were performed on Caco-2 cells, a human epithelial colorectal adenocarcinoma cell line. It demonstrated that the gastrointestinal (GI) digests of preserved eggs significantly accelerated (p<0.05) the apoptosis by upregulating caspase-3 in the Caco-2 cells. Besides, after treated with preserved eggs, the half maximal inhibitory concentration (IC50) of preserved eggs digests to Caco-2 cells was 5.75 mg/mL, indicating the significant inhibition of cell proliferation provided by preserved eggs (p<0.05). The results shown in this study demonstrated that preserved eggs may be a novel functional food involved with antilipemic, anti-inflammatory activity as well as the effect on accelarating the apoptosis of Caco-2 cells.Preserved egg, a well-received traditional egg product in China, is produced by marinating raw duck egg with CaO, H 2 O, and Na 2 CO 3 , and other spices (Hou, 1981) .Owing to its unique flavor and attractive color and texture, preserved egg is a popular marinated egg product in China. In addition, the percentage of nine amino acids in preserved egg yolk and egg white was higher than that of fresh egg yolk and egg white after pickling (Zhang et al., 1998) , the species of total fatty acids were increased from 23 to 27, as well as the species of free fatty acids were increased from 7 to 13 (Zhao et al., 2014a), which makes preserved eggs have a higher nutritional value. Abstract Since the progression of cirrhosis is accelerated each time a complication recurs, the management and treatment of the complication is critical in enhancement of the quality of life and expectation of life in patients. The use of model for end-stage liver disease with incorporation of serum-sodium (MELD-Na) with physiological indicators can be used to assess severity and differentiate therapeutic interventions.This study is aimed to determine the mean survival period and cumulative survival rate by classifying patients into high-risk and lowrisk groups based on MELD-Na, a predictor of mortality in liver disease, and to investigate the mortality prognostic factors.A retrospective cohort study, which follows the STROBE checklist, was performed. 263 patients who were diagnosed with liver cirrhosis complications for the first time and hospitalized were selected as the subjects of this study. The collected data were analyzed based on the survival package provided by the statistical program R version 3.4.2.Subjects were classified into high-risk and low-risk groups using MELD-Na 14 points where sensitivity and specificity crossed the cut-off point. Gender, age, and primary caregiver were significant variables in the mortality high-risk group, and AST, albumin, and primary caregiver were significant variables in the mortality low-risk group. Based on these mortality prognostic factors, it is possible to present the factors affecting mortality in patients who were diagnosed with liver cirrhosis complications for the first time. The classification of patients by risk level could be the foundation to provide accurate guidelines for management and it is necessary to modify prognostic factors and apply nursing interventions to manage complications.Abbreviations: AST = aspartate transferase, MELD-Na = model for end-stage liver disease with incorporation of serum-sodium. Abstract Middle East respiratory syndrome (MERS), an emerging infectious disease caused by MERS coronavirus (MERS-CoV), has garnered worldwide attention as a consequence of its continuous spread and pandemic potential, making the development of effective vaccines a high priority. We previously demonstrated that residues 377-588 of MERS-CoV spike (S) protein receptor-binding domain (RBD) is a very promising MERS subunit vaccine candidate, capable of inducing potent neutralization antibody responses. In this study, we sought to identify an adjuvant that optimally enhanced the immunogenicity of S377-588 protein fused with Fc of human IgG (S377-588-Fc). Specifically, we compared several commercially available adjuvants, including Freund's adjuvant, aluminum, Monophosphoryl lipid A, Montanide ISA51 and MF59 with regard to their capacity to enhance the immunogenicity of this subunit vaccine. In the absence of adjuvant, S377-588-Fc alone induced readily detectable neutralizing antibody and T-cell responses in immunized mice. However, incorporating an adjuvant improved its immunogenicity. Particularly, among the aforementioned adjuvants evaluated, MF59 is the most potent as judged by its superior ability to induce the highest titers of IgG, IgG1 and IgG2a subtypes, and neutralizing antibodies. The addition of MF59 significantly augmented the immunogenicity of S377-588-Fc to induce strong IgG and neutralizing antibody responses as well as protection against MERS-CoV infection in mice, suggesting that MF59 is an optimal adjuvant for MERS-CoV RBD-based subunit vaccines.Cellular & Molecular Immunology Abstract In the/S-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in B-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutivelike secretion of newly synthesized C-peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, Abstract Emerging virus diseases are a major threat to human and veterinary public health. With new examples occurring approximately one each year, the majority are viruses originating from an animal host. Of the many factors responsible, changes to local ecosystems that perturb the balance between pathogen and principal host species is one of the major drivers, together with increasing urbanization of mankind and changes in human behavior. Many emerging viruses have RNA genomes and as such are capable of rapid mutation and selection of new variants in the face of environmental changes in host numbers and available target species. This review summarizes recent work on aspects of virus emergence and the current understanding of the molecular and immunological basis whereby viruses may cross between species and become established in new ecological niches. Emergence is hard to predict, although mathematical modeling and spatial epidemiology have done much to improve the prediction of where emergence may occur. However, much needs to be done to ensure adequate surveillance is maintained of animal species known to present the greatest risk thus increasing general alertness among physicians, veterinarians and those responsible for formulating public health policy. Abstract A vaccine for equine coronavirus (ECoV) is so far unavailable. Bovine coronavirus (BCoV) is antigenically related to ECoV; it is therefore possible that BCoV vaccine will induce antibodies against ECoV in horses. This study investigated antibody response to ECoV in horses inoculated with BCoV vaccine. Virus neutralization tests showed that antibody titers against ECoV increased in all six horses tested at 14 days post inoculation, although the antibody titers were lower against ECoV than against BCoV. This study showed that BCoV vaccine provides horses with antibodies against ECoV to some extent. It is unclear whether antibodies provided by BCoV vaccine are effective against ECoV, and therefore ECoV challenge studies are needed to evaluate efficacy of the vaccine in the future. Abstract Background: Currently, diagnosis of equine coronavirus (ECoV) relies on the exclusion of other infectious causes of enteric disease along with molecular detection of ECoV in feces or tissue.Although this approach is complete, it is costly and may not always be achievable.Objective: We hypothesized that the overall fecal shedding of ECoV in hospitalized horses is low. Our objective was to determine whether systemically healthy horses and horses with gastrointestinal disorders shed ECoV in their feces at the time of admission to a referral hospital and after 48 hours of stress associated with hospitalization.Animals: One-hundred thirty adult horses admitted to the Washington State University Veterinary Teaching Hospital for gastrointestinal disease (n = 65) or for imaging under anesthesia (n = 65) that were hospitalized for 48 hours. Owner consent was obtained before sampling. Methods: Fecal samples were collected at admission and 48 hours later. Polymerase chain reaction (PCR) for ECoV and electron microscopy (EM) were performed on all samples. Results: Only 1 of 258 fecal samples was PCR-positive for ECoV. Electron microscopy identified ECoV-like particles in 9 of 258 samples, parvovirus-like particles in 4 of 258 samples, and rotavirus-like particles in 1 of 258 samples. Conclusions and Clinical Importance: The presence of ECoV in feces of hospitalized adult horses was low. Thus, fecal samples that are PCR-positive for ECoV in adult horses that have clinical signs consistent with this viral infection are likely to be of diagnostic relevance. The clinical relevance of the viruses observed using EM remains to be investigated. K E Y W O R D S anesthesia, anorexia, electron microscopy, fever, gastrointestinal disease, lethargy, PCR Abstract Objective To examine recent time trends in the incidence of osteonecrosis (ON) in Denmark and to investigate different common comorbidities association with ON in a population-based setting. Methods Using Danish medical databases, we included all patients with a first-time hospital diagnosis of ON during 1995-2012. Each ON case was matched with 10 randomly selected population control subjects from general population. For all participants, we obtained a complete hospital history of comorbidities included in the CharlsonComorbidity Index 5 years preceding the inclusion date. results 4107 ON cases and 41 063 controls were included. The incidence of ON increased from 3.9 in 1995 to 5.5 in 2012 per 100 000 inhabitants. Solid cancer was the most common comorbidity, associated with an adjusted OR (aOR) for ON of 2.0 (95% CI 1.7 to 2.2). For advanced metastatic cancer, leukaemia and lymphoma, aORs of ON were 3.4 (95% CI 2.5 to 4.5), 4.3 (95% CI 2.7 to 7.0) and 5.8 (95% CI 4.3 to 7.8), respectively. Among other chronic conditions, aORs were 3.5 (95% CI 3.0 to 4.1) for connective tissue diseases and 2.3 (95% CI 2.0 to 2.7) for chronic pulmonary diseases. aORs were also increased at 2.8 (95% CI 1.9 to 4.1) and 4.5 (95% CI 2.5 to 8.2) for mild and moderate-to-severe liver disease, respectively, and 4.2 (95% CI 3.4 to 5.2) for renal disease. Conclusion This large population-based study provides evidence for an increasing ON incidence in the general population and documents an association between several common comorbid conditions and risk of ON. Abstract The current study was intended to evaluate the knowledge and awareness toward Middle East respiratory syndrome coronavirus (MERS-CoV) of pilgrims from Saudi Arabia and from different Arabian countries.Methods: A prospective study was conducted among pilgrims from Saudi Arabia and those from other Arab nations. A total number of 2120 participants including 736 Saudi pilgrims (436 males and 300 females) and 1384 non-Saudi Arabian pilgrims (1384; 909 males and 475 females) were included in the study. The responses of the participants were descriptively analyzed. Pearson correlation coefficient was used to screen the possible correlations among different variables. The differences in the responses between the two groups were evaluated using Mann-Whitney analysis.The responses of the Saudi pilgrims showed statistically significant results in comparison to non-Saudi pilgrims in answering all questions except those related to the presence of efficient vaccination or treatment and the source of information. It was clear that the Saudi pilgrims were more oriented about different aspects of MERS-CoV including the nature of the causative agent, the signs, the severity of the disease, the animals that can transmit the infection to humans, the risk groups, and when one need to be screened for infection. In both Saudi and non-Saudi pilgrims, the official websites of health organizations constitute the main source of their information.It was concluded that Saudi pilgrims possess good knowledge about the MERS-CoV although more orientation is still required. Abstract Background: The Ebola virus has been responsible for numerous outbreaks since the 1970s, with the most recent outbreak taking place between 2014 and 2016 and causing an international public health emergency. Ebola virus disease (EVD) has a high mortality rate and no approved targeted treatment exists to date. A number of established drugs are being considered as potential therapeutic agents for the treatment of EVD. Objective: We aimed to identify potential drug repositioning candidates and to assess the scientific evidence available on their efficacy. Methods: We conducted a systematic literature search in MEDLINE, Embase, and other relevant trial registry platforms for studies published between January 1976 and January 2017. We included drug screening, preclinical studies, and clinical studies on repurposed drugs for the treatment of EVD. The risk of bias for animal studies and nonrandomized clinical studies was assessed. The quality of reporting for case series and case reports was evaluated. Finally, we selected drugs approved by established regulatory authorities, which have positive in vitro study outcomes and at least one additional animal or clinical trial. Results: We identified 3301 publications, of which 37 studies fulfilled our inclusion criteria. Studies were highly heterogeneous in terms of study type, methodology, and intervention. The risk of bias was high for 13 out of 14 animal studies. We selected 11 drugs with potential anti-EVD therapeutic effects and summarized their evidence. Conclusions: Several established drugs may have therapeutic effects on EVD, but the quality and quantity of current scientific evidence is lacking. This review highlights the need for well-designed and conducted preclinical and clinical research to establish the efficacy of potential repurposed drugs against EVD. Abstract Most human emerging infectious diseases originate from wildlife and bats are a major reservoir of viruses, a few of which have been highly pathogenic to humans. In some regions of Cameroon, bats are hunted and eaten as a delicacy. This close proximity between human and bats provides ample opportunity for zoonotic events. To elucidate the viral diversity of Cameroonian fruit bats, we collected and metagenomically screened eighty-seven fecal samples of Eidolon helvum and Epomophorus gambianus fruit bats. The results showed a plethora of known and novel viruses. Phylogenetic analyses of the eleven gene segments of the first complete bat rotavirus H genome, showed clearly separated clusters of human, porcine, and bat rotavirus H strains, not indicating any recent interspecies transmission events. Additionally, we identified and analyzed a bat bastrovirus genome (a novel group of recently described viruses, related to astroviruses and hepatitis E viruses), confirming their recombinant nature, and provide further evidence of additional recombination events among bat bastroviruses. Interestingly, picobirnavirus-like RNA-dependent RNA polymerase gene segments were identified using an alternative mitochondrial genetic code, and further principal component analyses suggested that they may have a similar lifestyle to mitoviruses, a group of virus-like elements known to infect the mitochondria of fungi. Although identified bat coronavirus, parvovirus, and cyclovirus strains belong to established genera, most of the identified partitiviruses and densoviruses constitute putative novel genera in their respective families. Finally, the results of the phage community analyses of these bats indicate a very diverse geographically distinct bat phage population, probably reflecting different diets and gut bacterial ecosystems. Abstract Background: The inflammatory response in pneumococcal infection is primarily driven by immunoreactive bacterial cell wall components [lipoteichoic acid (LTA)]. An acute release of these components occurs when pneumococcal infection is treated with b-lactam antibiotics.Objectives: We hypothesized that non-lytic rifampicin compared with lytic b-lactam antibiotic treatment would attenuate the inflammatory response in patients with pneumococcal pneumonia.Methods: In the PRISTINE (Pneumonia treated with RIfampicin aTtenuates INflammation) trial, a randomized, therapeutic controlled, exploratory study in patients with community-acquired pneumococcal pneumonia, we looked at LTA release and inflammatory and clinical response during treatment with both rifampicin and b-lactam compared with treatment with b-lactam antibiotics only. The trial is registered in the Dutch trial registry, number NTR3751 (European Clinical Trials Database number 2012-003067-22).Results: Forty-one patients with community-acquired pneumonia were included; 17 of them had pneumococcal pneumonia. LTA release, LTA-mediated inflammatory responses, clinical outcomes, inflammatory biomarkers and transcription profiles were not different between treatment groups.The PRISTINE study demonstrated the feasibility of adding rifampicin to b-lactam antibiotics in the treatment of community-acquired pneumococcal pneumonia, but, despite solid in vitro and experimental animal research evidence, failed to demonstrate a difference in plasma LTA concentrations and subsequent inflammatory and clinical responses. Most likely, an inhibitory effect of human plasma contributes to the low immune response in these patients. In addition, LTA plasma concentration could be too low to mount a response via Toll-like receptor 2 in vitro, but may nonetheless have an effect in vivo. Abstract Extracellular matrix stiffness (ECM) is one of the many mechanical forces acting on mammalian adherent cells and an important determinant of cellular function. While the effect of ECM stiffness on many aspects of cellular behavior has been studied previously, how ECM stiffness might mediate susceptibility of host cells to infection by bacterial pathogens is hitherto unexplored.To address this open question, we manufactured hydrogels of varying physiologically relevant stiffness and seeded human microvascular endothelial cells (HMEC-1) on them. We then infected HMEC-1 with the bacterial pathogen Listeria monocytogenes (Lm) and found that adhesion of Lm to host cells increases monotonically with increasing matrix stiffness, an effect that requires the activity of focal adhesion kinase (FAK). We identified cell surface vimentin as a candidate surface receptor mediating stiffness-dependent adhesion of Lm to HMEC-1 and found that bacterial infection of these host cells is decreased when the amount of surface vimentin is reduced. Our results provide the first evidence that ECM stiffness can mediate the susceptibility of mammalian host cells to infection by a bacterial pathogen.Monitoring Editor Abstract Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from >6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs are grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparing with external sequences, thus serving as an essential resource in the viral genomics community. Abstract In this two-part series of reviews, we have invited experts in their fields to contribute articles on the status of vaccine research and development for emerging pathogens. This topic has been brought into sharp focus in recent years following significant outbreaks of viral diseases such as those causing severe acute respiratory syndrome and Middle East respiratory syndrome, as well as devastating outbreaks of diseases caused by the Ebola, Marburg, Zika and Lassa fever viruses, to name only a few examples. Additionally, bacterial infections leading to bubonic and pneumonic plague, most notably in Madagascar in 2018, as well as malaria in many tropical countries, melioidosis in south east Asia and tularaemia in northern Europe and North America, have incurred significant morbidity and mortality. In this review series, the life cycle of these pathogens and the epidemiology of disease have been reviewed in the context of potential points of intervention for the prevention of human infection. Many of the emerging pathogens are zoonoses and, as such, there is scope for intervention at the animal/insect/environmental reservoir. Other pathogens covered in this review series are considered to be re-emerging, such as multi-drug resistant tuberculosis. Abstract Background: Cruise ships carry a large number of people in confined spaces with relative Abstract Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care. Abstract Graphical Abstract Highlights d Virolectins detect and distinguish between closely related O-Ac-Sias in situ d O-Ac-sialoglycans occur in nature in a diversity not appreciated so far d O-Ac-Sias are differentially expressed in a species-, tissue-, and cell-specific fashion d There is extensive cell-to-cell variability in O-Ac-Sia expression in vivo and in vitro SUMMARY Sialic acids (Sias), 9-carbon-backbone sugars, are among the most complex and versatile molecules of life. As terminal residues of glycans on proteins and lipids, Sias are key elements of glycotopes of both cellular and microbial lectins and thus act as important molecular tags in cell recognition and signaling events. Their functions in such interactions can be regulated by post-synthetic modifications, the most common of which is differential Sia-O-acetylation (O-Ac-Sias). The biology of O-Ac-Sias remains mostly unexplored, largely because of limitations associated with their specific in situ detection.Here, we show that dualfunction hemagglutinin-esterase envelope proteins of nidoviruses distinguish between a variety of closely related O-Ac-Sias. By using soluble forms of hemagglutinin-esterases as lectins and sialate-O-acetylesterases, we demonstrate differential expression of distinct O-Ac-sialoglycan populations in an organ-, tissue-and cell-specific fashion.Our findings indicate that programmed Sia-O-acetylation/de-O-acetylation may be critical to key aspects of cell development, homeostasis, and/or function. Abstract The present study aimed to investigate the effects of levodopa (LEV) on cellular apoptosis in a rabbit model of steroid-associated necrosis of the femoral head (SANFH). A total of 44 healthy adult Chinese rabbits were randomly divided into three groups: Group A (n=15), administered a combination of lipopolysaccharide and hormone to establish the SANFH animal model; group B (n=15), SANFH animal model as in group A orally administered LEV (0.4 g/kg/day) on the day of injection; and group C (n=14), the control group. On the 6th and 8th week of modeling, seven rabbits from each group were sacrificed to harvest bilateral femoral head specimens for hematoxylin and eosin staining and apoptosis detection by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay analysis, as well as for observing pathological changes and analyzing cellular apoptosis. Eight weeks after modeling, the serum insulin-like growth factor (IGF)-1 levels of the three groups were measured. The empty lacunae rate and apoptosis index of bone cells in the treatment group were significantly lower than that of the model group (P<0.01). Eight weeks after treatment, the serum levels of IGF-1 were significantly higher than that of the model group (P<0.01). These findings suggested that LEV was able to reduce steroid-induced bone cellular apoptosis, reduce the occurrence of necrosis of the femoral head and, through in vivo metabolism, it may promote the synthesis and release of IGF-1, which could be one of its biological pathways to prevent and treat SANFH.Levodopa attenuates cellular apoptosis in steroid-associated necrosis of the femoral head Abstract Background: Coronaviruses (CoVs) are large ribonucleic acid (RNA) viruses causing primarily respiratory disease in humans. A novel human coronavirus, subsequently named middle east respiratory syndrome coronavirus (MERS-CoV), was first reported in Saudi Arabia in September of 2012. With increasing numbers of infections and deaths from MERS-CoV, development of a rapid and reliable kit was crucial to prevent further spread of MERS-CoV.In this study, we present two real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays for in-house rapid and sensitive diagnostic testing of MERS-CoV, detecting the regions upstream of the envelope gene (upE) and open reading frame (ORF) 1b, respectively, for initial screening and final confirmation of MERS-CoV infection, as recommended by the world health organization (WHO).In this experimental study, acquiring patient samples was difficult; thus, according to WHO recommendations and standard protocols, we synthesized RNA sequences of upE and ORF1b genes as the template signatures and TaqMan based-diagnostic rRT-PCR assays were carried out using these synthetic genes for detection of MERS-CoV. In this research, we also inaugurated a cell-free system to transcribe these RNA sequences using the DNA templates synthesized.The upE and ORF1b based one-step rRT-PCR assays were optimized by testing several times via different synthetic RNAs, and validation results were highly successful. The sensitivity obtained for upE was fewer than ten copies of RNA template per reaction and for ORF1b was 50 or fewer copies per reaction.This study showed that the developed rRT-PCR assays are rapid, reliable, reproducible, specific, sensitive, and simple tools for detection of MERS-CoV. Finally, a kit consisting of two assay signatures and controls was assembled, which can be distributed to public health laboratories in Iran to support international MERS-CoV surveillance and public health response. Abstract Introduction: At Brigham and Women's Hospital, we identified the need for a comprehensive training program designed to prepare frontline staff to safely manage a patient with Ebola viral disease (EVD). The primary goal of this program was to ensure the safety of staff, patients, and the general public by training staff in the correct use of personal protective equipment (PPE) before, during, and after care of patients with EVD. Methods: We delivered a 4-hour experiential training program to frontline health care professionals who would be expected to care for a patient with EVD. The program occurred in a simulation center with multiple flexible spaces and consisted of demonstration, multiple skills practice sessions, and a patient simulation case. We analyzed completed pre-and posttraining questionnaires. The questionnaire assessed their subjective level of confidence in three key areas: donning and doffing PPE, performing clinical skills while wearing PPE, and management of a contamination breach. Results: This program was effectively deployed in the STRATUS Center for Medical Simulation over a 4-month period, with 220 health care professionals participating in the training and 195 participants completing the pre-/posttraining questionnaires. Our intervention significantly increased the confidence of participants on each primary objective (p = .001 for all three stations). Discussion: This interprofessional simulation-based program has been shown to be a well-received method of training clinicians to manage patients collaboratively during an EVD outbreak. Our intent is that the skills taught in this training program would also be transferable to management of other infectious diseases in the clinical setting.At the conclusion of the program, the participant will be able to:1. Demonstrate the appropriate step-by-step process of donning and doffing personal protective equipment (PPE) using a third-party trained observer for safety.2. Demonstrate the safe management of body-fluid spills and hospital waste in the setting of managing a patient with Ebola viral disease (EVD).3. List the major impediments to performing patient care procedures on a patient while clad in PPE.4. Perform clinical procedures correctly while clad in PPE, exhibiting appropriate management of physical and environmental challenges including dexterity, heat, stress, and communication.5. Demonstrate the team-enhanced collaboration necessary to safely care for a patient with EVD. Abstract There is an increasing recognition that detailed nucleic acid sequence information will be useful and even required in the diagnosis, treatment and surveillance of many significant pathogens. Because generating detailed information about pathogens leads to significantly larger amounts of data, it is necessary to develop automated analysis methods to reduce analysis time and to standardize identification criteria. This is especially important for multiple pathogen assays designed to reduce assay time and costs. In this paper, we present a successful algorithm for detecting pathogens and reporting the maximum level of detail possible using multipathogen resequencing microarrays. The algorithm filters the sequence of base calls from the microarray and finds entries in genetic databases that most closely match. Taxonomic databases are then used to relate these entries to each other so that the microorganism can be identified. Although developed using a resequencing microarray, the approach is applicable to any assay method that produces base call sequence information. The success and continued development of this approach means that a non-expert can now perform unassisted analysis of the results obtained from partial sequence data. Abstract A novel series of ribonucleosides of 1,2,3-triazolylbenzyl-aminophosphonates was synthesized through the Kabachnik-Fields reaction using I 2 as catalyst followed by copper-catalyzed cycloaddition of the azide-alkyne reaction (CuAAC). All structures of the newly prepared compounds were characterized by 1 H NMR, 13 C NMR, and HRMS spectra. The structures of 2e, 2f, 3d, and 3g were further confirmed by X-ray diffraction analysis. These compounds were tested against various strains of DNA and RNA viruses; compounds 4b and 4c showed a modest inhibitory activity against respiratory syncytial virus (RSV) and compound 4h displayed modest inhibitory activity against Coxsackie virus B4. Abstract The type H membrane protein p63 is a resi- Abstract A B S T R A C T Macrophages (Mφ) are central players in mediating proinflammatory and immunomodulatory functions. Unchecked Mφ activities contribute to pathology across many diseases, including those caused by infectious pathogens and metabolic disorders. A fine balance of Mφ responses is crucial, which may be achieved by enforcing appropriate bioenergetics pathways. Metabolism serves as the provider of energy, substrates, and byproducts that support differential Mφ characteristics. The metabolic properties that control the polarization and response of Mφ remain to be fully uncovered for use in managing infectious diseases. Here, we review the various metabolic states in Mφ and how they influence the cell function. Abstract Mig is a chemokine of the CXC subfamily that was discovered by differential screening of a cDNA library prepared from lymphokine-activated macrophages. The mig gene is inducible in macrophages and in other cells in response to interferon (IFN)-% We have transfected Chinese hamster ovary (CHO) cells with cDNA encoding human Mig and we have derived CHO cell lines from which we have purified recombinant human Mig (rHuMig). rHuMig induced the transient elevation of [Ca2+]i in human tumor-infiltrating T lymphocytes (TIL) and in cultured, activated human peripheral blood-derived lymphocytes. No responses were seen in human neutrophils, monocytes, or Epstein-Barr virus-transformed B lymphoblastoid cell lines. rHuMig was chemotactic for TIL by a modified Boyden chamber assay but rHuMig was not chemotactic for neutrophils or monocytes. The CHO cell lines, IFN-~/-treated human peripheral-blood monocytes, and IFN-~/-treated cells of the human monocytic cell line THP-1 all secreted multiple and identical HuMig species as revealed by SDS-PAGE. Using the CHO-derived rHuMig, we have shown that the species' heterogeneity is due to proteolytic cleavage at basic carboxy-terminal residues, and that the proteolysis occurs before and not after rHuMig secretion by the CHO cells. The major species of secreted rHuMig ranged from 78 to 103 amino acids in length, the latter corresponding to the full-length secreted protein predicted from the HuMig cDNA. Carboxy-terminal-truncated forms ofrHuMig were of lower specific activity compared to full-length rHuMig in the calcium flux assay, and the truncated species did not block the activity of the full-length species. It is likely that HuMig plays a role in T cell trafficking and perhaps in other aspects of the physiology of activated T cells. Abstract Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti-HBV agents, emergence of drug-resistant viruses highlights the need for new anti-HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti-HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection-susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while nonsusceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti-HBV potency, having a median inhibitory concentration <0.2 lM. Conclusion: This study provides a proof of concept for the novel strategy to identify anti-HBV agents by targeting the candidate HBV receptor, NTCP, using CsA as a structural platform. (HEPATOLOGY 2014;59:1726-1737 Abstract Objective: This study aimed to evaluate the prevalence and related factors of post-traumatic stress disorder (PTSD) symptoms among doctors and nurses who were exposed to H7N9 patients during the H7N9 influenza epidemic. To provide scientific basis for promoting the physical and psychological health of these staff members. Method: The 102 medical staff workers who were exposed to H7N9 patients were recruited through convenient sampling between January 2015 and May 2016. We used a self-reported questionnaire, the PTSD Checklist-Civilian Version (PCL-C), to evaluate the PTSD symptoms among doctors and nurses from an intensive care unit (n ¼ 61), a respiratory department (n ¼ 20), and an emergency department (n ¼ 21). We then analyzed the related factors. Results: Around 20.59% of the tested doctors and nurses showed PTSD symptoms. The sample had a mean PCL-C score of 30.00 ± 9.95. The differences in the scores of doctors and nurses with different genders, ages, professional titles, contact frequencies, trainings, and experiences were statistically significant (P < 0.05, P < 0.01). Moreover, t-tests and one-way analysis of variance showed that nurses received higher scores than doctors, female participants received higher scores than male participants, and the participants with low professional title and high contact frequency, aged between 20 years and 30 years, with less than five years of work experience, having not received related training and with no related experience obtained higher PCL-C scores than the others (P < 0.05, P < 0.01). Conclusion: The PTSD level of doctors and nurses after their exposure to H7N9 patients was high, which warrant further research. Health and medical institutions should pay attention to the physical and psychological health of these staff members. Abstract Respiratory diseases are a very common source of morbidity and mortality among children. Health care providers often face a dilemma when encountering a febrile infant or child with respiratory tract infection. The reason expressed by many clinicians is the trouble to confirm whether the fever is caused by a virus or a bacterium. The aim of this review is to update the current evidence on the virus-induced bacterial infection. We present several clinical as well in vitro studies that support the correlation between virus and secondary bacterial infections. In addition, we discuss the pathophysiology and prevention modes of the virus-bacterium coexistence. A search of the PubMed and MEDLINE databases was carried out for published articles covering bacterial infections associated with respiratory viruses. This review should provide clinicians with a comprehensive idea of the range of bacterial and viral coinfections or secondary infections that could present with viral respiratory illness. Abstract Bovine norovirus (BNoV) has emerged as a viral pathogen that causes a gastrointestinal illness and diarrhea in cattle. Despite its worldwide distribution, very little information is known about BNoV in Africa. In this study, BNoV was detected in 27.6% (8/29) of tested fecal materials, collected from sporadic cases of diarrheic calves, using the reverse transcription-polymerase chain reaction (RT-PCR) and primers that target RNA dependent RNA polymerase gene. Additionally, one primer pair was designed to flank the BNoV-VP2 (small capsid protein) gene for molecular analysis. Study VP2 sequences were phylogenetically-related to BNoV-GIII.2 (Newbury2-like) genotype, which is highly prevalent all over the world. However, they were separated within the cluster and one strain (41FR) grouped with recombinant GIII.P1/GIII.2 strains. Compared to reference VP2 sequences, 14 amino acid substitution mutations were found to be unique to our strains. The study confirms that BNoV is currently circulating among diarrheic calves of Egypt and also characterizes its ORF3 (VP2) genetically. The status of BNoV should be continuously evaluated in Egypt for effective prevention and control.T 1 The BNoV-RdRp targeting primers were used in one step RT-PCR for the purpose of BNoV detection. 2 The BNoV-VP2 targeting primers were used in one step RT-PCR for the purpose of sequencing and molecular analysis. 3 F; forward (sense) primer, R; reverse (antisense) primer. 4 The VP2 specific primers were designed based on the GenBank reference sequence (Bo/Newbury2/1976/UK/ AF097917). Abstract The M glycoprotein from the avian coronavirus, infectious bronchitis virus (IBV), contains information for localization to the cis -Golgi network in its first transmembrane domain. We hypothesize that localization to the Golgi complex may depend in part on specific interactions between protein transmembrane domains and membrane lipids. Because the site of sphingolipid synthesis overlaps the localization of IBV M, we asked whether perturbation of sphingolipids affected localization of IBV M. Short-term treatment with two inhibitors of sphingolipid synthesis had no effect on localization of IBV M or other Golgi markers. Thus, ongoing synthesis of these lipids was not required for proper localization. Surprisingly, a third inhibitor, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino- Abstract Since mid-December of 2019, coronavirus disease 2019 (COVID-19) has been spreading from Wuhan, China. The confirmed COVID-19 patients in South Korea are those who came from or visited China. As secondary transmissions have occurred and the speed of transmission is accelerating, there are rising concerns about community infections. The 54-year old male is the third patient diagnosed with COVID-19 in Korea. He is a worker for a clothing business and had mild respiratory symptoms and intermittent fever in the beginning of hospitalization, and pneumonia symptoms on chest computerized tomography scan on day 6 of admission. This patient caused one case of secondary transmission and three cases of tertiary transmission. Hereby, we report the clinical findings of the index patient who was the first to cause tertiary transmission outside China. Interestingly, after lopinavir/ritonavir (Kaletra, AbbVie) was administered, β-coronavirus viral loads significantly decreased and no or little coronavirus titers were observed. Abstract A 5-year-old male castrated Lhasa Apso cross was evaluated for a 1-month history of inappetence, lethargy, gagging, and progressive right thoracic limb lameness. Synovial fluid analysis revealed nonseptic suppurative inflammation, and a diagnosis of immune-mediated polyarthritis (IMPA) was made. After 3 months of treatment with prednisone and later cyclosporine, the dog developed multiple firm cutaneous and subcutaneous masses and a focal mass within the jejunum. Cultures of blood, urine, skin lesions, and the jejunal mass identified Nocardia veterana by matrix-absorption laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and allowed for earlier identification of the organism compared to more traditional secA1 gene sequencing. Immunosuppressive drug treatment was discontinued, and the dog was treated for 3 months by administration of trimethoprim-sulfamethoxazole (TMS). No recurrence of clinical signs was reported 1 year later. This case report highlights the clinical utility of MALDI-TOF MS, particularly for the rapid identification of slow-growing, fastidious organisms. Abstract The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS. Abstract is recognized as one of the most lethal pathogens in the planet. For RNA viruses, cellular or virusencoded RNA helicases play pivotal roles in viral life cycles by remodelling viral RNA structures and/or unwinding viral dsRNA produced during replication. However, no helicase or helicase-like activity has ever been found to associate with any NNSV-encoded proteins, and it is unknown whether the replication of NNSVs requires the participation of any viral or cellular helicase. Here, we show that despite of containing no conserved NTPase/helicase motifs, EBOV VP35 possesses the NTPase and helicase-like activities that can hydrolyse all types of NTPs and unwind RNA helices in an NTP-dependent manner, respectively. Moreover, guanidine hydrochloride, an FDAapproved compound and inhibitor of certain viral helicases, inhibited the NTPase and helicase-like activities of VP35 as well as the replication/transcription of an EBOV minigenome replicon in cells, highlighting the importance of VP35 helicase-like activity during EBOV life cycle. Together, our findings provide the first demonstration of the NTPase/helicase-like activity encoded by EBOV, and would foster our understanding of EBOV and NNSVs. Abstract Since its emergence in 2012, more than 900 laboratory-confirmed cases of Middle East respiratory syndrome (MERS) have been reported with a fatality rate of more than 30%. However, no antigen detection assay for commercial use is available for diagnosis. In this study, the full-length nucleocapsid protein (NP) gene of MERS coronavirus (MERS-CoV) was cloned and expressed in Escherichia coli. A MERS-CoV NP capture enzyme-linked immunosorbent assay (ELISA) using two MERS-CoV-NP-specific monoclonal antibodies (MAbs) generated was developed. The ELISA was evaluated using 129 nasopharyngeal aspirates (NPAs) positive for various respiratory viruses and simulated positive NPAs by adding serial dilutions of MERS-CoV. Using a cutoff OD of 0.19, all 129 NPAs positive for respiratory viruses showed very low OD, with a specificity of 100%. For the two simulated MERS-CoV-positive NPAs with serial dilutions of live MERS-CoV, all samples with o10 50% tissue culture infective dose (TCID 50 )/0.1 mL showed positive results. For the 10 additional NPAs with 20 and 200 TCID 50 /0.1 mL of live MERS-CoV added, all were positive. A highly sensitive and specific MAbs-based antigen capture ELISA has been developed for MERS. This sensitive and specific antigen capture ELISA should be useful for detection of MERS-CoV in human and dromedaries and in field studies. Abstract A novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCVinfected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus.Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine.Indiana and subsequently spread rapidly across the United States and Canada, causing significant economic losses. Here, we report the detailed genetic characterization, phylogeny, and virulence of emergent PdCV strains in the United States. We found that PdCV caused severe diarrhea, vomiting, and dehydration in gnotobiotic and conventional piglets, signs that were clinically indistinguishable from those caused by PEDV and TGEV. In addition to extensive intestinal lesions, PdCV caused significant lesions in the stomach and mild pulmonary lesions that have not been reported for TGEV and PEDV. The finding that PdCV is a significant enteric disease of swine highlights the need to develop effective measures to control this disease. . 2015. Origin, evolution, and virulence of porcine deltacoronaviruses in the United States. mBio 6(2): e00064-15. Abstract In the face of the worldwide threat of severe acute respiratory syndrome (SARS) to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA), these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potential epitope regions were located at Codons 469-882 in the S protein, and one epitope site was located at Codons 599-620. Identification of antigenic regions in the SARS-CoV S protein may be important for the functional studies of this virus or the development of clinical diagnosis. Abstract The lamin B receptor (LBR) is a polytopic integral membrane protein localized exclusively in the inner nuclear membrane domain of the nuclear envelope. Its cDNA deduced primary structure consists of a highly charged amino-terminal domain of 205 residues that faces the nucleoplasm followed by a hydrophobic domain with eight potential transmembrane segments. To identify determinants that sort LBR from its site of integration (RER and outer nuclear membrane) to the inner nuclear membrane, we prepared full-length, truncated, and chimeric cDNA constructs of chick LBR, transfected these into mammalian cells and detected the expressed protein by immunofluorescence microscopy using appropriate antibodies. Surprisingly, we found that the determinants for sorting of LBR to the inner nuclear membrane reside in a region comprising its first transmembrane sequence plus flanking residues on either side. The other transmembrane regions as well as the nucleoplasmic domain are not required for sorting. We propose that the first transmembrane segment of LBR interacts specifically with another transmembrane segment and consider several mechanisms by which such specific interaction could result in sorting to the inner nuclear membrane. Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) infects mainly the porcine alveolar macrophages (PAMs) and causes porcine reproductive and respiratory syndrome (PRRS). Previous studies have analyzed the global gene expression profiles of lung tissue in vivo and PAMs in vitro following infection with PRRSV, however, transcriptome-wide understanding of the interaction between highly pathogenic PRRSV (HP-PRRSV) and PAMs in vivo has not yet been established. In this study, we employed Affymetrix microarrays to investigate the gene expression patterns of PAMs isolated from Tongcheng piglets (a Chinese indigenous breed) after infection with HP-PRRSV. During the infection, Tongcheng piglets exhibited typical clinical signs, e.g. fever, asthma, coughing, anorexia, lethargy and convulsion, but displayed mild regional lung damage at 5 and 7 dpi. Microarray analysis revealed that HP-PRRSV infection has affected PAMs in expression of the important genes involved in cytoskeleton and exocytosis organization, protein degradation and folding, intracellular calcium and zinc homeostasis. Several potential antiviral strategies might be employed in PAMs, including upregulating IFN-induced genes and increasing intracellular zinc ion concentration. And inhibition of the complement system likely attenuated the lung damage during HP-PRRSV infection. Transcriptomic analysis of PAMs in vivo could lead to a better understanding of the HP-PRRSV-host interaction, and to the identification of novel antiviral therapies and genetic components of swine tolerance/susceptibility to HP-PRRS. Abstract Severe bronchiolitis is the most common reason for hospitalization among children younger than 2 years. This study analyzed the prevalence of community-acquired respiratory virus infection and the risk factors for hospitalization of Mexican children with severe bronchiolitis treated in an Emergency department.This retrospective study included 134 children 2 years or younger with severe viral bronchiolitis, and 134 healthy age-matched controls. The study period was September 2012 to January 2015. We determined the viral etiology and coinfections with multiple viruses and compared the risk factors detected in children with severe viral bronchiolitis with those in the control group.A total of 153 respiratory viruses in these 134 patients, single or mixed infections, were identified: respiratory syncytial virus (RSV) type A or B was the most frequently detected (23.6% and 17.6%, respectively), followed by rhinovirus (RV; 16.3%) and parainfluenza virus (PIV) type 3 (12.4%). Coinfections of 2 respiratory viruses were found in 14.2% of cases; all cases had either RSV type A or B with another virus, the most common being parainfluenza virus or rhinovirus. Exposure to cigarette smoking was independently associated with hospitalization for severe bronchiolitis (OR, 3.5; 95% CI, 1.99-6.18; P = .0001), and having completed the vaccination schedule for their age was a protective factor against adverse outcome (OR, 0.55; 95% CI, 0.35-0.87; P = .010).RSV is a common infection among young children with severe bronchiolitis; thus, developing a vaccine against RSV is essential. Campaigns to reinforce the importance of avoiding childhood exposure to cigarette smoke are also needed.Abbreviations: CI = confidence interval, OR = odds ratio, PCR = polymerase chain reaction, PIV = parainfluenza virus, RSV = respiratory syncytial virus, RT-PCR = reverse transcription-polymerase chain reaction. Abstract Dengue is the most important arboviral disease in the world. As chloroquine, an antimalarial agent, has shown some antiviral effects, this study evaluated its effect in patients with dengue. A randomised, double-blind study was performed by administering chloroquine or placebo for three days to 129 patients with dengue-related symptoms. Of these patients, 37 were confirmed as having dengue and completed the study; in total, 19 dengue patients received chloroquine and 18 received placebo. There was no significant difference in the duration of the disease or the degree and days of fever. However, 12 patients (63%) with confirmed dengue reported a substantial decrease in pain intensity and a great improvement in their ability to perform daily activities (p = 0.0004) while on the medication and the symptoms returned immediately after these patients stopped taking the medication. The same effect was not observed in patients with diseases other than dengue. Therefore, this study shows that patients with dengue treated with chloroquine had an improvement in their quality of life and were able to resume their daily activities. However, as chloroquine did not alter the duration of the disease or the intensity and days of fever, further studies are necessary to confirm the clinical effects and to assess the side effects of chloroquine in dengue patients. Abstract To identify and describe the hospital disaster preparedness (HDP) in major private hospitals in Riyadh, Saudi Arabia.Methods: This is an observational cross-sectional survey study performed in Riyadh city, Saudi Arabia between December 2015 and April 2016. Thirteen major private hospitals in Riyadh with more than 100 beds capacity were included in this investigation.The 13 hospitals had HDP plan and reported to have an HDP committee. In 12 (92.3%) hospitals, the HDP covered both internal and external disasters and HDP was available in every department of the hospital. There were agreements with other hospitals to accept patients during disasters in 9 facilities (69.2%) while 4 (30.8%) did not have such agreement. None of the hospitals conducted any unannounced exercises in previous year.Most of the weaknesses were apparent particularly in the education, training and monitoring of the hospital staff to the preparedness for disaster emergency occasion. Few hospitals had conducted an exercise with casualties, few had drilled evacuation of staff and patients in the last 12 months, and none had any unannounced exercise in the last year. Abstract Emerging infectious diseases (EIDs) pose a major threat to public health and security. Given the dynamic nature and significant impact of EIDs, the most effective way to prevent and protect against them is to develop vaccines in advance. Systems biology approaches provide an integrative way to understand the complex immune response to pathogens. They can lead to a greater understanding of EID pathogenesis and facilitate the evaluation of newly developed vaccine-induced immunity in a timely manner. In recent years, advances in high throughput technologies have enabled researchers to successfully apply systems biology methods to analyze immune responses to a variety of pathogens and vaccines. Despite recent advances, computational and biological challenges impede wider application of systems biology approaches. This review highlights recent advances in the fields of systems immunology and vaccinology, and presents ways that systems biology-based platforms can be applied to accelerate a deeper understanding of the molecular mechanisms of immunity against EIDs. Abstract Genome-wide association studies have indicated that human leukocyte antigen (HLA)-DP and HLA-DQ play roles in persistent hepatitis B virus (HBV) infection in Asia. To understand the evolution of HBV-related single nucleotide polymorphisms (SNPs) and to correlate these SNPs with chronic HBV infection among different populations, we conducted a global perspective study on hepatitis-related SNPs. We selected 12 HBV-related SNPs on the HLA locus and two HBV and three hepatitis C virus immune-related SNPs for analysis. Five nasopharyngeal carcinoma-related SNPs served as controls. All SNP data worldwide from 26 populations were downloaded from 1,000 genomes. We found a dramatic difference in the allele frequency in most of the HBV-and HLA-related SNPs in East Asia compared to the other continents. A sharp change in allele frequency in 8 of 12 SNPs was found between Bengali populations in Bangladesh and Chinese Dai populations in Xishuangbanna, China (P < 0.001); these areas represent the junction of South and East Asia. For the immune-related SNPs, significant changes were found after leaving Africa. Most of these genes shifted from higher expression genotypes in Africa to lower expression genotypes in either Europe or South Asia (P < 0.001). During this two-stage adaptation, immunity adjusted toward a weak immune response, which could have been a survival strategy during human migration to East Asia. The prevalence of chronic HBV infection in Africa is as high as in Asia; however, the HBV-related SNP genotypes are not present in Africa, and so the genetic mechanism of chronic HBV infection in Africa needs further exploration. Conclusion: Two stages of genetic changes toward a weak immune response occurred when humans migrated out of Africa. These changes could be a survival strategy for avoiding cytokine storms and surviving in new environments Abstract Over the past two decades a number of severe acute respiratory infection outbreaks such as the 2009 influenza A (H1N1) and the Middle East respiratory syndrome coronavirus (MERS-CoV) have emerged and presented a considerable global public health threat. Epidemiologic evidence suggests that diabetic subjects are more susceptible to these conditions. However, the prevalence of diabetes in H1N1 and MERS-CoV has not been systematically described. The aim of this study is to conduct a systematic review and meta-analysis of published reports documenting the prevalence of diabetes in H1N1 and MERS-CoV and compare its frequency in the two viral conditions. Meta-analysis for the proportions of subjects with diabetes was carried out in 29 studies for H1N1 (n=92,948) and 9 for MERS-CoV (n=308). Average age of H1N1 patients (36.2±6.0 years) was significantly younger than that of subjects with MERS-CoV (54.3±7.4 years, P<0.05). Compared to MERS-CoV patients, subjects with H1N1 exhibited 3-fold lower frequency of cardiovascular diseases and 2-and 4-fold higher prevalence of obesity and immunosuppression, respectively. The overall prevalence of diabetes in H1N1 was 14.6% (95% CI: 12.3-17.0%; P<0.001), a 3.6-fold lower than in P<0.001). The prevalence of diabetes among H1N1 cases from Asia and North America was ~two-fold higher than those from South America and Europe. The prevalence of diabetes in MERS-CoV cases is higher than in H1N1. Regional comparisons suggest that an etiologic role of diabetes in MERS-CoV may exist distinctive from that in H1N1. Abstract To understand viral disease, detection and identification of these viruses are essential. Although PCR is widely used for rapid virus identification due to its low cost and high sensitivity and specificity, very few online database resources have compiled PCR primers for RNA viruses. To effectively detect viruses, the MRPrimerV database (http://MRPrimerV.com) contains 152 380 247 PCR primer pairs for detection of 1818 viruses, covering 7144 coding sequences (CDSs), representing 100% of the RNA viruses in the most up-to-date NCBI RefSeq database. Due to rigorous similarity testing against all human and viral sequences, every primer in MRPrimerV is highly target-specific. Because MR-PrimerV ranks CDSs by the penalty scores of their best primer, users need only use the first primer pair for a single-phase PCR or the first two primer pairs for two-phase PCR. Moreover, MRPrimerV provides the list of genome neighbors that can be detected using each primer pair, covering 22 192 variants of 532 RefSeq RNA viruses. We believe that the public availability of MRPrimerV will facilitate viral metagenomics studies aimed at evaluating the variability of viruses, as well as other scientific tasks. Abstract Pulmonary complications, including acute respiratory distress syndrome (ARDS), are well described in P. falciparum (PF) and to a lesser extent in other malaria species. In non-endemic areas, malaria diagnosis may be overlooked; if a thorough travel history is not obtained on all patients with acute febrile illness. Three patients with malaria associated respiratory distress were admitted to our intensive care unit. The diagnosis was delayed; however, all patients received artesunate and intensive therapy with a satisfactory outcome. One patient presented with respiratory disease while the others developed ARDS during or following appropriate therapy. Similarly, level of parasitemia was variable ranging from undetectable to over 5%. Variability in timing and severity of illness is exciting and gives emphasis to the different pathological processes contemplated in this complication. Abstract Citation Klotz LC. 2015. Danger of potential-pandemic-pathogen research enterprises. mBio 6(3):e00815-15. Abstract Bluetongue virus (BTV) encodes a single capping protein, VP4, which catalyzes all reactions required to generate cap1 structures on nascent viral transcripts. Further, structural analysis by X-ray crystallography indicated each catalytic reaction is arranged as a discrete domain, including a nucleoside-2 0 -O-methyltransferase (2 0 -O MTase). In this study, we have exploited the structural information to identify the residues that are important for the catalytic activity of 2 0 -O MTase of VP4 and their influence on BTV replication. The effect of these mutations on GMP binding, guanylyltransferase (GTase) and methylase activities were analysed by a series of in vitro biochemical assays using recombinant mutant proteins; subsequently their effects on virus replication were assessed by introducing the same mutations in replicating viral genome using a reverse genetics system. Our data showed that single substitution mutations in the catalytic tetrad K-D-K-E were sufficient to abolish 2 0 -O MTase activity in vitro and to completely abrogate BTV replication in cells; although these mutants retained the upstream GMP binding, GTase and guanine-N7-methyltransferase activities. Mutations of the surrounding substrate-binding pocket (predicted to recruit cap0) had variable effects on in vitro VP4 capping activity. Only triple but not single substitution mutations of these residues in genome resulted in reduced virus replication kinetics. This is the first report investigating the importance of 2 0 -O MTase function for any member of the Reoviridae and highlights the significance of K-D-K-E tetrad and surrounding residues for the efficiency of 2 0 -O MTase activity and in turn, for virus fitness. Abstract Host protection against several intracellular pathogens requires the induction of CD8 + T cell responses. CD8 + T cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. However, a protective role for CD8 + T cells during Leishmania infections is still controversial and largely depends on the infection model. In this review, we discuss the role of CD8 + T cells during various types of Leishmania infections, following vaccination, and as potential immunotherapeutic targets. Abstract Abbreviations: CLSM, confocal laser scanning microscopy; HCC, hepatocellular carcinoma; HP1, heterochromatin protein 1; KO, knockout; lncRNA, long non-coding RNA; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; NEAT1, nuclear enriched abundant transcript 1; NSCLC, non-small cell lung cancer; Pc2: Polycomb 2 protein; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; RNAP, RNA polymerase; RPLP0, ribosomal protein, large subunit, P0; TRF2, telomere repeat factor 2; UBF, upstream binding factor; WT, wild-type; ZFN, zinc finger nuclease www.landesbioscience.com RNA Biology Abstract Infectious prions comprising abnormal prion protein, which is produced by structural conversion of normal prion protein, are responsible for transmissible spongiform encephalopathies including Creutzfeldt-Jakob disease in humans. Prions are infectious agents that do not possess a genome and the pathogenic protein was not thought to evoke any immune response. Although we previously reported that interferon regulatory factor 3 (IRF3) was likely to be involved in the pathogenesis of prion diseases, suggesting the protective role of host innate immune responses mediated by IRF3 signalling, this remained to be clarified. Here, we investigated the reciprocal interactions of type I interferon evoked by IRF3 activation and prion infection and found that infecting prions cause the suppression of endogenous interferon expression. Conversely, treatment with recombinant interferons in an ex vivo model was able to inhibit prion infection. In addition, cells and mice deficient in type I interferon receptor (subunit interferon alpha/beta receptor 1), exhibited higher susceptibility to 22L-prion infection. Moreover, in in vivo and ex vivo prion-infected models, treatment with RO8191, a selective type I interferon receptor agonist, inhibited prion invasion and prolonged the survival period of infected mice. Taken together, these data indicated that the interferon signalling interferes with prion propagation and some interferon-stimulated genes might play protective roles in the brain. These findings may allow for the development of new strategies to combat fatal diseases.Keywords: prion infection; type I interferon (I-IFN); innate immune system. Abbreviations: I-IFN = type I interferon; IRF = interferon regulatory factor; MEF = mouse embryonic fibroblast; TLR = Toll-like receptor Abstract Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation. Abstract Mycoplasma pneumoniae is responsible for approximately 20% to 30% of community-acquired pneumonia, and is well known for its diverse extrapulmonary manifestations. However, acute necrotizing pancreatits is an extremely rare extrapulmonary manifestation of M. pneumoniae infection. A 6-year-old girl was admitted due to abdominal pain, vomiting, fever, and confused mentality. Acute necrotizing pancreatitis was diagnosed according to symptoms, laboratory test results, and abdominal computed tomography scans. M. pneumoniae infection was diagnosed by a 4-fold increase in antibodies to M. pneumoniae between acute and convalescent sera by particle agglutination antibody assay. No other etiologic factors or pathogens were detected. Despite the occurrence of a large infected pseudocyst during the course, the patient was able to discharge without morbidity by early aggressive supportive care. This is the first case in Korea of a child with acute necrotizing pancreatitis associated with M. pneumoniae infection. Abstract Health has long been intertwined with the foreign policies of states. In recent years, however, global health issues have risen to the highest levels of international politics and have become accepted as legitimate issues in foreign policy. This elevated political priority is in many ways a welcome development for proponents of global health, and it has resulted in increased funding for and attention to select global health issues. However, there has been less examination of the tensions that characterize the relationship between global health and foreign policy and of the potential effects of linking global health efforts with the foreign-policy interests of states. In this paper, the authors review the relationship between global health and foreign policy by examining the roles of health across 4 major components of foreign policy: aid, trade, diplomacy, and national security. For each of these aspects of foreign policy, the authors review current and historical issues and discuss how foreign-policy interests have aided or impeded global health efforts. The increasing relevance of global health to foreign policy holds both opportunities and dangers for global efforts to improve health. Abstract There is extensive evidence that cultured macrophages from genetically resistant or susceptible strains of mice reflect the genetic resistance or susceptibility of the host. Yet the effects of cortisone, cytoxan, and epierythrozoon in vitro are minimal or absent. However, experiments reported here have shown that certain substances (lymphokines) in supernatant fluid from allogeneic mixed lymphocyte cultures (MLC) rendered the macrophages of C3H mice susceptible to MHV. Fluid derived from congenic MLC did not change the resistance of C3H macrophages to the virus.Mice. Three inbred strains of mice were used: PRI, a genetically susceptible strain (4); C3H, a genetically resistant strain (4); and C3Hss, a strain that carries a single gene for susceptibility. Both the PRI and C3H strains have been maintained in our laboratory through brother-sister mating since 1955 (4). The C3Hss was developed in this laboratory by backcrosses with PRI mice; progeny were selected on the basis of susceptibility of their cultured macrophagee to destruction by the Princeton strain of MHV (MHV.PRI). Mice from two other strains that are not histocompatible with the C3H strain were also used. These were C57 black/6 and DBA/2 stock mice, 4-6-wk old, from The Jackson Laboratory, Bar Harbor, Maine.Virus. The MHV-2 strain of mouse hepatitis virus was originally obtained from Dr. John Nelson of the Rockefeller Institute, Princeton, in 1952, and has since been passed in our laboratory by intraperitoneal inoculation of l-too-old PRI mice. This is the the MHV-PRI virus. A 10% (wt/ vol) liver homogenate was prepared by grinding up livers of moribund infected mice in Hanks' balanced salt solution (BSS) (Grand Island Biological Co., Grand Island, N. Y.). This was kept as stock virus and stored at -70°C.Macrophage Cultures. Methods of harvesting and preparing cultures of mouse peritoneal macrophages have been previously described (5). In the present experiments, macrophages were seeded in 13 x 100-ram Wassermann tubes and incubated at 37°C in a roller drum.MLC. Individual spleens from PRI, C3H, and C3Hss mice were removed and passed through a 60-mesh stainless steel wire cloth (Small Parts, Inc.). Cells were counted and suspended in a medium consisting of 80% RPMI-1640 (Grand Island Biological Co.), 20% Chang's medium (6) (90% horse serum, 8% Hanks' BSS and 2% beef embryo extract), and 100 U/rag per ml of penicillin and streptomycin. Equal amounts of PRI and C3Hss or C3H lymphocytes were mixed and cultivated in glass Petri dishes in a 37°C incubator maintained with 5% CO2. C57 black/6 and DBA/2 spleens were processed and cultivated in the same manner. Abstract Citation Imperiale MJ, Casadevall A. 2015. The importance of virology at a time of great need and great jeopardy. mBio 6(2):e00236-15. Abstract Use of pecan shell fiber in human food is presently limited, but could increase pending demonstration of safety. In a 91-day rat study, pecan shell fiber was administered at dietary concentrations of 0 (control), 50 000, 100 000 or 150 000 ppm. There was no effect of the ingredient on body weight of males or females or food consumption of females. Statistically significant increases in food consumption were observed throughout the study in 100 000 and 150 000 ppm males, resulting in intermittent decreases in food efficiency (150 000 ppm males only) that were not biologically relevant. All animals survived and no adverse clinical signs or functional changes were attributable to the test material. There were no toxicologically relevant changes in hematology, clinical chemistry or urinalysis parameters or organ weights in rats ingesting pecan shell fiber. Any macroscopic or microscopic findings were incidental, of normal variation and/or of minimal magnitude for test substance association. Pecan shell fiber was non-mutagenic in a bacterial reverse mutation test and non-clastogenic in a mouse peripheral blood micronucleus test. Based on these results, pecan shell fiber has an oral subchronic (13-week) no observable adverse effect level (NOAEL) of 150 000 ppm in rats and is not genotoxic at the doses analyzed. Abstract A novel human coronavirus (HCoV-EMC/2012) was isolated from a man with acute pneumonia and renal failure in June 2012. This report describes the complete genome sequence, genome organization, and expression strategy of HCoV-EMC/ 2012 and its relation with known coronaviruses. The genome contains 30,119 nucleotides and contains at least 10 predicted open reading frames, 9 of which are predicted to be expressed from a nested set of seven subgenomic mRNAs. Phylogenetic analysis of the replicase gene of coronaviruses with completely sequenced genomes showed that HCoV-EMC/2012 is most closely related to Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4) and Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), which prototype two species in lineage C of the genus Betacoronavirus. In accordance with the guidelines of the International Committee on Taxonomy of Viruses, and in view of the 75% and 77% amino acid sequence identity in 7 conserved replicase domains with BtCoV-HKU4 and BtCoV-HKU5, respectively, we propose that HCoV-EMC/2012 prototypes a novel species in the genus Betacoronavirus. HCoV-EMC/2012 may be most closely related to a coronavirus detected in Pipistrellus pipistrellus in The Netherlands, but because only a short sequence from the most conserved part of the RNA-dependent RNA polymerase-encoding region of the genomewas reported for this bat virus, its genetic distance from HCoV-EMC remains uncertain. HCoV-EMC/2012 is the sixth coronavirus known to infect humans and the first human virus within betacoronavirus lineage C.show that coronaviruses are able to cause severe, sometimes fatal disease in humans. We have determined the complete genome of HCoV-EMC/2012 using an unbiased virus discovery approach involving next-generation sequencing techniques, which enabled subsequent state-of-the-art bioinformatics, phylogenetics, and taxonomic analyses. By establishing its complete genome sequence, HCoV-EMC/2012 was characterized as a new genotype which is closely related to bat coronaviruses that are distant from SARS-CoV. We expect that this information will be vital to rapid advancement of both clinical and vital research on this emerging pathogen.Citation van Boheemen S, et al. 2012. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mBio 3(6): e00473-12. Abstract Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, in- Abstract Unlike the wild-type asialoglycoprotein receptor subunit H1 which is transported to the cell surface, endocytosed and recycled, a mutant lacking residues 4-33 of the 40--amino acid cytoplasmic domain was found to be retained intracellularly upon expression in different cell lines. The mutant protein accumulated in the trans-Golgi, as judged from the acquisition of trans-Golgi-specific modifications of the protein and from the immunofluorescence staining pattern. It was localized to juxtanuclear, tubular structures that were also stained by antibodies against galactosyltransferase and -y-adaptin. The results of further mutagenesis in the cytoplasmic domain indicated that the size rather than the specific sequence of the cytoplasmic domain deter-mines whether H1 is retained in the trans-Golgi or transported to the cell surface. Truncation to less than 17 residues resulted in retention, and extension of a truncated tail by an unrelated sequence restored surface transport. The transmembrane segment of H1 was not sufficient for retention of a reporter molecule and it could be replaced by an artificial apolar sequence without affecting Golgi localization. The cytoplasmic domain thus appears to inhibit interaction(s) of the exoplasmic portion of H1 with trans-Golgi component(s)for example by steric hindrance or by changing the positioning of the protein in the membrane. This mechanism may also be functional in other proteins.1. Abbreviations used in this paper: ASGP, asialoglycoprotein; M6P, mannose-6-phosphate. Abstract Viral respiratory infections are frequently eliminated from human bodies without any sequelae. Secondary serious bacterial infection (SBI) in children with acute bronchiolitis has been an apprehension expressed by health care providers. Several published studies have shown an association between acute bronchiolitis and secondary bacterial infection, including urinary tract infections (UTI). However, the proposed mechanism by which a virus can induce UTIs is not yet known. The aim of this commentary is to update the current evidence of risk of UTI in children with bronchiolitis. We present several clinical studies related to the topic as well as a brief review of the potential pathophysiology of secondary infections that could present with viral respiratory illness. Abstract Oxidative stress and chronic low-grade inflammation in the lungs are associated with aging and may contribute to age-related immune dysfunction. To maintain lung homeostasis, chronic inflammation is countered by enhanced expression of proresolving/antiinflammatory factors. Here, we show that age-dependent increases of one such factor in the lungs, a phospholipase A 2 (PLA 2 ) group IID (PLA 2 G2D) with antiinflammatory properties, contributed to worse outcomes in mice infected with severe acute respiratory syndrome-coronavirus (SARS-CoV). Strikingly, infection of mice lacking PLA 2 G2D expression (Pla2g2d / mice) converted a uniformly lethal infection to a nonlethal one (>80% survival), subsequent to development of enhanced respiratory DC migration to the draining lymph nodes, augmented antivirus T cell responses, and diminished lung damage. We also observed similar effects in influenza A virus-infected middle-aged Pla2g2d / mice. Furthermore, oxidative stress, probably via lipid peroxidation, was found to induce PLA 2 G2D expression in mice and in human monocyte-derived macrophages. Thus, our results suggest that directed inhibition of a single inducible phospholipase, PLA 2 G2D, in the lungs of older patients with severe respiratory infections is potentially an attractive therapeutic intervention to restore immune function. Abstract Peroxisomes are components of virtually all eukaryotic cells. While much is known about peroxisomal matrix protein import, our understanding of how peroxisomal membrane proteins (PMPs) are targeted and inserted into the peroxisome membrane is extremely limited. Here, we show that PEX19 binds a broad spectrum of PMPs, displays saturable PMP binding, and interacts with regions of PMPs required for their targeting to peroxisomes. Furthermore, mislocalization of PEX19 to the nucleus leads to nuclear accumulation of newly synthesized PMPs. At steady state, PEX19 is bimodally distributed between the cytoplasm and peroxisome, with most of the protein in the cytoplasm. We propose that PEX19 may bind newly synthesized PMPs and facilitate their insertion into the peroxisome membrane. This hypothesis is supported by the observation that the loss of PEX19 results in degradation of PMPs and/or mislocalization of PMPs to the mitochondrion. Abstract A number of emerging and re-emerging viruses have caused epidemics or pandemics of infectious diseases leading to major devastations throughout human history. Therefore, developing effective and safe vaccines against these viruses is clearly important for the protection of at-risk populations. Our previous studies have shown that the receptor-binding domain (RBD) in the spike protein of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is a key target for the development of SARS vaccines. In this review, we highlight some key advances in the development of antiviral vaccines targeting the RBDs of spike proteins of emerging and re-emerging viruses, using SARS-CoV, influenza virus, Hendra virus (HeV) and Nipah virus (NiV) as examples. Figure 1 The interaction between the receptor-binding domain in the spike of an emerging virus and the corresponding receptor on the target cell. Abstract The characteristics of human genomics and cellular immune function between clinically symptomatic venous thromboembolism (VTE) and controls were systematically compared to explore the immunologic pathogenesis of VTE. Microarray assay showed the mRNA expressions of genes related to non-specific cellarer immune and cytokines were significantly down-regulated. Abnormal expressions of CD3+, CD4+, CD8+, NK marker CD16+56+, CD19 and aberrant CD4+/CD8+ ratio were detected in 54 among 56 patients. In PE patients, microarray assay revealed the imbalance in the expressions of genes related to the immune system. The expressions of genes related to non-specific immune cells and cytokines were markedly up-regulated and those associated with cellular immune were dramatically down-regulated. In VTE patients, cytological examination indicated the functions of NK cells were significantly compromised, and the antigen recognition and killing function of T cells markedly decreased. The consistence between genomic and cytological examination suggests the symptomatic VTE is closely associated with the infection and immune dysfunction. Abstract RNA interference (RNAi) is a powerful approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication. However, HIV-1 can escape from RNAimediated antiviral therapy by selection of mutations in the targeted sequence. To prevent viral escape, multiple small interfering RNAs (siRNAs) against conserved viral sequences should be combined. Ideally, these RNA inhibitors should be expressed simultaneously from a single transgene transcript.In this study, we tested a multiplex microRNA (miRNA) expression strategy by inserting multiple effective anti-HIV siRNA sequences in the miRNA polycistron mir-17-92. Individual anti-HIV miRNAs that resemble the natural miRNA structures were optimized by varying the siRNA position in the hairpin stem to obtain maximal effectiveness against luciferase reporters and HIV-1. We show that an antiviral miRNA construct can have a greater intrinsic inhibitory activity than a conventional short hairpin (shRNA) construct. When combined in a polycistron setting, the silencing activity of an individual miRNA is strongly boosted. We demonstrate that HIV-1 replication can be efficiently inhibited by simultaneous expression of four antiviral siRNAs from the polycistronic miRNA transcript. These combined results indicate that a multiplex miRNA strategy may be a promising therapeutic approach to attack escape-prone viral pathogens. Abstract Dengue virus receptors are relatively poorly characterized, but there has been recent interest in 2 C-type lectin molecules, dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) and its close homologue liver/lymph node-specific ICAM-3-grabbing integrin (L-SIGN), which can both bind dengue and promote infection. In this report we have studied the interaction of dengue viruses produced in insect cells, tumor cell lines, and primary human dendritic cells (DCs) with DC-SIGN and L-SIGN. Virus produced in primary DCs is unable to interact with DC-SIGN but remains infectious for L-SIGNexpressing cells. Skin-resident DCs may thus be a site of initial infection by insect-produced virus, but DCs will likely not participate in large-scale virus replication during dengue infection. These results reveal that differential glycosylation of dengue virus envelope protein is highly dependent on cell state and suggest that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution. Abstract SJG), and John.Skehel@crick.ac.uk (JJS)SUPPLEMENTAL EXPERIMENTAL PROCEDURES:Monoclonal antibody isolation was perfomed as previously described (Pappas et al., 2014; Traggiai et al., 2004) . In brief, peripheral blood mononuclear cell (PBMC) samples were obtained from a single healthy donor after vaccination following written consent. Memory B cells were isolated from cryopreserved or fresh PBMCs using CD22 microbeads or anti-FITC (fluorescein isothiocyanate) microbeads (Miltenyi Biotec) after staining of PBMCs with CD22-FITC, and were immortalized with Epstein-Barr virus (EBV) and CpG in multiple wells. Supernatants of immortalized B cell clones containing monoclonal antibodies were screened for binding to HA proteins representing two different HA subtypes. Using this methodology, we identified a monoclonal from primary screening in ELISA assay that showed crossreactive binding to recombinant HA proteins of A/Vietnam/2005 H5N1 and A/ Netherlands/2003 H7N7. Variable gene sequences were isolated from this cross-reactive clone by RT-PCR, cloned and expressed as a full-length IgG transiently in 293 T cells. The lead FY1 antibody was further modified to revert the non-germline framework amino acid changes and affinity optimize the CDRs. For the affinity optimization, full-length IgG constructs containing the different variants created by CDR based parsimonious mutagenesis were expressed transiently in 293 T cells and supernatants were screened by ELISA to select clones that resulted in increased binding activity to H3 while maintaining or increasing the binding activity to H1 HA proteins. Coating concentrations of 0.15 µg/ml of rabbit anti-human IgG were used to capture and normalize variant IgG constructs from supernatants, and binding was probed using 0.5 µg/ml of biotinylated HA proteins A/Perth/2009 H3N2 and A/California/7/2009 H1N1 followed by the addition of streptavidin-HRP (1:5000). Beneficial single mutations were combined and cloned into a combinatorial library, which were expressed and screened by ELISA as described above, leading to the generation of MEDI8852.The DNA Maximum Likelihood program (Dnaml) of the PHYLIP package, version 3.69, was used to estimate immunoglobulin phylogenies from nucleotide sequences that were first aligned using ClustalW2, as previously described12. IgHCDR3 regions were defined with their Kabat numbering using the software available on the Abnum website (http://www.bioinf.org.uk/abs/abnum/). The V, D and J genes of the IgH DNA sequences were identified using the IMGT database as a reference (Brochet et al., 2008; Ye et al., 2013) . The UCA sequences were inferred with Antigen Receptor Probabilistic Parser (ARPP) UA Inference software (Kepler, 2013) , and produced by gene synthesis (Genscript). Branchpoint antibodies were determined by the antibody phylogenetic tree derived by dnaml, and produced by gene synthesis (Genscript).Recombinant HA proteins were expressed and purified as previously described (Benjamin et al., 2014) ELISA binding assays were performed using 384 well plates (Maxisorp, Nunc) with 0.5 µg/ml of purified HA protein as described in Table S2 . ELISA plates were washed, blocked 1% (w/v) casein (Thermo Scientific), and serially diluted antibodies were incubated for 1 hr at room temperature. Bound antibodies were detected using a peroxidase-conjugated mouse anti-human IgG antibody (KPL 1:10,000), followed by development with TMB solution (KPL). The absorbance at 450nm was measured, and EC 50 values were calculated using a non-linear regression of log (inhibitor) vs response in Graph Pad Prism. Cell surface expressed protein binding assays were conducted using flow cytometry to detect binding of antibodies to HA transfected cells. HEK 293 cells were transiently transfected with full-length wild type HA expressing plasmids. Forty-eight hours after transfection, cells were detached with trypsin, and incubated with 5 µg/ml of FY1 or MEDI8852 on ice for 1 hour. Antibody remaining bound to surface expressed HA protein after washing was then stained with a goat anti-human IgG Daylight 649 (Jackson ImmunoResearch), and detected by flow cytometry. Abstract Given the side effects of vaccination against infectious laryngotracheitis (ILT), novel strategies for ILT control and therapy are urgently needed. The modulation of host-virus interactions is a promising strategy to combat the virus; however, the interactions between the host and avian ILT herpesvirus (ILTV) are unclear. Using genome-wide transcriptome studies in combination with a bioinformatic analysis, we identified proto-oncogene tyrosine-protein kinase Src (Src) to be an important modulator of ILTV infection. Src controls the virulence of ILTV and is phosphorylated upon ILTV infection. Functional studies revealed that Src prolongs the survival of host cells by increasing the threshold of virus-induced cell death. Therefore, Src is essential for viral replication in vitro and in ovo but is not required for ILTV-induced cell death. Furthermore, our results identify a positive-feedback loop between Src and the tyrosine kinase focal adhesion kinase (FAK), which is necessary for the phosphorylation of either Src or FAK and is required for Src to modulate ILTV infection. To the best of our knowledge, we are the first to identify a key host regulator controlling host-ILTV interactions. We believe that our findings have revealed a new potential therapeutic target for ILT control and therapy.IMPORTANCEtions is considered to be a promising strategy for controlling ILTV infections. However, little is known about the mechanisms governing host-ILTV interactions. The results from our study advance our understanding of host-ILTV interactions on a molecular level and provide experimental evidence that it is possible to control ILT via the manipulation of host-virus interactions. S. 2016. Genome-wide gene expression analysis identifies the proto-oncogene tyrosine-protein kinase Src as a crucial virulence determinant of infectious laryngotracheitis virus in chicken cells. J Virol 90:9 -21.We are greatly indebted to our colleagues who shared valuable reagents with us, and we are grateful to Abstract Balanced transmembrane signals maintain a competent peripheral B cell pool limited in selfreactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22 /[B6] ) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag-specific B cells in Ig Tg hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance. Abstract Hemodialysis (HD) patients had a high rate of infection transmission and mortality during the middle east respiratory syndrome coronavirus (MERS-CoV) outbreak in Saudi Arabia. A standardized guideline on isolation technique for exposed HD patients is not available. Thus, this study aimed to evaluate the effect of different isolation strategies on the prevention of secondary viral transmission and clinical outcomes among exposed HD patients.During the 2015 MERS-CoV outbreak in Korea, 116 patients in 3 HD units were incidentally exposed to individuals with confirmed MERS-CoV infection and underwent different types of isolation, which were as follows: single-room isolation (n = 54, 47%), cohort isolation (n = 46, 40%), and self-imposed quarantine (n = 16, 13%). The primary outcome was rate of secondary viral transmission. The secondary outcome measures were changes in clinical and biochemical markers during the isolation period, difference in clinical and biochemical markers according to the types of isolation practice, and effect of isolation practice on patient survival.During a mean isolation period of 15 days, no further cases of secondary transmission were detected among HD patients. Plasma hemoglobin, serum calcium, and serum albumin levels and single-pool Kt/V decreased during the isolation period but normalized thereafter. Patients who were subjected to self-imposed quarantine had higher systolic and diastolic blood pressure, lower total cholesterol level, and lower Kt/V than those who underwent single-room or cohort isolation. During the 24-month follow-up period, 12 patients died. However, none of the deaths occurred during the isolation period, and no differences were observed in patient survival rate according to different isolation strategies.Although 116 participants in 3 HD units were incidentally exposed to MERS-CoV during the 2015 outbreak in Korea, strict patient surveillance and proper isolation practice prevented secondary transmission of the virus. Thus, a renal disaster protocol, which includes proper contact surveillance and isolation practice, must be established in the future to accommodate the needs of HD patients during disasters or outbreaks.Abbreviations: BP = blood pressure, ELISA = enzyme-linked immunosorbent assay, Hb = hemoglobin, HD = hemodialysis, IFA = immunofluorescence assay, MERS-CoV = middle east respiratory syndrome coronavirus, ppNT = pseudoparticle neutralization assay. Abstract Little is known about clinical factors associated with undetectable pneumonic shadows on chest radiographs (CRs) for diagnosing pneumonia in the primary care setting.A retrospective assessment of CRs was conducted to compare chest computed tomography (CT) images of patients admitted to Kesennuma City Motoyoshi Municipal Hospital who were diagnosed with pneumonia from Results: Eighty-three patients were included, and their average age was 83.8 years. Sixtyeight patients (81.9%) were officially certified as requiring long-term care or support.Twenty-nine of the 83 patients (34.9%) had either negative or normal findings on CRs, and positive findings consistent with pneumonia on CT. There were no significant differences in gender, age, cardiothoracic ratio on CR, or severity between the CR-negative and CRpositive groups. The proportion of negative CRs was significantly higher in patients with certified care level 5 under the long-term care system in Japan and tube feeding.The failure rate of CRs for detecting pneumonic shadows was significantly higher in patients with certified care level 5 and tube feeding.certified care level, chest radiograph, computed tomography, pneumonia, tube feeding | 399 SUGISHITA eT Al.We retrospectively studied patients admitted to our hospital who were diagnosed with pneumonia from April 2014 through June 2016.The diagnoses were based on the consensus between our hospital doctors at admission. We selected patients who underwent chest radiography (posterior-anterior or anterior-posterior) and CT on the same day, excluding those for whom a diagnosis of active pneumonia was difficult due to nonpneumonic or interstitial shadows. We analyzed each patients' characteristics, clinical parameters, and radiographic findings at the time of diagnosis.We obtained CRs with the use of a KONICA MINOLTA FPD digital radiography with a 100 kV tube voltage and 1.5 m receptor distance.For sequential CT scans, we used a 16-detector-row CT scanner (Alexon TSX-032A, Toshiba Medical Systems, Tochigi, Japan) with a slice thickness of 2 mm, scanning parameter of 120 kVp, and field of view of 32 cm.We divided the patients into two groups according to their radiographic findings. Patients whose CRs as well as CT images noted diagnostic findings were classified in the CR-positive group. Those whose CRs were negative or nondiagnostic and whose CT images solely noted diagnostic findings consistent with pneumonia were classified in the CR-negative group. Gender, age, cardiothoracic ratio (CTR) on CRs, severity of pneumonia, level of long-term care or support, and form of feeding (oral or tube feeding) were compared between the two groups.The severity of pneumonia was evaluated with the A-DROP (age, dehydration, respiratory failure, orientation disturbance, and low blood pressure): scoring system used in Japan proposed by the Japan Respiratory Society (JRS).We evaluated patients' performance status (PS) using certified care or support level under the long-term care system in Japan. The Long-term Care Insurance Act in Japan assigns seven levels in accordance with the degree of care required for insured persons: support levels 1 and 2, and care levels 1, 2, 3, 4, and 5 in order. People certified to be in high-care levels such as level 4 or 5 are severely disabled or have severe dementia.Statistical analysis was performed using the chi-square test for categorical variables and Mann-Whitney test for quantitative variables. A value of P<.05 was considered significant. A statistical software package (MedCalc version 16.4.3, Belgium) was used for the analysis. Abstract Viruses differ markedly in their specificity toward host organisms. Here, we test the level of general sequence adaptation that viruses display toward their hosts. We compiled a representative data set of viruses that infect hosts ranging from bacteria to humans. We consider their respective amino acid and codon usages and compare them among the viruses and their hosts. We show that bacteriainfecting viruses are strongly adapted to their specific hosts, but that they differ from other unrelated bacterial hosts. Viruses that infect humans, but not those that infect other mammals or aves, show a strong resemblance to most mammalian and avian hosts, in terms of both amino acid and codon preferences. In groups of viruses that infect humans or other mammals, the highest observed level of adaptation of viral proteins to host codon usages is for those proteins that appear abundantly in the virion. In contrast, proteins that are known to participate in host-specific recognition do not necessarily adapt to their respective hosts. The implication for the potential of viral infectivity is discussed. Abstract Contact networks are convenient models to investigate epidemics, with nodes and links representing potential hosts and infection pathways, respectively. The outcomes of outbreak simulations on networks are driven both by the underlying epidemic model, and by the networks' structural properties, so that the same pathogen can generate different epidemic dynamics on different networks. Here we ask whether there are general properties that make a contact network intrinsically vulnerable to epidemics (that is, regardless of specific epidemiological parameters). By conducting simulations on a large set of modelled networks, we show that, when a broad range of network topologies is taken into account, the effect of specific network properties on outbreak magnitude is stronger than that of fundamental pathogen features such as transmission rate, infection duration, and immunization ability. Then, by focusing on a large set of real world networks of the same type (potential contacts between field voles, Microtus agrestis), we showed how network structure can be used to accurately assess the relative, intrinsic vulnerability of networks towards a specific pathogen, even when those have limited topological variability. These results have profound implications for how we prevent disease outbreaks; in many real world situations, the topology of host contact networks can be described and used to infer intrinsic vulnerability. Such an approach can increase preparedness and inform preventive measures against emerging diseases for which limited epidemiological information is available, enabling the identification of priority targets before an epidemic event. Abstract Multiple sclerosis (MS) is believed to be an autoimmune disease in which autoreactive T cells infiltrate the central nervous system (CNS). Animal models of MS have shown that CNS-specific T cells are present in the peripheral T cell repertoire of healthy mice and cause autoimmune disease only when they are activated by immunization. T cell entry into the CNS is thought to require some form of peripheral activation because the blood-brain barrier prohibits trafficking of this tissue by naive cells. We report here that naive T cells can traffic to the CNS without prior activation. Comparable numbers of T cells are found in the CNS of both healthy recombinase activating gene (Rag) Ϫ / Ϫ T cell receptor (TCR) transgenic mice and nontransgenic mice even when the transgenic TCR is specific for a CNS antigen. Transgenic T cells isolated from the CNS that are specific for non-CNS antigens are phenotypically naive and proliferate robustly to antigenic stimulation in vitro. Strikingly, transgenic T cells isolated from the CNS that are specific for myelin basic protein (MBP) are also primarily phenotypically naive but are unresponsive to antigenic stimulation in vitro. Mononuclear cells from the CNS of MBP TCR transgenic but not nontransgenic mice can suppress the response of peripheral MBP-specific T cells in vitro. These results indicate that naive MBP-specific T cells can traffic to the CNS but do not trigger autoimmunity because they undergo tolerance induction in situ. Abstract Objective: Pleural effusion is common problem, but the rapid and reliable diagnosis for specific pathogenic effusions are lacking. This study aimed to identify the diagnosis based on clinical variables to differentiate pleural tuberculous exudates from other pleural effusions. We also investigated the role of renin-angiotensin system (RAS) and matrix metalloproteinase (MMPs) in the pathogenesis of pleural exudates. Experimental design: The major components in RAS and extracellular matrix metabolism, including angiotensin converting enzyme (ACE), ACE2, MMP-2 and MMP-9 activities, were measured and compared in the patients with transudative (n = 45) and exudative (n = 80) effusions. The exudative effusions were come from the patients with tuberculosis (n = 20), pneumonia (n = 32), and adenocarcinoma (n = 28). Results: Increased ACE and equivalent ACE2 activities, resulting in a significantly increased ACE/ACE2 ratio in exudates, were detected compared to these values in transudates. MMP-9 activity in exudates was significantly higher than that in transudates. The significant correlation between ACE and ACE2 activity that was found in transudates was not found in exudates. Advanced analyses showed significantly increased ACE and MMP-9 activities, and decreased ACE2 activity in tuberculous pleural effusions compared with those in pneumonia and adenocarcinoma effusions. The results indicate that increased ACE and MMP-9 activities found in the exudates were mainly contributed from a higher level of both enzyme activities in the tuberculous pleural effusions. Conclusion: Interplay between ACE and ACE2, essential functions in the RAS, and abnormal regulation of MMP-9 probably play a pivotal role in the development of exudative effusions. Moreover, the ACE/ACE2 ratio combined with MMP-9 activity in pleural fluid may be potential biomarkers for diagnosing tuberculous pleurisy. Abstract By pulse-chase labeling with [35S]methionine and long-term labeling with 3H-sugars, the E1 glycoprotein of coronavirus MHV-A59 has been shown to acquire O-linked oligosaccharides in a two-step process. About 10 min after synthesis of the E1 protein, N-acetyl-galactosamine was added. This was followed ~10 min later by the addition of both galactose and sialic acid to give the mature oligosaccharides. This sequence of additions was confirmed by analyzing the 3H-labeled oligosaccharides bound to each of the El forms using gel filtration on P4 columns. The intracellular location of the first step was determined by ex- Abstract Sialic acids (Sias), 9-carbon-backbone sugars, are among the most complex and versatile molecules of life. As terminal residues of glycans on proteins and lipids, Sias are key elements of glycotopes of both cellular and microbial lectins and thus act as important molecular tags in cell recognition and signaling events. Their functions in such interactions can be regulated by postsynthetic modifications, the most common of which is differential Sia-O-acetylation (O-Ac-Sias). The biology of O-Ac-Sias remains mostly unexplored, largely because of limitations associatedThis is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). with their specific in situ detection. Here, we show that dual-function hemagglutinin-esterase envelope proteins of nidoviruses distinguish between a variety of closely related O-Ac-Sias. By using soluble forms of hemagglutinin-esterases as lectins and sialate-O-acetylesterases, we demonstrate differential expression of distinct O-Ac-sialoglycan populations in an organ-, tissueand cell-specific fashion. Our findings indicate that programmed Sia-O-acetylation/de-Oacetylation may be critical to key aspects of cell development, homeostasis, and/or function.Graphical AbstractCells are covered by a dense thicket of glycans. Though attached to proteins and lipids, these oligo-and polysaccharides are more than decoration. With a structural complexity vastly exceeding that of proteins and nucleic acids, they act as molecular tags in cell recognition and signaling events. Mammalian glycans form linear and branched chains of variable size and composition yet commonly with sialic acids (Sias) as terminal, surface-exposed residues. In consequence of this topology, Sias are key elements of the glycotopes of many regulatory cellular lectins (Crocker et al., 2007; Schauer, 2009 ). For the same reason, they have become attachment factors of choice for a range of pathogens including protozoa, fungi, bacteria, and viruses. Abstract Double-stranded RNA (dsRNA) structures form triplexes and RNA-protein complexes through binding to single-stranded RNA (ssRNA) regions and proteins, respectively, for diverse biological functions. Hence, targeting dsRNAs through majorgroove triplex formation is a promising strategy for the development of chemical probes and potential therapeutics. Short (e.g., 6-10 mer) chemicallymodified Peptide Nucleic Acids (PNAs) have been developed that bind to dsRNAs sequence specifically at physiological conditions. For example, a PNA incorporating a modified base thio-pseudoisocytosine (L) has an enhanced recognition of a G-C pair in an RNA duplex through major-groove L·G-C base triple formation at physiological pH, with reduced pH dependence as observed for C + ·G-C base triple formation. Currently, an unmodified T base is often incorporated into PNAs to recognize a Watson-Crick A-U pair through major-groove T·A-U base triple formation. A substitution of the 5-methyl group in T by hydrogen and halogen atoms (F, Cl, Br, and I) causes a decrease of the pK a of N3 nitrogen atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions. Here, we synthesized a series of PNAs incorporating uracil and halouracils, followed by binding studies by non-denaturing polyacrylamide gel electrophoresis, circular dichroism, and thermal melting. Our results suggest that replacing T with uracil and halouracils may enhance the recognition of an A-U pair by PNA·RNA 2 triplex formation in a sequence-dependent manner, underscoring the importance of local stacking interactions. Incorporating bromouracils and chlorouracils into a PNA results in a significantly reduced pH dependence of triplex formation even for PNAs containing C bases, likely due to an upshift of the apparent pK a of N3 atoms of C bases. Thus, halogenation and other chemical modifications may be utilized to enhance hydrogen bonding of the adjacent base triples and thus triplex formation. Furthermore, our experimental and computational modelling data suggest that PNA·RNA 2 triplexes may be stabilized by incorporating a Br UL step but not an L Br U step, in dsRNAbinding PNAs. Abstract Current data indicate that the "bronchiolitis" diagnosis comprises more than one condition. Clinically, pathophysiologically, and even genetically three main clusters of patients can be identified among children suffering from severe bronchiolitis (or first wheezing episode): (a) respiratory syncytial virus (RSV)-induced bronchiolitis, characterized by young age of the patient, mechanical obstruction of the airways due to Abstract Background. The characteristics, determinants, and potential contribution to transmission of asymptomatic cases of respiratory syncytial virus (RSV) infection have not been well described.Methods. A cohort of 47 households (493 individuals) in coastal Kenya was recruited and followed for a 26-week period spanning a complete RSV season. Nasopharyngeal swab specimens were requested weekly, during the first 4 weeks, and twice weekly thereafter from all household members, regardless of illness status. The samples were screened for a range of respiratory viruses by multiplex real-time polymerase chain reaction.Results. Tests on 16 928 samples yielded 205 RSV infection episodes in 179 individuals (37.1%) from 40 different households. Eighty-six episodes (42.0%) were asymptomatic. Factors independently associated with an increased risk of asymptomatic RSV infection episodes were higher age, shorter duration of infection, bigger household size, lower peak viral load, absence of concurrent RSV infections within the household, infection by RSV group B, and no prior human rhinovirus infections. The propensity of RSV spread in households was dependent on symptom status and amount (duration and load) of virus shed.Conclusions. While asymptomatic RSV was less likely to spread, the high frequency of symptomless RSV infection episodes highlights a potentially important role of asymptomatic infections in the community transmission of RSV. Abstract The E1 glycoprotein of the avian coronavims infectious bronchitis virus contains a short, glycosylated amino-terminal domain, three membranespanning domains, and a long carboxy-terminal cytoplasmic domain. We show that E1 expressed from cDNA is targeted to the Golgi region, as it is in infected cells. E1 proteins with precise deletions of the first and second or the second and third membranespanning domains were glycosylated, thus suggesting that either the first or third transmembrane domain can function as an internal signal sequence. The mutant protein with only the first transmembrane domain accumulated intracellulady like the wild-type protein, but the mutant protein with only the third transmembrane domain was transported to the cell surface. This result suggests that information specifying accumulation in the Golgi region resides in the first transmembrane domain, and provides the first example of an intracellular membrane protein that is transported to the plasma membrane after deletion of a specific domain. Abstract Today, accredited zoos are not just places for entertainment, they are actively involved in research for conservation and health. During recent decades in which the challenges for biodiversity conservation and public health have escalated, zoos have made significant changes to address these difficulties. Zoos increasingly have four key areas of focus: education, recreation, conservation, and research. These key areas are important in addressing an interrelated global conservation (i.e. habitat and wildlife loss) and public health crisis. Zoo and public health professionals working together within a One Health framework represent a powerful alliance to address current and future conservation and public health problems around the world. For researchers, practitioners, and students, the collaboration between zoos and public health institutions offers the opportunity to both teach and operationalize this transdisciplinary approach. Using examples from our programs, we give a template for moving forward with collaborative initiatives and sustainable solutions involving partners in both zoos and public health institutions. We provide examples of cooperative programs and suggest a model for consideration in the development of further activities in this area. Abstract Background: Human metapneumovirus (hMPV) is a Paramyxovirus known to cause acute respiratory tract infections in children and young adults. To date, there is no study from the Aseer region of Saudi Arabia determining the proportion and severity of hMPV infection among pediatric hospitalized patients with respiratory infections. Objectives: The objective of this study is to determine the presence of hMPV antigens in the nasopharyngeal secretions of pediatric patients hospitalized with respiratory tract infections in the Aseer region of Saudi Arabia. Materials and Methods: This prospective, serological hospital-based study included all pediatric patients who were admitted to Aseer Central Hospital, Abha, Saudi Arabia, from July 2016 to November 2017 with upper and/or lower respiratory tract infections. Basic demographics of patients and their clinical data on and after admission were recorded. Direct fluorescent antibody assay was used to detect the presence of hMPV antigens in the obtained nasopharyngeal secretion specimens. Results: During the study, 91 pediatric patients were hospitalized due to upper and/or lower respiratory tract infections, of which 9.9% were positive for hMPV. These patients were aged 9 months to 16 years, were from Abha city or its surrounding localities and were mostly (77.8%) hospitalized during autumn or winter. The most common diagnosis on admission was bronchopneumonia (55.5%) and aspiration pneumonia (22.2%), and some patients also had underlying chronic conditions such as chronic heart disease (22.2%) and bronchial asthma (11.1%).The results obtained indicated that hMPV is a potential etiologic factor for the commonly occurring acute respiratory infections in hospitalized children from the Aseer region of Saudi Arabia. hMPV infection was also found to be associated with complicated respiratory conditions such as bronchopneumonia, chronic heart disease and bronchial asthma. Abstract Objective: This study presents a preliminary report on the chest radiographic and computed tomography (CT) findings of the 2019 novel coronavirus disease (COVID-19) pneumonia in Korea. Materials and Methods: As part of a multi-institutional collaboration coordinated by the Korean Society of Thoracic Radiology, we collected nine patients with COVID-19 infections who had undergone chest radiography and CT scans. We analyzed the radiographic and CT findings of COVID-19 pneumonia at baseline. Fisher's exact test was used to compare CT findings depending on the shape of pulmonary lesions. Results: Three of the nine patients (33.3%) had parenchymal abnormalities detected by chest radiography, and most of the abnormalities were peripheral consolidations. Chest CT images showed bilateral involvement in eight of the nine patients, and a unilobar reversed halo sign in the other patient. In total, 77 pulmonary lesions were found, including patchy lesions (39%), large confluent lesions (13%), and small nodular lesions (48%). The peripheral and posterior lung fields were involved in 78% and 67% of the lesions, respectively. The lesions were typically ill-defined and were composed of mixed ground-glass opacities and consolidation or pure ground-glass opacities. Patchy to confluent lesions were primarily distributed in the lower lobes (p = 0.040) and along the pleura (p < 0.001), whereas nodular lesions were primarily distributed along the bronchovascular bundles (p = 0.006). Conclusion: COVID-19 pneumonia in Korea primarily manifested as pure to mixed ground-glass opacities with a patchy to confluent or nodular shape in the bilateral peripheral posterior lungs. A considerable proportion of patients with COVID-19 pneumonia had normal chest radiographs. Abstract The World Health Organization (WHO) have been in collaborative efforts with the Republic of Korea in keeping of and for better health for all for the past decades. From the control of parasites to building of community health system in rural places, the works has now resulted in healthier Korea than ever, and has transformed the role of engaging as the world leader in contribution of health and development. Seventy years of independence, war, and poverty, transforming from a recipient country of official development assistance to a significant donor to the global society, we have emphasized the importance of international cooperation and the role of WHO in the past years in Korea and neighboring countries. Looking back of the past is meaningful to diagnose the present problems, and to foresee the future of our world. Abstract b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 876-881 h t t p : / / w w w . b j m i c r o b i o l . c o m . b r / a b s t r a c t Three dog shelters in Rio Grande do Sul were investigated for associations between the occurrence of respiratory viruses and shelter environmental conditions. Nasal secretions randomly collected during the cold season were tested via PCR, and this data collection was followed by nucleotide sequencing of the amplicons. In shelter #1 (poor sanitary and nutritional conditions, high animal density and constant contact between dogs), 78% (58/74) of the nasal samples were positive, 35% (26/74) of which were in single infections and 44% (32/74) of which were in coinfections. Shelters #2 and #3 had satisfactory sanitary and nutritional conditions, outdoors exercise areas (#2) and animal clustering by groups (#3).In shelter #2, 9% (3/35) of the samples were positive for Canine parainfluenza virus (CPIV), and 6% (2/35) were positive for Canid herpesvirus 1 (CaHV-1). In shelter #3, 9% (7/77) of the samples were positive for Canine adenovirus type 2 (CAdV-2), and 1% (1/77) were positive for Canine distemper virus (CDV). The amplicon sequences (CPIV and CDV nucleoprotein gene;CAdV-2 E3 gene; CaHV-1 glycoprotein B gene) showed 94-100% nucleotide identity with Gen-Bank sequences. Our results demonstrate that CPIV, CAdV-2 and CDV are common in dog shelters and that their frequencies appear to be related with environmental and nutritional conditions. These results indicate the need for control/prevention measures, including vaccination and environmental management, to minimize these infections and improve dog health. Abstract Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV). RVF is mainly prevalent on the Arabian Peninsula, the African continent, and several islands in the Indian Ocean near southeast Africa. RVFV has been classified by the World Organisation for Animal Health (OIE) as a category A pathogen. To avoid biological safety concerns associated with use of the pathogen in RVFV neutralization assays, the present study investigated and established an RVFV pseudovirus-based neutralization assay. This study used the human immunodeficiency virus (HIV) lentiviral packaging system and RVFV structural proteins to successfully construct RVFV pseudoviruses. Electron microscopy observation and western blotting indicated that the size, structure, and shape of the packaged pseudoviruses were notably similar to those of HIV lentiviral vectors. Infection inhibition assay results showed that an antibody against RVFV inhibited the infective ability of the RVFV pseudoviruses, and an antibody neutralization assay for RVFV detection was then established. This study has successfully established a neutralization assay based on RVFV pseudoviruses and demonstrated that this method can be used to effectively evaluate antibody neutralization. Abstract Programmed -1 ribosomal frameshifting (-1PRF) is tightly regulated by messenger RNA (mRNA) sequences and structures in expressing two or more proteins with precise ratios from a single mRNA. Using single-molecule fluorescence resonance energy transfer (smFRET) between (Cy5)EF-G and (Cy3)tRNA Lys , we studied the translational elongation dynamics of -1PRF in the Escherichia coli dnaX gene, which contains three frameshifting signals: a slippery sequence (A AAA AAG), a Shine-Dalgarno (SD) sequence and a downstream hairpin. The frameshift promoting signals mostly impair the EF-G-catalyzed translocation step of the two tRNA Lys and the slippery codons from the A-and P-sites. The hairpin acts as a road block slowing the translocation rate. The upstream SD sequence together with the hairpin promotes dissociation of futile EF-G and thus causes multiple EF-G driven translocation attempts. A slippery sequence also helps dissociation of the EF-G by providing alternative base-pairing options. These results indicate that frameshifting takes place during the repetitive ribosomal conformational changes associated with EF-G dissociation upon unsuccessful translocation attempts of the second slippage codon from the A-to the P-sites. Abstract Objective: To uncover the potential effect of metformin on proliferation and apoptosis of thyroid cancer TPC-1 cell line, and the underlying mechanism. Methods: Viability, apoptosis and LRP2 level in TPC-1 cells treated with different doses of metformin for different time points were determined. Besides, protein levels of p-JNK1 and c-Jun N-terminal kinases (JNK) in metformin-treated TPC-1 cells were detected by Western blot. Regulatory effects of LRP2 on the JNK pathway and cell viability in metformin-treated TPC-1 cells were assessed. Results: Viability in TPC-1 cells gradually decreased with the treatment of increased doses of metformin either for 24 h or 48 h. The apoptotic rate was concentration-dependently elevated by metformin treatment. Relative levels of LRP2 and p-JNK1 were concentrationdependently downregulated by metformin treatment. In addition, overexpression of LRP2 partially abolished the inhibitory effect of metformin on the viability of TPC-1 cells. Conclusion: Metformin treatment suppresses the proliferative ability and induces apoptosis of TPC-1 cells by downregulating LRP2 to block the JNK pathway. Abstract Cross-species transmission of viruses poses a sustained threat to public health. Due to increased contact between humans and other animal species the possibility exists for cross-species transmissions and ensuing disease outbreaks. By using conventional PCR amplification and next generation sequencing, we obtained 130 partial or full genome kobuvirus sequences from humans in a sentinel cohort in Vietnam and various mammalian hosts including bats, rodents, pigs, cats, and civets. The evolution of kobuviruses in different hosts was analysed using Bayesian phylogenetic methods. We estimated and compared time of origin of kobuviruses in different host orders; we also examined the cross-species transmission of kobuviruses within the same host order and between different host orders. Our data provide new knowledge of rodent and bat kobuviruses, which are most closely related to human kobuviruses. The novel bat kobuviruses isolated from bat roosts in Southern Vietnam were genetically distinct from previously described bat kobuviruses, but closely related to kobuviruses found in rodents. We additionally found evidence of frequent cross-species transmissions of kobuviruses within rodents. Overall, our phylogenetic analyses reveal multiple cross-species transmissions both within and among mammalian species, which increases our understanding of kobuviruses genetic diversity and the complexity of their evolutionary history. Abstract Background. UpToDate is an online clinical decision support resource that is used extensively by clinicians around the world. Digital surveillance techniques have shown promise to aid with the detection and monitoring of infectious disease outbreaks. We sought to determine whether UpToDate searches for Middle East respiratory syndrome (MERS) could be used to detect and monitor MERS outbreaks in Saudi Arabia.Methods. We analyzed daily searches related to MERS in Jeddah and Riyadh, Saudi Arabia during 3 outbreaks in these cities in 2014 and 2015 and compared them with reported cases during the same periods. We also compared UpToDate MERS searches in the affected cities to those in a composite of 4 negative control cities for the 2 outbreaks in 2014.Results. UpToDate MERS searches during all 3 MERS outbreaks in Saudi Arabia showed a correlation to reported cases. In addition, UpToDate MERS search volume in Jeddah and Riyadh during the outbreak periods in 2014 was significantly higher than the concurrent search volume in the 4 negative control cities. In contrast, during the baseline periods, there was no difference between UpToDate searches for MERS in the affected cities compared with the negative control cities.Conclusions. UpToDate search activity seems to be useful for detecting and monitoring outbreaks of MERS in Saudi Arabia. Abstract Zika virus (ZIKV) has become a global public health emergency due to its rapidly expanding range and its ability to cause severe congenital defects such as microcephaly. However, there are no FDA-approved therapies or vaccines against ZIKV infection. Through our screening of viral entry inhibitors, we found that chloroquine (CQ), a commonly used antimalarial and a FDA-approved drug that has also been repurposed against other pathogens, could significantly inhibit ZIKV infection in vitro, by blocking virus internalization. We also demonstrated that CQ attenuates ZIKV-associated morbidity and mortality in mice. Finally, we proved that CQ protects fetal mice from microcephaly caused by ZIKV infection. Our methodology of focusing on previously identified antivirals in screens for effectiveness against ZIKV proved to be a rapid and efficient means of discovering new ZIKV therapeutics. Selecting drugs that were previously FDA-approved, such as CQ, also improves the likelihood that they may more quickly reach stages of clinical testing and use by the public. Abstract Aim: Pharmacoeconomic cost-effectiveness analysis of two different dosage regimens of levofloxacin in the treatment of acute lower respiratory tract infection in elderly patients. Methods: A total of 108 elderly patients with acute lower respiratory tract infection who visited by our hospital between September 2013 and September 2014 were randomly divided into Group A and Group B, with 54 patients in each group. In Group A, levofloxacin injection was given for continuous intravenous infusion treatment, whereas in Group B, levofloxacin injection and levofloxacin capsule were given as sequential therapy (ST). The period of treatment for both the groups was 10 days, and minimum cost analysis was used to analyze the treatment. Results: Groups A and B had cure rates of 61.1% and 59.3% (P0.05), effective rates of 88.9% and 83.3% (P0.05), bacterial clearance rates of 96.3% and 92.6% (P0.05), and incidence rates of adverse reactions of 7.4% and 3.7% (P0.05), respectively. Treatment costs of Groups A and B were 1,588 RMB and 1,150 RMB, respectively, whereas the cost-effectiveness of the two groups was at 17.86 and 13.81, respectively (P0.05). Conclusion: Levofloxacin ST had relatively higher cost-effectiveness ratio for the treatment of acute lower respiratory tract infection in elderly patients, especially Chinese. Abstract In the Anthropocene context, changes in climate, land use and biodiversity are considered among the most important anthropogenic factors affecting parasites-host interaction and wildlife zoonotic diseases emergence. Transmission of vector borne pathogens are particularly sensitive to these changes due to the complexity of their cycle, where the transmission of a microparasite depends on the interaction between its vector, usually a macroparasite, and its reservoir host, in many cases represented by a wildlife vertebrate. The scope of this paper focuses on the effect of some major, fast-occurring anthropogenic changes on the vectorial capacity for tick and mosquito borne pathogens. Specifically, we review and present the latest advances regarding two emerging vector-borne viruses in Europe: Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV). In both cases, variation in vector to host ratio is critical in determining the intensity of pathogen transmission and consequently infection hazard for humans. Forecasting vector-borne disease hazard under the global change scenarios is particularly challenging, requiring long term studies based on a multidisciplinary approach in a One-Health framework. Abstract Objectives: The present study aimed to examine the trends and characteristics of fall-related attendance in accident and emergency department (AED) by injury type and the trend in associated average length of stay (LOS) among children and adolescents in Hong Kong.Design: A retrospective approach was adopted. Setting: AED, involving all local public emergency departments from 2001 to 2012. Participants: 63 557 subjects aged 0-19 years with fall injury record were included in the analysis.Primary outcome measures: Fall-related injury number and rates were calculated and reported. Poisson and negative binomial regression models were used to study the trends of injury incidence rate at different body regions.Results: AED fall-related attendance rate increased significantly with an annual percentage change of 4.45 (95% CI 3.43 to 5.47%, p<0.0001). The attendance number of male subjects was persistently higher than female subjects. The standardised rate of fracture injury increased by 1.31% (95% CI 0.56 to 2.05%, p<0.0001) and that of non-fracture injury increased by 9.23% (95% CI 7.07 to 11.43%, p<0.0001) annually. Upper limb was the most frequently fractured location. It included forearm/elbow, shoulder/upper arm and wrist/hand with descending order of frequency. On the contrary, head was the most frequent non-fracture location, followed by forearm/elbow.The rates of fall-related attendance have been increasing and still remain high. There were significant increases in non-fracture injuries. Fractures were most frequently found in the upper extremity of a child while the most common non-fracture location was head. It appears that more efforts should be made and preventive measures should be implemented for children and adolescents in Hong Kong.Lee JC-Y, et al. Abstract A B S T R A C T Porcine Circovirus type 2 (PCV2) infections and associated diseases have been rarely studied in Africa. There is no report of PCV2 infection-associated morbidity and the level of awareness of stakeholders has never been investigated in Uganda. This cross sectional survey investigated the occurrence of Porcine Circovirus type 2systemic disease (PCV2-SD) among pigs and the associated level of awareness of stakeholders in Central Uganda. Data were collected using questionnaires, Focus Group Discussions (FGDs), key informant interviews and laboratory investigations. All respondents (n = 131) and farmers attending FGDs (n = 31) had never heard of PCV2-SD and only 16.7% (n = 2) of the interviewed animal health workers (n = 12) knew about the disease. Among the farms, 20 piglets presenting with a chronic wasting and a persistent diarrhea were detected and sampled for laboratory investigations. Severe lymphoid depletion with histiocytic and macrophage infiltration in lymphoid organs (n = 8), shortening of intestinal villi (n = 9), abscesses in various organs (n = 15) and granulomatous pneumonia (n = 2) were the major histopathological lesions described. Immunohistochemistry and PCR assays on organs with implicating lesions confirmed PCV2 infection in 25% (n = 5) of the 20 pigs. The study confirmed the occurrence of PCV2 infections among piglets with persistent diarrhea on pig farms in central Uganda and revealed a low level of associated knowledge among farmers and veterinary practitioners. The study arouses the need for systematic studies on prevalence of PCV2 infections and sensitization of stakeholders on occurrence of PCV2 infections in Uganda. Abstract New protein-coding genes can originate either through modification of existing genes or de novo. Recently, the importance of de novo origination has been recognized in eukaryotes, although eukaryotic genes originated de novo are relatively rare and difficult to identify. In contrast, viruses contain many de novo genes, namely those in which an existing gene has been "overprinted" by a new open reading frame, a process that generates a new protein-coding gene overlapping the ancestral gene. We analyzed the evolution of 12 experimentally validated viral genes that originated de novo and estimated their relative ages. We found that young de novo genes have a different codon usage from the rest of the genome. They evolve rapidly and are under positive or weak purifying selection. Thus, young de novo genes might have strain-specific functions, or no function, and would be difficult to detect using current genome annotation methods that rely on the sequence signature of purifying selection. In contrast to young de novo genes, older de novo genes have a codon usage that is similar to the rest of the genome. They evolve slowly and are under stronger purifying selection. Some of the oldest de novo genes evolve under stronger selection pressure than the ancestral gene they overlap, suggesting an evolutionary tug of war between the ancestral and the de novo gene. Abstract In this study, we evaluate a method for the early diagnosis of radiodermatitis for use in the prevention and therapy of this condition. Hairless mice (SKH1-hr) were used to study the early diagnosis of radiodermatitis. Lactate dehydrogenase (LDH, EC 1.1.1.27) isozymes were analyzed using nativepolyacrylamide gel electrophoresis and western blotting of blood serum and tissues collected from SKH1hr mice. Radiodermatitis developed 24 days after the first X-irradiation. Reduced spleen weight was observed after the last X-irradiation (P<0.05). Thereafter the weight increased until 24 days after the first irradiation, finally reaching levels comparable to those in the sham-irradiated control group. LDH activity was the highest in skeletal muscle and lowest in blood serum. LDH C 4 , A 4 , A 3 B, A 2 B 2 , AB 3 , and B 4 isozymes were detected, in the mentioned order, from the cathode. This result was similar in other mouse strains. In the irradiated group, LDH A 4 isozyme levels were reduced in the serum until inflammation occurred, whereas those of B 4 isozyme were elevated. The subunits A and B followed a similar trend to that of LDH A 4 and B 4 isozyme, respectively. Importantly, antibodies against LDH B 4 isozyme could prove useful in the early diagnosis of radiodermatitis. Abstract Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in doublestranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5 0 end and fluorophore attached to the 3 0 end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3 0 end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection. Abstract A high percentage (up to 90%) of dromedary camels in the Middle East as well as eastern and central Africa have antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV). Here we report comparably high positivity of MERS-CoV antibodies in dromedary camels from northern Mali. This extends the range of MERS-CoV further west in Africa than reported to date and cautions that MERS-CoV should be considered in cases of severe respiratory disease in the region. Abstract Introduction: Transport of patients undergoing extracorporeal membrane oxygenation is currently available in 5 referral centers in our country. Methods: Retrospective case series of patients managed by our mobile extracorporeal membrane oxygenation team and transferred to San Gerardo University Hospital from December 2004 to December 2012. Results: 42 patients were transported. The mean age was 42.11 (standard deviation ±18.11) years, with a range between 2 years and 70. 14 patients were females (33%) and 28 males (67%). The average transport distance was 121.69 km (±183.08) with a range between 9 km and 1044 Km. The mission's mean time was equal to 508 minutes (±185) with range of 120-960 minutes. 29 patients (69%) were transported with extracorporeal membrane oxygenation support, while 13 patients (31%) were transported with conventional ventilation. In 28 patients (97%) a veno-venous bypass was utilized, while in one case (3%) a Veno-Arterial cannulation was performed. 32 patients survived (76%) and have been discharged alive from hospital. No major clinical or technical issues were observed during the transport. Conclusions: According to our data, we conclude that a dedicated mobile team allowed safe ground transportation of patients with severe acute lung injury to our tertiary care institution. Abstract Objectives 40 This qualitative study attempts to understand the perspectives and experiences of United 41 States-based global health physicians and program leaders on how their experiences abroad 42 influence their healthcare practices in the United States. 43 44 Design 45 We administered online questionnaires and open-ended, semi-structured interviews with 46 global health physicians and program leaders affiliated with United States-based academic 47 medical centers. We utilized open coding procedures and content analysis to derive relevant 48 themes from the data. Abstract CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200 tr ) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200. Abstract Viral respiratory infections are among the most common causes of disease in humans, particularly in young children, and remain a major public health problem worldwide. For many geographic regions, there is limited epidemiological information on the main causative agents of these diseases. In this article, we investigated, in a prospective study, the viral agents leading to acute respiratory disease in children younger than 15 years of age who were admitted to the pediatric emergency unit of a major teaching hospital in Erbil City, capital of the Kurdistan region, Iraq. Nasopharyngeal samples obtained from 269 hospitalized children were analyzed for viral respiratory pathogens using the xTAG Respiratory Virus Panel Fast assay, and the data were correlated with the clinical and demographic information available for these patients. One or more respiratory virus(es) were detected in 203 out of 269 (75.5%) samples. The most frequent viruses were enterovirus/rhinovirus (n = 88; 32.7%), respiratory syncytial virus (n = 55; 20.4%), and human metapneumovirus (n = 36; 13.4%). In 42 samples (15.6%), coinfections with 2 or more respiratory viruses were detected, with enterovirus/rhinovirus, respiratory syncytial virus, human metapneumovirus, and adenovirus being identified as the most common agents in viral coinfections in these patients. Abstract Investigating gene expression of immune cells of whole blood or peripheral blood mononuclear cells (PBMC) under polyinosinic:polycytidylic acid (poly I:C) stimulation is valuable for understanding the immune response of organism to RNA viruses. Quantitative real-time PCR (qRT-PCR) is a standard method for quantification of gene expression studies. However, the reliability of qRT-PCR data critically depends on proper selection of reference genes. In the study, using two different analysis programs, geNorm and NormFinder, we systematically evaluated the gene expression stability of six candidate reference genes (GAPDH, ACTB, B2M, RPL4, TBP, and PPIA) in samples of whole blood and PBMC with or without poly I:C stimulation. Generally, the six candidate genes performed a similar trend of expression stability in the samples of whole blood and PBMC, but more stably expressed in whole blood than in PBMC. geNorm ranked B2M and PPIA as the best combination for gene expression normalization, while according to NormFinder, TBP was ranked as the most stable reference gene, followed by B2M and PPIA. Comprehensively considering the results from the two programs, we recommended using the geometric mean of the three genes, TBP, PPIA and B2M, to normalize the gene expression of whole blood and PBMC with poly I:C stimulation. Our study is the first detailed survey of the gene expression stability in whole blood and PBMC with or without poly I:C stimulation and should be helpful for investigating the molecular mechanism involved in porcine whole blood and PBMC in response to poly I:C stimulation. ( Abstract Leukemia/lymphoma-related factor (LRF), a zinc-finger transcription factor encoded by Zbtb7a, is a protooncogene that regulates differentiation in diverse cell lineages, and in the CNS, its function is relatively unexplored. This study is the first to examine the role of LRF in CNS pathology. We first examined LRF expression in a murine viral model of spinal cord demyelination with clinically relevant lesion characteristics. LRF was rarely expressed in oligodendrocyte progenitors (OP) yet, was detected in nuclei of the majority of oligodendrocytes in healthy adult CNS and during remyelination. Plp/CreER T :Zbtb7a fl/fl mice were then used with cuprizone demyelination to determine the effect of LRF knockdown on oligodendrocyte repopulation and remyelination. Cuprizone was given for 6 weeks to demyelinate the corpus callosum. Tamoxifen was administered at 4, 5, or 6 weeks after the start of cuprizone. Tamoxifen-induced knockdown of LRF impaired remyelination during 3 or 6-week recovery periods after cuprizone. LRF knockdown earlier within the oligodendrocyte lineage using NG2CreER T :Zbtb7a fl/fl mice reduced myelination after 6 weeks of cuprizone.LRF knockdown from either the Plp/CreER T line or the NG2CreER T line did not significantly change OP or oligodendrocyte populations. In vitro promoter assays demonstrated the potential for LRF to regulate transcription of myelin-related genes and the notch target Hes5, which has been implicated in control of myelin formation and repair. In summary, in the oligodendrocyte lineage, LRF is expressed mainly in oligodendrocytes but is not required for oligodendrocyte repopulation of demyelinated lesions. Furthermore, LRF can modulate the extent of remyelination, potentially by contributing to interactions regulating transcription.demyelination, cuprizone, notch, differentiation, oligodendrocyte progenitor Abstract Background Person-to-person transmission of respiratory pathogens, including Pseudomonas aeruginosa, is a challenge facing many cystic fibrosis (CF) centres. Viable P aeruginosa are contained in aerosols produced during coughing, raising the possibility of airborne transmission. Methods Using purpose-built equipment, we measured viable P aeruginosa in cough aerosols at 1, 2 and 4 m from the subject (distance) and after allowing aerosols to age for 5, 15 and 45 min in a slowly rotating drum to minimise gravitational settling and inertial impaction (duration). Aerosol particles were captured and sized employing an Anderson Impactor and cultured using conventional microbiology. Sputum was also cultured and lung function and respiratory muscle strength measured. Results Nineteen patients with CF, mean age 25.8 (SD 9.2) years, chronically infected with P aeruginosa, and 10 healthy controls, 26.5 (8.7) years, participated. Viable P aeruginosa were detected in cough aerosols from all patients with CF, but not from controls; travelling 4 m in 17/18 (94%) and persisting for 45 min in 14/18 (78%) of the CF group. Marked inter-subject heterogeneity of P aeruginosa aerosol colony counts was seen and correlated strongly (r=0.73-0.90) with sputum bacterial loads. Modelling decay of viable P aeruginosa in a clinic room suggested that at the recommended ventilation rate of two air changes per hour almost 50 min were required for 90% to be removed after an infected patient left the room. Conclusions Viable P aeruginosa in cough aerosols travel further and last longer than recognised previously, providing additional evidence of airborne transmission between patients with CF. Abstract Purpose: Community-acquired pneumonia is a common illness worldwide. In adults, community-acquired bacterial pneumonia has been well studied, but viral pneumonia is less well understood. We designed this study to identify respiratory pathogens, including common pneumonia-causing bacteria, viruses and atypical pneumonia pathogens, using reverse transcription-polymerase chain reaction. Patients and methods: We conducted a retrospective study of outpatients with community-acquired pneumonia at the Fever Clinic of Peking University Third Hospital. We collected sputum or throat swabs from patients diagnosed with community-acquired pneumonia. Multiplex real-time reverse transcription-polymerase chain reaction was performed for 20 pathogens, including 9 viruses, 3 atypical pathogens and 8 bacteria.Results: There were 232 outpatients enrolled in our study, and 153 patients (65.9%) had positive test results, of which 26.7% were viruses, 19.4% were atypical pathogens and 19.8% were bacteria. Mycoplasma pneumoniae infection was detected at the highest frequency (19.0%), exceeding Streptococcus pneumoniae infection. The most commonly identified viral pathogens were IFVs (15.1%), PIVs (3.4%) and RhV (2.6%). The most commonly identified bacteria were Streptococcus pneumoniae (9.1%), Haemophilus influenza (6.5%) and Klebsiella pneumoniae (2.6%). Conclusion: Our study suggests that viruses were commonly detected in outpatients with CAP, and IFVs were the most common viruses, especially during flu season. Patients with viral infection were prone to viral-bacterial coinfection. Mycoplasma pneumoniae was the leading pathogen in the outpatients with CAP. Viral infection occurs in a large number of outpatients with CAP, and it should receive greater attention in clinical work. Abstract The past decade and a half has been characterized by numerous emerging infectious diseases. With each new threat, there has been a call for rapid vaccine development. Pathogens such as the Middle East Respiratory Syndrome coronavirus (MERS-CoV) and the Zika virus represent either new viral entities or viruses emergent in new geographic locales and characterized by novel complications. Both serve as paradigms for the global spread that can accompany new pathogens. In this paper, we review the epidemiology and pathogenesis of MERS-CoV and Zika virus with respect to vaccine development. The challenges in vaccine development and the approach to clinical trial design to test vaccine candidates for disease entities with a changing epidemiology are discussed.In 2012, cases of a progressive pulmonary infection related to individuals who reside in or traveled to the Arabian Peninsula were determined as caused by a novel Group C, b-coronavirus MERS-CoV. 1,2 In contrast to the majority of human pathogenic coronaviruses that cause self-limited upper-respiratory illness, the mortality rate of early MERS-CoV cases was approximately 60%, 3 and has remained greater than 35% -approximating that seen during the West African Ebola virus outbreak. In contrast, the mortality rate during Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) epidemic was 10%.Following an incubation period of about 1 week, MERS-CoV causes a rapidly progressive lower respiratory infection with a prodromal illness characterized by fever, cough, and mild shortness of breath. Clinical deterioration is typical leading to the need for intensive care and ventilator support Abstract The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, caliciand lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: 'breadth first' beginning with whole-genome and proteome curation using standardized protocols, a 'targeted' approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website (https:// patric.vbi.vt.edu) will continually grow with the addition of data, analysis and functionality over the course of the project. Abstract Aim: Infection control in the emergency department is important for hospital risk management; however, few clinical guidelines have been established. This study aimed to determine whether hospitals in Japan have infection control manuals, and investigate the contents of manuals, consulting systems, and isolation facilities for emergency departments.Methods: A total of 517 hospitals certified as educational institutions for board-certified acute care physicians in Japan were requested between March and May 2015 to provide a written evaluation of the infection control in the emergency department.Results: A total of 51 of 303 (16.8%) hospitals had no manuals regarding infection control in the emergency department. Among 250 hospitals having emergency department manuals, 115 (46.0%) did not include contents regarding disinfection and sterilization for imaging examination rooms, and only 44 (17.6%) had criteria for contacting the emergency medical service when patients are suspected of, or diagnosed with, communicable diseases. Of the 303 hospitals, 277 (91.4%) prepared specific manuals for the 2009 pandemic influenza. Of the 303 hospitals, 80 (26.4%) did not prepare manuals for the Ebola virus disease outbreak in West Africa in 2014. Furthermore, 92 (30.4%) of the 303 hospitals did not have any negative-pressure isolation rooms.Conclusions: Practices and guidelines necessary for infection control in the emergency department were not sufficiently covered in the hospitals studied. Education, information sharing, and a checklist for preparing manuals are needed to establish better infection control systems in emergency departments. Abstract The review intends to present and recapitulate the current knowledge on the roles and importance of regulatory RNAs, such as microRNAs and small interfering RNAs, RNA binding proteins and enzymes processing RNAs or activated by RNAs, in cells infected by RNA viruses. The review focuses on how non-coding RNAs are involved in RNA virus replication, pathogenesis and host response, especially in retroviruses HIV, with examples of the mechanisms of action, transcriptional regulation, and promotion of increased stability of their targets or their degradation. Abstract Effective vaccination is based on three critical aspects of the B-cell response towards infectious agents: (i) that B-cells can generate specific antibodies towards a vast molecular diversity of antigens; proteins, sugars, DNA and lipids. There seems to be no limit to the ability to raise antibodies to everything. (ii) once stimulated, B-cells can perfect their antibodies through affinity maturation to complement every nook and cranny of the epitope and (iii) that the pathogen remains genetically stable and does not change to any great extent. Thus, antibodies produced against the vaccine and subsequent boosts recognize the viral virulent field isolates in future encounters and effectively knock them out. However, some vaccine targets, such as flu virus and HIV, are extremely genetically dynamic. The rapid genetic drift of these viruses renders them moving targets which assist in their ability to evade immune surveillance. Here we postulate that in the case of hypervariable pathogens the B-cell response actually might be "too good". We propose that restricting B-cell activities may prove effective in counteracting the genetic diversity of variant viruses such as flu and HIV. We suggest two levels of "B-cell restriction": (i) to focus the B-cell response exclusively towards neutralizing epitopes by creating epitope-based immunogens; (ii) to restrict affinity maturation of B-cells to prevent the production of overly optimized exquisitely specific antibodies. Together, these "B-cell restrictions" provide a new modality for vaccine design.ARTICLE HISTORY Abstract Changes occurred in the patterns of utilization of emergency medical services during the Middle East respiratory syndrome (MERS) outbreak. The purpose of this study was to analyze the patterns of adult and pediatric patients who visited the emergency department (ED) during the outbreak. This retrospective study was conducted by analyzing changes in the patterns of visits among adult and pediatric patients in the ED at one tertiary teaching hospital in Korea. The study was performed from June 1, 2013 to July 31, 2015. The MERS outbreak period was from June 1 to July 31, 2015, and we compared that period to the same periods in 2013 and 2014. We compared and analyzed the patients' characteristics, emergency severity index (ESI) level at the visit, cause of visit, diagnosis, final dispositions, injury/non-injury, length of stay at the ED (EDLOS), and hospitalization rate. A total of 9,107 patients visited the ED during this period. Of these patients, 2,572 (28.2%) were pediatric patients and 6,535 (71.8%) were adult patients. The most common cause of an ED visit was fever (adult patients: 21.6%, pediatric patients: 56.2%). The proportion of non-urgent visits involving an ESI level of 4 or 5 and the EDLOS decreased significantly in pediatric and adult patients in comparison to that during the past two years. This change was significant in pediatric patients. Among adult patients, the rate of injury decreased, whereas it increased among pediatric patients. During the MERS outbreak period, pediatric ED visits due to non-urgent cases decreased significantly and there were more pronounced differences in ED utilization patterns in pediatric patients than in adult patients. Abstract Big infectious diseases do harm to the whole society, and it is highly crucial to control them on time. The major purpose of this article is to theoretically demonstrate that the Chinese government's intervention in large-scale infectious diseases is successful and efficient. Two potential strategies were considered: strategy 1 was infectious disease without government intervention, and strategy 2 was infectious disease with government intervention. By evolution model, this article illustrates the efficiency of big infectious disease reimbursement policy in China. Without government reimbursement, this article finds that high expenditures accelerate the disease infection. The number of infected persons decreases under big infectious disease reimbursement policy in China. The higher the treatment costs, the more important the government intervention. Big infectious disease reimbursement policy in China can serve as an efficient example to cope with big infectious diseases.Big infectious diseases do harm to the whole society, and it is highly crucial to control them on time. We have done some data collection and research on big infectious diseases from an economic angle.This research is to theoretically demonstrate that the Chinese government's intervention in large-scale infectious diseases is successful and efficient. And this article intends to draw on the traditional infectious disease model to analyze the Chinese government's compensation system for major diseases to reduce the harm of infectious diseases. What are your research's implications toward theory, practice, or policy? It is necessary to launch full reimbursement policy for infectious diseases under high expenditures incurred by treatment condition. The higher the treatment costs, the more important the government intervention. Abstract In mononegaviruses, cap-MTase activities have been involved in the 2'O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2'O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting internal adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2'O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original capindependent methylation opens new research opportunities to elucidate the role of RNA internal methylations in the viral replication. Abstract Evidence is accumulating that a distinct compartment(s) exists in the secretory pathway interposed between the rough ER (RER) and the Golgi stack. In this study we have defined a novel post-RER, pre-Golgi compartment where unassembled subunits of rubella virus (RV) E1 glycoprotein accumulate. When RV E1 is expressed in CHO cells in the absence of E2 glycoprotein, transport of E1 to the Golgi complex is arrested. The compartment in which E1 accumulates consists of a tubular network of smooth membranes which is in continuity with the RER but has distinctive properties from either the RER, Golgi, or previously characterized intermediate compartments. It lacks RER and Golgi membrane proteins and is not disrupted by agents which disrupt either the RER (thap-sigargin, ionomycin) or Golgi (nocodazole and brefeldin A). However, luminal ER proteins bearing the KDEL signal have access to this compartment. Kinetically the site of E1 arrest lies distal to or at the site where palmitylation occurs and proximal to the low temperature 15~ block. Taken together the findings suggest that the site of E1 arrest corresponds to, or is located close to the exit site from the ER. This compartment could be identified morphologically because it is highly amplified in cells overexpressing unassembled E1 subunits, but it may have its counterpart among the transitional elements of non-transfected cells. We conclude that the site of E1 arrest may represent a new compartment or a differentiated proximal moiety of the intermediate compartment. Abstract Background. The high burden of respiratory syncytial virus (RSV)-associated morbidity and mortality makes vaccine development a priority.Methods. As part of an efficacy trial of pandemic influenza vaccines (NCT01051661), RSV epidemiology in healthy children aged 6 months to <10 years at first vaccination with influenza-like illness (ILI) was evaluated in Australia, Brazil, Colombia, Costa Rica, Mexico, the Philippines, Singapore, and Thailand between February 2010 and August 2011. Active surveillance for ILI was conducted for approximately 1 year, with nasal and throat swabs analyzed by polymerase chain reaction. The prevalence and incidence of RSV among ILI episodes were calculated.Results. A total of 6266 children were included, of whom 2421 experienced 3717 ILI episodes with a respiratory sample available. RSV was detected for 359 ILI episodes, a prevalence of 9.7% (95% confidence interval: 8.7-10.7). The highest prevalence was in children aged 12-23 or 24-35 months in all countries except the Philippines, where it was in children aged 6-11 months. The incidence of RSV-associated ILI was 7.0 (6.3-7.7) per 100 person-years (PY). Eighty-eight ILI episodes resulted in hospitalization, of which 8 were associated with RSV ( prevalence 9.1% [4.0-17.1]; incidence 0.2 [0.1-0.3] per 100 PY). The incidence of RSV-associated ILI resulting in medical attendance was 6.0 (5.4-6.7) per 100 PY. RSV B subtypes were observed more frequently than A subtypes.Conclusions. Active surveillance demonstrated the considerable burden of RSV-associated illness that would not be identified through hospital-based surveillance, with a substantial part of the burden occurring in older infants and children. Abstract Introduction: Even in developed economies infectious diseases remain the most common cause of illness in early childhood. Our current understanding of the epidemiology of these infections is limited by reliance on data from decades ago performed using low-sensitivity laboratory methods, and recent studies reporting severe, hospital-managed disease.in Childhood Infectious Diseases (ORChID) study is an ongoing study enrolling a dynamic birth cohort to document the community-based epidemiology of viral respiratory and gastrointestinal infections in early childhood. Women are recruited antenatally, and their healthy newborn is followed for the first 2 years of life. Parents keep a daily symptom diary for the study child, collect a weekly anterior nose swab and dirty nappy swab and complete a burden diary when a child meets pre-defined illness criteria. Specimens will be tested for a wide range of viruses by real-time PCR assays. Primary analyses involves calculating incidence rates for acute respiratory illness (ARI) and acute gastroenteritis (AGE) for the cohort by age and seasonality. Control material from children when they are without symptoms will allow us to determine what proportion of ARIs and AGE can be attributed to specific pathogens. Secondary analyses will assess the incidence and shedding duration of specific respiratory and gastrointestinal pathogens. Abstract Porcine epidemic diarrhea (PED) is a highly contagious enteric swine disease. The large economic impact of PED on the swine industry worldwide has made the development of an effective PED vaccine a necessity. S0, a truncated region of the porcine epidemic diarrhea virus (PEDV) spike protein, has been suggested as a candidate antigen for PED subunit vaccines; however, poor solubility problems when the protein is expressed in Escherichia coli, and the inherent problems of subunit vaccines, such as low immunogenicity, remain. Flagellin has been widely used as a fusion partner to enhance the immunogenicity and solubility of many difficult-to-express proteins; however, the conjugation effect of flagellin varies depending on the target antigen or the position of the fusion placement. Here, we conjugated flagellin, Vibrio vulnificus FlaB, to the N-and C-termini of S0 and evaluated the ability of the fusion to enhance the solubility and immunogenicity of S0. Flagellin conjugation in the presence of the trigger factor chaperone tig greatly improved the solubility of the fusion protein (up to 99%) regardless of its conjugation position. Of importance, flagellin conjugated to the N-terminus of S0 significantly enhanced S0-specific humoral immune responses compared to other recombinant antigens in Balb/c mice. The mechanism of this phenomenon was investigated through in vitro and in vivo studies. These findings provide important information for the development of a novel PED vaccine and flagellinbased immunotherapeutics. Abstract In vitro, nitric oxide (NO) has been shown to have antimicrobial activity against a wide range of viruses, including influenza A virus. Therefore, we hypothesized that inhaled nitric oxide (iNO) would increase survival in vivo by reducing the viral load in C57Bl/6 mice infected with a lethal dose of influenza A/WSN/33 (H1N1; WSN/33) virus. NO was delivered to influenza-infected mice either continuously or intermittently at 80 or 160 ppm, respectively, using both prophylactic and post-infection treatment strategies. Murine survival and weight loss were assessed, and lung viral load was quantified via plaque assay. Here, we report that iNO administered prophylactically or post-influenza infection failed to improve survival of infected mice. No difference in lung viral load was observed between experimental groups. Although NO has antiviral activity against influenza A virus in vitro, iNO therapy provided no apparent benefit when used for treatment of influenza A virus infection in vivo. Abstract In theoretical physics, there exist two basic mathematical approaches, algebraic and geometrical methods, which, in most cases, are complementary. In the area of genome sequence analysis, however, algebraic approaches have been widely used, while geometrical approaches have been less explored for a long time. The Z-curve theory is a geometrical approach to genome analysis. The Z-curve is a three-dimensional curve that represents a given DNA sequence in the sense that each can be uniquely reconstructed given the other. The Z-curve, therefore, contains all the information that the corresponding DNA sequence carries. The analysis of a DNA sequence can then be performed through studying the corresponding Z-curve. The Z-curve method has found applications in a wide range of areas in the past two decades, including the identifications of protein-coding genes, replication origins, horizontally-transferred genomic islands, promoters, translational start sides and isochores, as well as studies on phylogenetics, genome visualization and comparative genomics. Here, we review the progress of Z-curve studies from aspects of both theory and applications in genome analysis. Abstract Since the mid-19 th century, human activities have increased greenhouse gases such as carbon dioxide, methane, and nitrous oxide in the Earth's atmosphere that resulted in increased average temperature. The effects of rising temperature include soil degradation, loss of productivity of agricultural land, desertification, loss of biodiversity, degradation of ecosystems, reduced fresh-water resources, acidification of the oceans, and the disruption and depletion of stratospheric ozone. All these have an impact on human health, causing non-communicable diseases such as injuries during natural disasters, malnutrition during famine, and increased mortality during heat waves due to complications in chronically ill patients. Direct exposure to natural disasters has also an impact on mental health and, although too complex to be quantified, a link has even been established between climate and civil violence.Over time, climate change can reduce agricultural resources through reduced availability of water, alterations and shrinking arable land, increased pollution, accumulation of toxic substances in the food chain, and creation of habitats suitable to the transmission of human and animal pathogens. People living in low-income countries are particularly vulnerable.Climate change scenarios include a change in distribution of infectious diseases with warming and changes in outbreaks associated with weather extreme events. After floods, increased cases of leptospirosis, campylobacter infections and cryptosporidiosis are reported. Global warming affects water heating, rising the transmission of water-borne pathogens. Pathogens transmitted by vectors are particularly sensitive to climate change because they spend a good part of their life cycle in a cold-blooded host invertebrate whose temperature is similar to the environment. A warmer climate presents more favorable conditions for the survival and the completion of the life cycle of the vector, going as far as to speed it up as in the case of mosquitoes. Diseases transmitted by mosquitoes include some of the most widespread worldwide illnesses such as malaria and viral diseases. Tick-borne diseases have increased in the past years in cold regions, because rising temperatures accelerate the cycle of development, the production of eggs, and the density and distribution of the tick population. The areas of presence of ticks and diseases that they can transmit have increased, both in terms of geographical extension than in altitude. In the next years the engagement of the health sector would be working to develop prevention and adaptation programs in order to reduce the costs and burden of climate change. Abstract Citation Lipkin WI. 2015. Middle East respiratory syndrome coronavirus recombination and the evolution of science and public health in China. mBio 6(5):e01381-15. Abstract Graphical Abstract Highlights d Binding to all influenza A subtypes neutralizing seasonal and pandemic strains d Utilizes a rare VH (VH6-1) and carries a low level of somatic mutations d Highly conserved epitope encompassing fusion peptide and hydrophobic groove d Superior therapeutic window compared to oseltamivir in animals Correspondence zhuq@medimmune.com (Q.Z.), john.skehel@crick.ac.uk (J.J.S.) In Brief Identification of a human monoclonal antibody that reacts effectively with all influenza A hemagglutinin subtypes paves the way for developing immunotherapy for people infected with the flu virus. Accession Numbers 5JW5 5JW4 5JW3 KX398429 KX398468 SUMMARYInfluenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans. Abstract Inflammatory responses contribute to the morbidity and mortality of severe influenza. Current antiviral therapy offers limited success in treating severe influenza infection with both H1N1 and H5N1 viruses. We evaluated the effect of a neuraminidase inhibitor in combination with immunomodulatory drugs in vitro and in a mouse model of influenza A H1N1 infection by determining survival rate, lung inflammation markers and histopathology. Sertraline and rolipram significantly improved survival in mice infected with a lethal dose of influenza A H1N1 virus. Prophylactic treatment resulted in survival rates of 40% (rolipram), 30% (oseltamivir), 0% (sertraline), 100% (rolipram/oseltamivir) and 70% (sertraline/oseltamivir). Treatment in a therapeutic setting (24 h post-infection) resulted in 80% (rolipram/oseltamivir) and 40% (sertraline/oseltamivir) survival. Sertraline and rolipram had no effect on virus replication in vitro and in vivo, but significantly reduced lung inflammation. A significant reduction in cellular infiltration (10-fold) along with inflammatory cytokines monocyte chemotactic protein-1 (10-fold), interleukin-6 (5-fold) and regulated on activation normal T cell expressed and secreted (5-fold) was observed in the animals treated with the combination compared to oseltamivir alone. Lung histopathology of mice treated with combinations revealed significantly reduced consolidation, infiltration and alveolitis compared to oseltamivir alone. Rolipram and sertraline reduced H1N1 virus-induced lung inflammation and mortality. These data support further development of immunomodulatory agents for severe influenza. Abstract Epidemiological determinants of successful vaccine development were explored using measurable biological variables including antigenic stability and requirement of T-cell immunity. Employing a logistic regression model, we demonstrate that a high affinity with blood and immune cells and pathogen interactions (e.g. interference) would be the risk factors of failure for vaccine development. Abstract Nipah virus and Hendra virus are emerging, highly pathogenic, zoonotic paramyxoviruses that belong to the genus Henipavirus. They infect humans as well as numerous mammalian species. Both viruses use ephrin-B2 and -B3 as cell entry receptors, and following initial entry into an organism, they are capable of rapid spread throughout the host. We have previously reported that Nipah virus can use another attachment receptor, different from its entry receptors, to bind to nonpermissive circulating leukocytes, thereby promoting viral dissemination within the host. Here, this attachment molecule was identified as heparan sulfate for both Nipah virus and Hendra virus. Cells devoid of heparan sulfate were not able to mediate henipavirus trans-infection and showed reduced permissivity to infection. Virus pseudotyped with Nipah virus glycoproteins bound heparan sulfate and heparin but no other glycosaminoglycans in a surface plasmon resonance assay. Furthermore, heparin was able to inhibit the interaction of the viruses with the heparan sulfate and to block cell-mediated trans-infection of henipaviruses. Moreover, heparin was shown to bind to ephrin-B3 and to restrain infection of permissive cells in vitro. Consequently, treatment with heparin devoid of anticoagulant activity improved the survival of Nipah virus-infected hamsters. Altogether, these results reveal heparan sulfate as a new attachment receptor for henipaviruses and as a potential therapeutic target for the development of novel approaches against these highly lethal infections.and open novel perspectives for the development of heparan sulfate-targeting therapeutic approaches for these emerging infections. Abstract Human adenovirus 52 (HAdV-52) is one of only three known HAdVs equipped with both a long and a short fiber protein. While the long fiber binds to the coxsackie and adenovirus receptor, the function of the short fiber in the virus life cycle is poorly understood. Here, we show, by glycan microarray analysis and cellular studies, that the short fiber knob (SFK) of HAdV-52 recognizes long chains of α-2,8-linked polysialic acid (polySia), a large posttranslational modification of selected carrier proteins, and that HAdV-52 can use polySia as a receptor on target cells. X-ray crystallography, NMR, molecular dynamics simulation, and structure-guided mutagenesis of the SFK reveal that the nonreducing, terminal sialic acid of polySia engages the protein with direct contacts, and that specificity for polySia is achieved through subtle, transient electrostatic interactions with additional sialic acid residues. In this study, we present a previously unrecognized role for polySia as a cellular receptor for a human viral pathogen. Our detailed analysis of the determinants of specificity for this interaction has general implications for proteincarbohydrate interactions, particularly concerning highly charged glycan structures, and provides interesting dimensions on the biology and evolution of members of Human mastadenovirus G.human adenovirus | short fiber | polysialic acid | glycan receptor | glycan microarray Abstract Activation of a galactosidase-specific murine T hybridoma clone and of a human tetanus toxoid-specific T clone by antigen-presenting cells (APC) was used to evaluate the regulatory function ofantibodies complexed with the relevant antigen . Complexed antigen, in fact, is taken up with high efficiency thanks to Fc receptors borne by APC . Antibody/antigen ratio in the complexes proved to be a critical parameter in enhancing antigen presentation . Complexes in moderate antibody excess provided optimal T cell activation independently of the physical state of the complexes {precipitated by a second antibody or solubilized by complement) . Complexes in extreme antibody excess, on the contrary, did not yield T cell activation although taken up by APC efficiently. The effect of antibodies at extreme excess was observed with substimulatory dose of antigen (loss of potentiation) and with optimal dose of antigen (loss of stimulation). An excess of specific polyclonal antibodies hampers proteolytic degradation of antigen in vitro, supporting the view that a similar mechanism may operate within the APC that have internalized immune complexes in extreme antibody excess. The possibility that immune complex forming in extreme antibody excess may turn off the T cell response is proposed as a regulatory mechanism . Abstract A severe respiratory ongoing outbreak of pneumonia associated with 2019 novel coronavirus was recently emerged in China. Here, we reported the epidemiological, clinical, laboratory and radiological characteristics of 19 suspect cases. We compared J o u r n a l P r e -p r o o f Abstract PLATES 45 ~o 50 Abstract This study was aiming to investigate the knowledge, practice and attitudes of secondary school and university students toward MERS-CoV infection. This is a cross-sectional study conducted in Riyadh, Saudi Arabia. Study participants were recruited from several constituent colleges of King Saud University and secondary schools in Riyadh. Data were collected using selfadministered, closed-ended questionnaires. Frequencies and proportions were computed for descriptive purposes. Chi square test was utilized to depict statistical difference between groups. Among the 1109 students who answered the questionnaires, 53.1% were male, and 46.9% were female. Level of knowledge about clinical presentation of MERS is generally similar among university and school students. The most frequently reported source of transmission is entering crowded spaces and being exposed to coughing and sneezing. Additionally, hand washing was the most commonly reported method of protection against the infection. The localized spread of MERS in Saudi Arabia and the number of fatalities associated with it might have increased public interest in understanding how to maintain proper precautionary measures both on a community and on an Abstract well as in important, prespecified subgroups. ALIC 4 E is likely therefore to enhance the evidence base supporting and important and common area of clinical practice. Abstract Macrophage polarization is a process by which macrophages acquire a distinct phenotypic and functional profile in response to microenvironmental signals. The classically and alternatively activated (M1 and M2, respectively) macrophage phenotypes are defined by the specific molecular characteristics induced in response to prototypic pro-and anti-inflammatory cues. In this study, we used LPS/IFN-γ and IL-4 to stimulate porcine alveolar macrophages (PAMs) in vitro and investigated the expression changes of several novel markers during macrophage polarization. Notably, we found that LPS/IFN-γ-stimulated PAMs express prototypical M1 molecules, whereas IL-4-stimulated PAMs express M2 molecules. We also demonstrated that replication of the highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) strain HuN4 was effectively suppressed in LPS/IFN-γ-stimulated M1 PAMs (M1 type), but not IL-4 stimulated M2 PAMs. However, this was not observed with the classic, less pathogenic CH-1a strain. Moreover, we found that M2 marker expression gradually increased after PAM infection with PRRSV, whereas no significant changes were found with M1 marker expression, suggesting that PRRSV infection may skew macrophage polarization towards an M2 phenotype. Finally, we found that anti-viral cytokine expression was significantly higher in M1 macrophages than in M2 macrophages or nonpolarized controls. In summary, our results show that PRRSV replication was significantly impaired in M1 PAMs, which may serve as a foundation for further understanding of the dynamic phenotypic changes during macrophage polarization and their effects on viral infection.KEY WORDS: M1/M2 polarization, porcine alveolar macrophages, porcine reproductive and respiratory syndrome virus, viral replication Abstract Ribosome-mediated transcription attenuation is a basic posttranscriptional regulation mechanism in bacteria. Liberated attenuator RNAs arising in this process are generally considered nonfunctional. In Sinorhizobium meliloti, the tryptophan (Trp) biosynthesis genes are organized into three operons, trpE(G), ppiD-trpDC-moaC-moeA, and trpFBA-accD-folC, of which only the first one, trpE(G), contains a short ORF (trpL) in the 5 -UTR and is regulated by transcription attenuation. Under conditions of Trp sufficiency, transcription is terminated between trpL and trpE(G), and a small attenuator RNA, rnTrpL, is produced. Here, we show that rnTrpL base-pairs with trpD and destabilizes the polycistronic trpDC mRNA, indicating rnTrpL-mediated downregulation of the trpDC operon in trans. Although all three trp operons are regulated in response to Trp availability, only in the two operons trpE(G) and trpDC the Trp-mediated regulation is controlled by rnTrpL. Together, our data show that the trp attenuator coordinates trpE(G) and trpDC expression posttranscriptionally by two fundamentally different mechanisms: ribosome-mediated transcription attenuation in cis and base-pairing in trans. Also, we present evidence that rnTrpL-mediated regulation of trpDC genes expression in trans is conserved in Agrobacterium and Bradyrhizobium, suggesting that the small attenuator RNAs may have additional conserved functions in the control of bacterial gene expression. Abstract The distribution of secretory leukocyte protease inhibitor (SLPI) at entry portals indicates its involvement in defending the host from pathogens, consistent with the ability of SLPI to inhibit human immunodeficiency virus (HIV)-1 infection by an unknown mechanism. We now demonstrate that SLPI binds to the membrane of human macrophages through the phospholipidbinding protein, annexin II. Based on the recent identification of human cell membrane phosphatidylserine (PS) in the outer coat of HIV-1, we define a novel role for annexin II, a PS-binding moiety, as a cellular cofactor supporting macrophage HIV-1 infection. Moreover, this HIV-1 PS interaction with annexin II can be disrupted by SLPI or other annexin II-specific inhibitors. The PS-annexin II connection may represent a new target to prevent HIV-1 infection.Abbreviations used in this paper: LC-MS/MS, nano-capillary HPLC ion trap mass spectrometry; PS, phosphatidylserine; rhSLPI, recombinant human secretory leukocyte protease inhibitor; RNAi, RNA-mediated interference; siRNA, small interfering RNA; SLPI, secretory leukocyte protease inhibitor. Abstract The world has experienced an increased incidence and transboundary spread of emerging infectious diseases over the last four decades. We divided emerging infectious diseases into four categories, with subcategories in categories 1 and 4. The categorization was based on the nature and characteristics of pathogens or infectious agents causing the emerging infections, which are directly related to the mechanisms and patterns of infectious disease emergence. The factors or combinations of factors contributing to the emergence of these pathogens vary within each category. We also classified public health laboratories into three types based on function, namely, research, reference and analytical diagnostic laboratories, with the last category being subclassified into primary (community-based) public health and clinical (medical) analytical diagnostic laboratories. The frontline/leading and/or supportive roles to be adopted by each type of public health laboratory for optimal performance to establish the correct etiological agents causing the diseases or outbreaks vary with respect to each category of emerging infectious diseases. We emphasize the need, especially for an outbreak investigation, to establish a harmonized and coordinated national public health laboratory system that integrates different categories of public health laboratories within a country and that is closely linked to the national public health delivery system and regional and international high-end laboratories. Abstract Arginase I (Arg I) and inducible nitric oxide synthase (iNOS) are important in regulating immune functions through their metabolites. Previous studies have revealed that the expression of Arg I is increased and the expression of iNOS is reduced in the serum and peripheral blood mononuclear cells of human immunodeficiency virus (HIV)-infected patients. As one of the most important immune organs and HIV replication sites, whether similar changes are present in the lymph nodes following HIV infection remains to be elucidated. To investigate this, the present study collected lymph node and blood specimens from 52 HIV-infected patients to measure the expression levels of Arg I and iNOS by immunohistochemistry and fluoresence-based flow cytometry. Compared with control subjects without HIV infection, the patients with HIV had significantly higher expression levels of Arg I in the lymph nodes and higher frequencies of Arg I + CD4 + T cells and CD8 + T cells in the blood and lymph nodes, and these results were contrary the those of iNOS in the corresponding compartments. The expression levels of Arg I in the lymph nodes and blood were negatively associated with peripheral CD4 + T cell count and positively associated with viral load. However, the expression levels of iNOS in the lymph nodes and blood were positively associated with peripheral CD4 + T cell count and negatively associated with viral load. These results showed that alterations in the expression levels of Arg I and iNOS in the peripheral T cells and peripheral nodes of HIV infected patients are associated with disease progression in these patients. These results indicate a potential to therapeutic strategy for delaying disease progression through regulating and manipulating the expression levels of Arg I and iNOS in patients infected with HIV. Abstract Nearly all licensed vaccines confer protection against infectious diseases by stimulating the production of pathogen-specific Abs by B cells. Understanding the nature of a successful Ab response is therefore a fundamental step to providing new tools for the design of novel or better vaccines. The isolation and characterization of the Ab repertoire produced by antigen-specific B cells has acquired a central importance in the last decade to unravel the response to vaccine antigens.Dissecting the basic mechanisms that define the dynamics of the Ab responses to vaccination and deepening the Traditionally, vaccines have been developed by cultivating infectious agents and isolating the inactivated whole pathogen or some of its purified components. 20 years ago, reverse vaccinology enabled vaccine discovery and design based on information deriving from the sequence of microbial genomes rather than via the growth of pathogens. Today, the high throughput discovery of protective human antibodies, sequencing of the B cell repertoire, and the increasing structural characterization of protective antigens and epitopes provide the molecular and mechanistic understanding to drive the discovery of novel vaccines that were previously impossible. We are entering a "reverse vaccinology 2.0" era. Abstract The novel coronavirus pneumonia (COVID-19) epidemic has brought serious social psychological impact to the Chinese people, especially those quarantined and thus with limited access to face-to-face communication and traditional social psychological interventions. To better deal with the urgent psychological problems of people involved in the COVID-19 epidemic, we developed a new psychological crisis intervention model by utilizing internet technology. This new model, one of West China Hospital, integrates physicians, psychiatrists, psychologists and social workers into Internet platforms to carry out psychological intervention to patients, their families and medical staff. We hope this model will make a sound basis for developing a more comprehensive psychological crisis intervention response system that is applicable for urgent social and psychological problems. Abstract Life-threatening pulmonary influenza can be caused by inborn errors of type I and III IFN immunity. We report a 5-yr-old child with severe pulmonary influenza at 2 yr. She is homozygous for a loss-of-function IRF9 allele. Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/ IRF9) in response to IFN-α2b. The transcriptome induced by IFN-α2b in the patient's cells is much narrower than that of control cells; however, induction of a subset of IFN-stimulated gene transcripts remains detectable. In vitro, the patient's cells do not control three respiratory viruses, influenza A virus (IAV), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). These phenotypes are rescued by wild-type IRF9, whereas silencing IRF9 expression in control cells increases viral replication. However, the child has controlled various common viruses in vivo, including respiratory viruses other than IAV. Our findings show that human IRF9-and ISGF3-dependent type I and III IFN responsive pathways are essential for controlling IAV. Abstract In the history of vaccine development, the synthetic vaccine is a milestone that is in stark contrast with traditional vaccines based on live-attenuated or inactivated microorganisms. Synthetic vaccines not only are safer than attenuated or inactivated microorganisms but also provide the opportunity for vaccine design for specific purposes. The first generation of synthetic vaccines has been largely based on DNA recombination technology and genetic manipulation. This de novo generation is occasionally time consuming and costly, especially in the era of genomics and when facing pandemic outbreaks of infectious diseases. To accelerate and simplify the R&D process for vaccines, we developed an improved method of synthetic vaccine construction based on protein assembly. We optimized and employed the recently developed SpyTag/ SpyCatcher technique to establish a protein assembly system for vaccine generation from pre-prepared subunit proteins. As proof of principle, we chose a dendritic cell (DC)-targeting molecule and specific model antigens to generate desired vaccines. The results demonstrated that a new vaccine generated in this way does not hamper the individual function of different vaccine components and is efficient in inducing both T and B cell responses. This protein assembly strategy may be especially useful for high-throughput antigen screening or rapid vaccine generation.S ince the creation of the first vaccine, for cowpox, by Edward Jenner in the late eighteenth century 1 , immunological research on vaccines has focused on deconstruction analysis, or evaluation of the importance and mechanisms of each component of a vaccine that may determine its effect. This research strategy has led to the discovery of a large, increasing number of functional elements of different categories including antigens, immune modulators and adjuvants, and delivery systems, among others 2 . A successful vaccine is usually composed of multiple elements, such as those listed above. Given the multitude of choices, the construction of different elements into an integrated, functional whole has become a new challenge in the field. Although genebased synthetic and recombinant DNA technologies provide great flexibility for construction, certain limitations still exist: (1) large fusion proteins containing multiple functional elements are occasionally technically difficult to express or purify, and (2) de novo generation is usually a tedious and long process that is especially inadequate in the face of emergent pandemics of infectious diseases, when screening and identification of antigens are crucial for vaccine development 3,4 . Facing such difficulties and demands, instead of making complex fusion-protein candidate vaccines de novo every time, it would be easier, faster, more flexible and more efficient to prepare the smaller building blocks first and then to assemble them into a whole, as needed.To achieve this goal, in the present study, we have developed a new method for synthetic vaccine construction based on a novel protein-protein conjugation technique. The SpyTag/SpyCatcher conjugation technique was recently developed based on the split protein CnaB2 from Streptococcus pyogenes 5-7 . This protein contains two fragments: one named SpyTag (13aa), and the other named SpyCatcher (138aa). Once combined under nearly any common conditions, SpyTag and SpyCatcher can rapidly and efficiently covalently conjugate to each other through an isopeptide bond 5 . We hypothesized that this conjugation technique could allow us to achieve our goal and assemble vaccines based on different pre-prepared functional components.Dendritic cell (DC) targeting has emerged as an important strategy for vaccine development due to the increasing recognition of this small population of cells in both cellular and humoral immune responses 8-13 . DEC205 is a C-type lectin endocytic receptor that is highly expressed on CD8a 1 DCs in mice 14 and on CD141 1 DCs in humans 15 . An antibody against DEC205 has been developed as a useful targeted delivery molecule. When conjugated to this antibody, an antigen can be efficiently delivered to DCs, an approach that has been found to be superior in mediating both cytotoxic T cell responses [16] [17] [18] and antibody responses 19,20 . Abstract Background: To compare the differences between elderly and non-elderly patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) due to viral infections.Methods: Patients with chronic obstructive pulmonary disease (COPD) exacerbation were recruited and classified as elderly (>65 years) and non-elderly (≤ 65 years). Sputum and oropharyngeal samples were assessed, PCR for respiratory viruses and cultures for common pathogens were performed.Results: 247 patients (median age: 69.3±9.5 years) were recruited and categorized into group A: non-elderly patients [n=81 (32.8%), median age 58±5.99] and group B: elderly patients [n=166 (67.2%), median age 74.8±4.8] years. In 133 (53.8%) patients a viral infection was identified and in 34 (13.8%) a bacterial pathogen was isolated from cultures. In 18 (7.3%) patients a double infection (bacterial+viral) was identified. In group B, the presence of cardiac failure (46.6% vs 28.3%, p<0.001), renal failure (10.5% vs 4%, p=0.03), bacterial co-infection (13.8% vs 7.4%, p=0.04), influenza vaccination rates (45.5% vs 215, p<0.001), and longer hospital stay (8.4±4.4 vs 7.5±3.2 days, p=0.02) were higher than group A. The overall rate of viral infections did not differ according to age. A trend to higher rates of infection with parainfluenza 3 [19 (20%) patients in group B vs3 (7.5%) patients in group A, p=0.04] was observed in older patients.No differences on the rate and type of viral infections were noted for elderly vs non elderly patients. However, they tended to have more bacterial co-infections that led to AECOPD and longer hospitalization stays compared to non-elderly patients. Abstract Feline bocavirus (FBoV) is a newly identified bocavirus of cats in the family Parvoviridae. A novel FBoV HRB2015-LDF was first identified from the cat with severe enteritis in Northeast China, with an overall positive rate of 2.78% (1/36). Phylogenetic and homologous analysis of the complete genome showed that FBoV HRB2015-LDF was clustered into the FBoV branch and closely related to other FBoVs, with 68.7-97.5% identities. And the genes of VP1, NPA and NS1 shared 70.7-97.6, 72.4-98.6 and 67.2-98.0% nucleotide identities with other FBoVs, respectively. The results suggested that the FBoVs could be divided into two distinct lineages, and the difference of nucleotide identities was >20-30% between the lineages. Abstract Objectives The aim of this study was to determine the safety and efficacy of the nucleoside analog GS-441524 for cats suffering from various forms of naturally acquired feline infectious peritonitis (FIP). Methods Cats ranged from 3.4-73 months of age (mean 13.6 months); 26 had effusive or dry-to-effusive FIP and five had non-effusive disease. Cats with severe neurological and ocular FIP were not recruited. The group was started on GS-441524 at a dosage of 2.0 mg/kg SC q24h for at least 12 weeks and increased when indicated to 4.0 mg/kg SC q24h. Results Four of the 31 cats that presented with severe disease died or were euthanized within 2-5 days and a fifth cat after 26 days. The 26 remaining cats completed the planned 12 weeks or more of treatment. Eighteen of these 26 cats remain healthy at the time of publication (OnlineFirst, February 2019) after one round of treatment, while eight others suffered disease relapses within 3-84 days. Six of the relapses were non-neurological and two neurological. Three of the eight relapsing cats were treated again at the same dosage, while five cats had the dosage increased from 2.0 to 4.0 mg/kg q24h. The five cats treated a second time at the higher dosage, including one with neurological disease, responded well and also remain healthy at the time of publication. However, one of the three cats re-treated at the original lower dosage relapsed with neurological disease and was euthanized, while the two remaining cats responded favorably but relapsed a second time. These two cats were successfully treated a third time at the higher dosage, producing 25 long-time survivors. One of the 25 successfully treated cats was subsequently euthanized due to presumably unrelated heart disease, while 24 remain healthy. Conclusions and relevance GS-441524 was shown to be a safe and effective treatment for FIP. The optimum dosage was found to be 4.0 mg/kg SC q24h for at least 12 weeks. Abstract a b s t r a c tRenin angiotensin system (RAS) is an endocrine system widely known for its physiological roles in electrolyte homeostasis, body fluid volume regulation and cardiovascular control in peripheral circulation. However, brain RAS is an independent form of RAS expressed locally in the brain, which is known to be involved in brain functions and disorders. There is strong evidence for a major involvement of excessive brain angiotensin converting enzyme (ACE)/Angiotensin II (Ang II)/Angiotensin type-1 receptor (AT-1R) axis in increased activation of oxidative stress, apoptosis and neuroinflammation causing neurodegeneration in several brain disorders. Numerous studies have demonstrated strong neuroprotective effects by blocking AT1R in these brain disorders. Additionally, the angiotensin converting enzyme 2 (ACE2)/Angiotensin (1-7)/Mas receptor (MASR), is another axis of brain RAS which counteracts the damaging effects of ACE/Ang II/AT1R axis on neurons in the brain. Thus, angiotensin II receptor blockers (ARBs) and activation of ACE2/Angiotensin (1-7)/MASR axis may serve as an exciting and novel method for neuroprotection in several neurodegenerative diseases. Here in this review article, we discuss the expression of RAS in the brain and highlight how altered RAS level may cause neurodegeneration. Understanding the pathophysiology of RAS and their links to neurodegeneration has enormous potential to identify potentially effective pharmacological tools to treat neurodegenerative diseases in the brain. Abstract The PLEX-ID/Flu assay has been recently developed to enable the detection and typing of influenza viruses based on the RT-PCR/electrospray ionization mass spectrometry technology.This novel assay was evaluated for typing performance on 201 positive influenza A or B nasopharyngeal swab specimens (NPS) detected by real-time RT-PCR during the 2010-2011 season. The PLEX-ID/Flu assay detected and characterized 91.3% and 95.3% of all influenza A and B samples, respectively. All non-typeable influenza A and B specimens by the assay showed low viral loads with threshold cycle values 33. Taken together, and although our results need to be confirmed by further prospective studies, the PLEX-ID/Flu assay detected positively and gave a typing result for 93% of all NPS detected positively by real-time RT-PCR, thus suggesting a potential role for influenza virus surveillance among other techniques. Abstract Objective: We explored the prescribing patterns of physicians in North Trinidad in treating upper respiratory tract infections (URTI) in paediatric patients and the appropriateness of drugs prescribed. Methods: A retrospective observational study was conducted, with a sample size of 523 paediatric patients, diagnosed with an URTI during the period of June 2003 to 22 June 2005. The study was conducted at five Primary Health Care Facilities in North Trinidad. Results: The three most frequent URTIs diagnosed were non-specific URTI, common cold, and acute tonsillitis in rank order. Four patterns of prescribing were identified, (1) no drug therapy [1.9%]; (2) antibiotic therapy alone [6.1%]; (3) antibiotic and symptomatic therapy [53.0%]; and (4) symptomatic therapy alone [39.0%]. The, most frequently prescribed antibiotics were penicillins (amoxicillin [46.3%] and amoxicillin/clavulanate [5.3%]) and a macrolide (erythromycin [6.1%]). The three symptomatic agents most frequently prescribed were paracetamol [40.1%]; diphenhydramine [29.1%]; and normal saline nasal drops [14.2%]. In 112 cases with swab analyses done, of these, 98.2% revealed a growth of commensals only, while 1.8% grew pathogenic micro-organisms. Of the cases showing commensal growth only, 84.6%were treated with an antibiotic, 14.5% were treated with symptomatic agents alone and 0.9% received no drug therapy at all. Conclusions: A large proportion of paediatric patients diagnosed with an URTI in North Trinidad was prescribed antibiotics although not indicated The inappropriate use of antibiotics can potentiate the worldwide trend of antimicrobial resistance. Abstract Cell death plays a crucial role for a myriad of physiological processes, and several human diseases such as cancer are characterized by its deregulation. There are many methods available for both quantifying and qualifying the accurate process of cell death which occurs. Choosing the right assay tool is essential to generate meaningful data, provide sufficient information for clinical applications, and understand cell death processes. In vitro cell death assays are important steps in the search for new therapies against cancer as the ultimate goal remains the elaboration of drugs that interfere with specific cell death mechanisms. However, choosing a cell viability or cytotoxicity assay among the many available options is a daunting task. Indeed, cell death can be approached by several viewpoints and require a more holistic approach. This review provides an overview of cell death assays usually used in vitro for assessing cell death so as to elaborate new potential chemotherapeutics and discusses considerations for using each assay. Abstract The pathogenesis of Japanese encephalitis virus (JEV) is complex and unclearly defined, and in particular, the effects of the JEV receptor (JEVR) on diverse susceptible cells are elusive. In contrast to previous studies investigating JEVR in rodent or mosquito cells, in this study, we used primate Vero cells instead. We noted that few novel proteins co-immunoprecipitated with JEV, and discovered that one of these was heat shock protein 90β (HSP90β), which was probed by mass spectrometry with the highest score of 60.3 after questing the monkey and human protein databases. The specific HSP90β-JEV binding was confirmed by western blot analysis under non-reducing conditions, and this was significantly inhibited by an anti-human HSP90β monoclonal antibody in a dose-dependent manner, as shown by immunofluorescence assay and flow cytometry. In addition, the results of confocal laser scanning microscopic examination demonstrated that the HSP90β-JEV binding occurred on the Vero cell surface. Finally, JEV progeny yields determined by plaque assay were also markedly decreased in siRNA-treated Abstract Case summary A 15-year-old male neutered domestic shorthair cat was presented with a recent history of seizures, diarrhoea, lethargy, fever and jaundice. Marked elevation of liver enzyme activity was present and ultrasound examination was suggestive of cholecystitis and hepatitis. Neutrophilic cholangitis was confirmed on histopathology of liver biopsies. Bile culture identified a monomicrobial infection with Providencia rettgeri, which was resistant to multiple antimicrobial agents. The cat was treated with oral pradofloxacin for 4 weeks and remained well 4 months later.Relevance and novel information Providencia species are rarely reported in the veterinary literature and are an uncommon cause of disease in humans. The significance of this species in humans relates to the high prevalence of antimicrobial resistance. This is the first report of P rettgeri causing clinical illness in a cat and highlights the importance of bile cultures in hepatic disease. Abstract The presence of canine parvovirus type 2 (CPV-2), 2a and 2b has been described in Brazil, however, the type 2c had not been reported until now. In the current study, seven out of nine samples from dogs with diarrhea were characterized as CPV-2c, indicating that this virus is already circulating in the Brazilian canine population.Key words: canine parvovirus type 2c, sequence analysis, Brazil.The canine parvovirus type 2 (CPV-2) emerged as a novel pathogen in the late 70`s (1) and rapidly spread worldwide. Within few years, the virus underwent a rapid evolution and, new antigenic types, termed CPV-2a and 17) , completely replaced the original type 2 (21, Abstract Viral gain-of-function mutations frequently evolve during laboratory experiments. Whether the specific mutations that evolve in the lab also evolve in nature and whether they have the same impact on evolution in the real world is unknown. We studied a model virus, bacteriophage λ, that repeatedly evolves to exploit a new host receptor under typical laboratory conditions. Here, we demonstrate that two residues of λ's J protein are required for the new function. In natural λ variants, these amino acid sites are highly diverse and evolve at high rates. Insertions and deletions at these locations are associated with phylogenetic patterns indicative of ecological diversification. Our results show that viral evolution in the laboratory mirrors that in nature and that laboratory experiments can be coupled with protein sequence analyses to identify the causes of viral evolution in the real world. Furthermore, our results provide evidence for widespread host-shift evolution in lambdoid viruses. K E Y W O R D S : Experimental evolution, gain-of-function, host shift, genomic epidemiology, natural variation, synthetic biology. Abstract A 3-month-old female Maltese puppy was hospitalized with persistent diarrhea in a local veterinary clinic. Blood chemistry and hematology profile were analyzed and fecal smear was examined. Diarrheal stools were examined in a diagnostic laboratory, using multiplex real-time polymerase chain reaction (PCR) against 23 diarrheal pathogens. Sequence analysis was performed using nested PCR amplicon of 18S ribosomal RNA. Coccidian oocysts were identified in the fecal smear. Although multiplex real-time PCR was positive for Cyclospora cayetanensis, the final diagnosis was Cystoisospora ohioensis infection, confirmed by phylogenetic analysis of 18S rRNA. To our knowledge, this the first case report of C. ohioensis in Korea, using microscopic examination and phylogenetic analysis. Abstract The sharp increase in antibiotic resistance imposes a global threat to human health and the discovery of effective antimicrobial alternatives is needed. The use of probiotics to combat bacterial pathogens has gained a rising interest. Pathogenic Escherichia coli is causative of multiple clinical syndromes such as diarrheal diseases, meningitis and urinary tract infections. In this work, we evaluated the efficacy of probiotics to control multidrug-resistant E. coli and reduce their ability to form biofilms. Six E. coli resistant to at least five antibiotics (Ceftazidime, Ampicillin, Clarithromycin, Amoxicillin + Clavulanic Acid and Ceftriaxone) were isolated in this work. Preparations of cell-free spent media (CFSM) of six probiotics belonging to the genus Bifidobacterium and Lactobacillus which were grown in Man-Rogosa-Sharpe (MRS) broth exhibited strong antibacterial activity (inhibition zones of 11.77-23.10 mm) against all E. coli isolates. Two E. coli isolates, namely E. coli WW1 and IC2, which were most resistant to all antibiotics were subjected to antibiofilm experiments. Interestingly, the CFSM of MRS fermented by all probiotics resulted in inhibition of biofilm formation while B. longum caused highest inhibition (57.94%) in case of E. coli IC2 biofilms and L. plantarum was responsible for 64.57% reduction of E. coli WW1 biofilms. On the other hand, CFSM of skim milk fermented by L. helveticus and L. rhamnosus exhibited a slight inhibitory activity against IC2 isolate (inhibition percentage of 31.52 and 17. 68, respectively) while WW1 isolate biofilms was reduced by CFSM of milk fermented by B. longum and L. helveticus (70.81 and 69.49 reduction percentage, respectively). These results support the effective use of probiotics as antimicrobial alternatives and to eradicate biofilms formed by multidrug-resistant E. coli. Abstract Objectives The objectives were to collect baseline data on the occurrence, testing and vaccination practices, and clinical outcomes of feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) in New Zealand Methods A cross-sectional survey of 423 veterinary practices in New Zealand was performed to collect data on FeLV and FIV testing and vaccination during the 2015 calendar year. Clinical records from 572 cats tested using a point-of-care ELISA at a first-opinion veterinary practice between 7 April 2010 and 23 June 2016 were also obtained and multivariable logistic regression models were constructed to identify risk factors for test positivity. Survival times were estimated using Kaplan-Meier methods. Results The survey was completed by 112 clinics (26.4%) of which 72 performed in-house testing. Of the 2125 tests performed, 56 (2.6%) were positive for FeLV and 393 (18.5%) were positive for FIV. Fewer than 1% of cats were vaccinated for FeLV, with veterinarians citing low perceived prevalence as the primary reason for not vaccinating. Being male compared with being female and having clinical evidence of immunosuppression were significant risk factors for both FeLV and FIV test positivity. The median survival times of FeLV and FIV test-positive cats were 10 days (95% confidence interval [CI] 0-16) and 650 days (95% CI 431-993), respectively. Conclusions and relevance Testing and vaccination for FeLV and FIV in New Zealand appears targeted towards high-risk animals, which may bias prevalence estimates. Baseline data should be monitored for changes in FeLV epidemiology now commercial vaccines are no longer available. Abstract Context: During the Ebola outbreak in West Africa in 2014-2015, close cooperation between the curative sector and the public health sector in the Netherlands was necessary for timely identification, referral, and investigation of patients with suspected Ebola virus disease (EVD). Objective: In this study, we evaluated experiences in preparedness among stakeholders of both curative and public health sectors to formulate recommendations for optimizing preparedness protocols. Timeliness of referred patients with suspected EVD was used as indicator for preparedness. Design: In focus group sessions and semistructured interviews, experiences of curative and public health stakeholders about the regional and national process of preparedness and response were listed. Timeliness recordings of all referred patients with suspected EVD (13) were collected from first date of illness until arrival in the referral academic hospital. Results: Ebola preparedness was considered extensive compared with the risk of an actual patient, however necessary. Regional coordination varied between regions. More standardization of regional preparation and operational guidelines was requested, as well as nationally standardized contingency criteria, and the National Centre for Infectious Disease Control was expected to coordinate the development of these guidelines. For the timeliness of referred patients with suspected EVD, the median delay between first date of illness until triage was 2.0 days (range: 0-10 days), and between triage and arrival in the referral hospital, it was 5.0 hours (range: 2-7.5 hours). In none of these patients Ebola infection was confirmed. Conclusions: Coordination between the public health sector and the curative sector needs improvement to reduce delay in patient management in emerging infectious diseases. Standardization of preparedness and response practices, through guidelines for institutional preparedness and blueprints for regional and national coordination, is necessary, as preparedness for emerging infectious diseases needs a multidisciplinary approach overarching both the public health sector and the curative sector. In the Netherlands a national platform for preparedness is established, in which both the curative sector and public health sector participate, in order to implement the outcomes of this study. Abstract Gram-negative pathogen-induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF), a traditional Chinese medicine (TCM) formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosainduced acute pneumonia in mice were evaluated. The mechanisms by which four typical antiinflammatory ingredients from QF, arctigenin (ATG), cholic acid (CLA), chlorogenic acid (CGA) and sinapic acid (SPA), regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6) and chemokines (IL-8 and RANTES), reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In ;6(3):212-221 addition, ATG was identified as the primary anti-inflammatory agent with action on the PI3K/AKT and Ras/MAPK pathways. CLA and CGA enhanced the actions of ATG and exhibited synergistic NF-κB inactivation effects possibly via the Ras/MAPK signaling pathway. Moreover, CLA is speculated to target FGFR and MEK firstly. Overall, QF regulated the PI3K/AKT and Ras/MAPK pathways to inhibit pathogenic bacterial infections effectively. Abstract Mathematical models provide a quantitative framework with which scientists can assess hypotheses on the potential underlying mechanisms that explain patterns in observed data at different spatial and temporal scales, generate estimates of key kinetic parameters, assess the impact of interventions, optimize the impact of control strategies, and generate forecasts. We review and illustrate a simple data assimilation framework for calibrating mathematical models based on ordinary differential equation models using time series data describing the temporal progression of case counts relating, for instance, to population growth or infectious disease transmission dynamics. In contrast to Bayesian estimation approaches that always raise the question of how to set priors for the parameters, this frequentist approach relies on modeling the error structure in the data. We discuss issues related to parameter identifiability, uncertainty quantification and propagation as well as model performance and forecasts along examples based on phenomenological and mechanistic models parameterized using simulated and real datasets. Abstract 191 SM Moghadas, M Haworth-Brockman, H Isfeld-Kiely, J Kettner. Improving public health policy through infection transmission modelling: Guidelines for creating a Community of Practice. Can J Infect Dis Med Microbiol 2015;26(4):191-195. BACKGROUND: Despite significant research efforts in Canada, real application of modelling in public health decision making and practicehas not yet met its full potential. There is still room to better address the diversity of the Canadian population and ensure that research outcomes are translated for use within their relevant contexts. OBJECTIVES: To strengthen connections to public health practice and to broaden its scope, the Pandemic Influenza Outbreak Research Modelling team partnered with the National Collaborating Centre for Infectious Diseases to hold a national workshop. Its objectives were to: understand areas where modelling terms, methods and results are unclear; share information on how modelling can best be used in informing policy and improving practice, particularly regarding the ways to integrate a focus on health equity considerations; and sustain and advance collaborative work in the development and application of modelling in public health.Decision Making for Infectious Diseases workshop brought together research modellers, public health professionals, policymakers and other experts from across the country. Invited presentations set the context for topical discussions in three sessions. A final session generated reflections and recommendations for new opportunities and tasks. CONCLUSIONS: Gaps in content and research include the lack of standard frameworks and a glossary for infectious disease modelling. Consistency in terminology, clear articulation of model parameters and assumptions, and sustained collaboration will help to bridge the divide between research and practice. Abstract Toll-like receptor 9 (TLR9) triggering is a promising novel strategy to combat cancer as it induces innate and adaptive immunity responses. B-cell lymphoma is unique in this context as tumor cells express TLR9 and may harbor latent Epstein-Barr virus (EBV), a gamma-herpesvirus with remarkable oncogenic potential when latent. Latent EBV may be promoted by TLR9 triggering via suppression of lytic EBV. Here, we elaborated an initial assessment of the impact of TLR9 triggering on EBV-positive and EBV-negative B-cell lymphoma using Burkitt's lymphoma (BL) cell lines as an in vitro model. We show that, independent of the presence of EBV, the TLR9 ligand oligodeoxynucleotide (ODN) CpG-2006 may or may not induce caspase-dependent cell death in BL cells. Moreover, ODN CpG-2006-induced cell death responses of BL cells were associated with TLR9 single-nucleotide polymorphisms (SNPs) rs5743836 or rs352140, which we detected in primary BL tumors and in peripheral blood from healthy individuals at similar frequencies. Thus, our findings suggest that the effect of TLR9 agonists on BL cells should be tested in vitro before installment of therapy and TLR9 SNPs in BL patients should be determined as potential biological markers for the therapeutic response to treatment targeting innate immunity. Abstract BACKGROUND: Despite the fact that traditional Chinese medicine (TCM) has been developed and used to treat acute and urgent illness for many thousands of years. TCM has been widely perceived in western societies that TCM may only be effective to treat chronic diseases. The aim of this article is to provide some scientifi c evidence regarding the application of TCM in emergency medicine and its future potential.Multiple databases (PubMed, ProQuest, Academic Search Elite and Science Direct) were searched using the terms: Traditional Chinese Medicine/ Chinese Medicine, Emergency Medicine, China. In addition, three leading TCM Journals in China were searched via Oriprobe Information Services for relevant articles (published from 1990-2012). Particular attention was paid to those articles that are related to TCM treatments or combined medicine in dealing with intensive and critical care.RESULTS: TCM is a systematic traditional macro medicine. The clinical practice of TCM is guided by the TCM theoretical framework -a methodology founded thousands of years ago. As the methodologies between TCM and Biomedicine are signifi cantly different, it provides an opportunity to combine two medicines, in order to achieve clinical effi cacy. Nowadays, combined medicine has become a common clinical model particular in TCM hospitals in China.CONCLUSIONS: It is evident that TCM can provide some assistance in emergency although to combine them in practice is still its infant form and is mainly at TCM hospitals in China. The future effort could be put into TCM research, both in laboratories and clinics, with high quality designs, so that TCM could be better understood and then applied in emergency medicine. Abstract Flying foxes have been considered to be involved in the transmission of serious infectious diseases to humans. Using questionnaires, we aimed to determine the direct and/or indirect contacts of flying foxes in an Indonesian nature conservation area with domestic animals and humans living in the surrounding area. We surveyed 150 residents of 10 villages in West Java. Villages were classified into 3 groups: inside and/or within 1 km from the outer border of the conservation area and 1-5 km or 5-10 km away from the reserve's outer border. Data were collected by direct interview using a structured questionnaire consisting of the respondent characteristics (age, sex and occupation); histories of contacts between flying foxes and humans, dogs and other domestic animals; and knowledge about infectious diseases, mainly rabies, in flying foxes. We found that flying foxes from the nature conservation area often enter residential areas at night to look for food, especially during the fruit season. In these residential areas, flying foxes had direct contacts with humans and a few contacts with domestic animals, especially dogs. People who encounter flying foxes seldom used personal protective equipment, such as leather gloves, goggles and caps. The residents living around the conservation area mostly had poor knowledge about flying foxes and disease transmission. This situation shows that the population in this region is at a quite high risk for contracting infectious diseases from flying foxes. Abstract Facing the threats of infectious diseases, we take various actions to protect ourselves, but few studies considered an evolving system with competing strategies. In view of that, we propose an evolutionary epidemic model coupled with human behaviors, where individuals have three strategies: vaccination, self-protection and laissez faire, and could adjust their strategies according to their neighbors' strategies and payoffs at the beginning of each new season of epidemic spreading. We found a counter-intuitive phenomenon analogous to the well-known Braess's Paradox, namely a better condition may lead to worse performance. Specifically speaking, increasing the successful rate of self-protection does not necessarily reduce the epidemic size or improve the system payoff. The range and degree of the Braess's Paradox are sensitive to both the parameters characterizing the epidemic spreading and the strategy payoff, while the existence of the Braess's Paradox is insensitive to the network topologies. This phenomenon can be well explained by a mean-field approximation. Our study demonstrates an important fact that a better condition for individuals may yield a worse outcome for the society. Abstract Background Influenza causes substantial morbidity and mortality despite available treatments. Anecdotal reports suggest that plasma with high antibody titres to influenza might be of benefit in the treatment of severe influenza.Methods In this randomised, open-label, multicentre, phase 2 trial, 29 academic medical centres in the USA assessed the safety and efficacy of anti-influenza plasma with haemagglutination inhibition antibody titres of 1:80 or more to the infecting strain. Hospitalised children and adults (including pregnant women) with severe influenza A or B (defined as the presence of hypoxia or tachypnoea) were randomly assigned to receive either two units (or paediatric equivalent) of anti-influenza plasma plus standard care, versus standard care alone, and were followed up for 28 days. The primary endpoint was time to normalisation of patients' respiratory status (respiratory rate of ≤20 breaths per min for adults or age-defined thresholds of 20-38 breaths per min for children) and a room air oxygen saturation of 93% or more. This study is registered with ClinicalTrials.gov, number NCT01052480.Between Jan 13, 2011, and March 2, 2015, 113 participants were screened for eligibility and 98 were randomly assigned from 20 out of 29 participating sites. Of the participants with confirmed influenza (by PCR), 28 (67%) of 42 in the plasma plus standard care group normalised their respiratory status by day 28 compared with 24 (53%) of 45 participants on standard care alone (p=0·069). The hazard ratio (HR) comparing plasma plus standard care with standard care alone was 1·71 (95% CI 0·96-3·06). Six participants died, one (2%) from the plasma plus standard care group and five (10%) from the standard care group (HR 0·19 [95% CI 0·02-1·65], p=0·093). Participants in the plasma plus standard care group had non-significant reductions in days in hospital (median 6 days [IQR 4-16] vs 11 days [5-25], p=0·13) and days on mechanical ventilation (median 0 days [IQR 0-6] vs 3 days [0-14], p=0·14). Fewer plasma plus standard care participants had serious adverse events compared with standard care alone recipients (nine [20%] of 46 vs 20 [38%] of 52, p=0·041), the most frequent of which were acute respiratory distress syndrome (one [2%] vs two [4%] patients) and stroke (one [2%] vs two [4%] patients).Interpretation Although there was no significant effect of plasma treatment on the primary endpoint, the treatment seemed safe and well tolerated. A phase 3 randomised trial is now underway to further assess this intervention. Abstract Suicide is the outcome of the interaction of biological, personal, and social risk factors. The purpose of this study was to verify the effects of strain due to individual risk factors and social risk factors on suicidality, and the mediating effect of depressive symptoms in relationship between strain related to individual risk factors and social risk factors and suicidality. The data from sociopsychological anxiety survey of Korea society conducted by the Korea Institute for Health and Social Affairs in 2015 were used in verifying the model. We analyzed the data of 7000 adults aged 19 to 79 years using Structural Equation Modeling. Strain due to individual risk factors was positively related to depressive symptoms and suicidality. Interestingly, strain induced by social risk factors was positively associated with depressive symptoms and suicidality. Social support is significantly associated with depressive symptoms and suicidality. Depressive symptoms directly affected suicidality. In addition, strain due to individual risk factors and social risk factors indirectly affected suicidality mediating depressive symptoms. These findings suggest that not only individual efforts such as social interaction and depression prevention but also government efforts such as preparation for aging may be needed to decrease suicide rate.Abbreviations: CESD-11 = Center for Epidemiologic Studies-Depression Scale 11, KIHASA = Korea Institute for Health and Social Affairs, MERSC = Middle East Respiratory Syndrome Coronavirus, OECD = Organization for Economic Co-operation and Development, SARS = severe acute respiratory syndrome. Abstract Coccidiosis and necrotic enteritis (NE) are among the most significant diseases affecting the poultry industry. These diseases have become more prominent in the wake of policies to reduce the use of antibiotics in animal production. This has led to more research focused on better understanding the immune system and its responses to pathogen challenge, and thus developing informed strategies to exploit immune responses that can support enhanced disease resistance and growth performance. Some chicken breeds and lines show greater resistance or susceptibility to various diseases, and thus these birds maybe able to shed light on immune processes or pathways that contribute to the more resistant/susceptible state. This review attempts to identify potentially important genes that show some consistency in (relative) up or downregulation in key tissues between the resistant and susceptible chickens. For coccidiosis and NE, relative downregulation of IL-10 and (slightly less consistently) upregulation of IFNγ appear to be features of more resistant birds. Data for IFN-α, IL-12, and IL-17D are currently less consistent. Gene expression data from NE studies have identified some potentially interesting, perhaps less well understood, immune-related genes (e.g., TCF12, BCL2, IRF2, TRAF3, TAB3, etc.,) that maybe associated with the resistant and/or susceptible phenotype. Salmonella and Campylobacter are important foodborne pathogens harbored by the chicken intestinal tract, while infectious bursal disease and infectious bronchitis are also important viral diseases of poultry. We, therefore, consider whether there are consistent features from resistant/susceptible disease models with these pathogens that relate to findings from the coccidiosis and NE studies. It is not anticipated that ideal immune responses to these pathogens will be identical but rather that consistent elements maybe identified that could help inform breeding or alternative strategies to support general disease resistance and enhanced (and efficient) flock productivity. Abstract The ongoing epidemic of chronic wasting disease (CWD) within cervid populations indicates the need for novel approaches for disease management. A vaccine that either reduces susceptibility to infection or reduces shedding of prions by infected animals, or a combination of both, could be of benefit for disease control. The development of such a vaccine is challenged by the unique nature of prion diseases and the requirement for formulation and delivery in an oral format for application in wildlife settings. To address the unique nature of prions, our group targets epitopes, termed disease specific epitopes (DSEs), whose exposure for antibody binding depends on diseaseassociated misfolding of PrP C into PrP Sc . Here, a DSE corresponding to the rigid loop (RL) region, which was immunogenic following parenteral vaccination, was translated into an oral vaccine. This vaccine consists of a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein G recombinant fusion with the RL epitope (hAd5:tgG-RL). Oral immunization of white-tailed deer with hAd5:tgG-RL induced PrP Sc -specific systemic and mucosal antibody responses with an encouraging safety profile in terms of no adverse health effects nor prolonged vector shedding. By building upon proven strategies of formulation for wildlife vaccines, these efforts generate a particular PrP Sc -specific oral vaccine for CWD as well as providing a versatile Correspondence to: Scott Napper; 120 Veterinary Road, platform, in terms of carrier protein and biological vector, for generation of other oral, peptide-based CWD vaccines. Abstract The human genome contains about 1.5 million Alu elements, which are transcribed into Alu RNAs by RNA polymerase III. Their expression is upregulated following stress and viral infection, and they associate with the SRP9/14 protein dimer in the cytoplasm forming Alu RNPs. Using cell-free translation, we have previously shown that Alu RNPs inhibit polysome formation. Here, we describe the mechanism of Alu RNP-mediated inhibition of translation initiation and demonstrate its effect on translation of cellular and viral RNAs. Both cap-dependent and IRES-mediated initiation is inhibited. Inhibition involves direct binding of SRP9/14 to 40S ribosomal subunits and requires Alu RNA as an assembly factor but its continuous association with 40S subunits is not required for inhibition. Binding of SRP9/14 to 40S prevents 48S complex formation by interfering with the recruitment of mRNA to 40S subunits. In cells, overexpression of Alu RNA decreases translation of reporter mRNAs and this effect is alleviated with a mutation that reduces its affinity for SRP9/14. Alu RNPs also inhibit the translation of cellular mR-NAs resuming translation after stress and of viral mRNAs suggesting a role of Alu RNPs in adapting the translational output in response to stress and viral infection. Abstract Background: A novel coronavirus (2019-nCoV) causing an outbreak of pneumonia in Wuhan, Hubei province of China was isolated in January 2020. This study aims to investigate its epidemiologic history, and analyze the clinical characteristics, treatment regimens, and prognosis of patients infected with 2019-nCoV during this outbreak. Methods: Clinical data from 137 2019-nCoV-infected patients admitted to the respiratory departments of the respiratory departments of nine tertiary hospitals in Hubei province from December 30, 2019 to January 24, 2020 were retrospectively collected, including general status, clinical manifestations, laboratory test results, imaging characteristics, and treatment regimens. Results: None of the 137 patients (61 males, 76 females, aged 20-83 years, median age 57 years) had a definite history of exposure to Huanan Seafood Wholesale Market. Major initial symptoms included fever (112/137, 81.8%), coughing (66/137, 48.2%), and muscle pain or fatigue (44/137, 32.1%), with other, less typical initial symptoms observed at low frequency, including heart palpitations, diarrhea, and headache. Nearly 80% of the patients had normal or decreased white blood cell counts, and 72.3% (99/137) had lymphocytopenia. Lung involvement was present in all cases, with most chest computed tomography scans showing lesions in multiple lung lobes, some of which were dense; ground-glass opacity co-existed with consolidation shadows or cordlike shadows. Given the lack of effective drugs, treatment focused on symptomatic and respiratory support. Immunoglobulin G was delivered to some critically ill patients according to their conditions. Systemic corticosteroid treatment did not show significant benefits. Notably, early respiratory support facilitated disease recovery and improved prognosis. The risk of death was primarily associated with age, underlying chronic diseases, and median interval from the appearance of initial symptoms to dyspnea. Conclusions: The majority of patients with 2019-nCoV pneumonia present with fever as the first symptom, and most of them still showed typical manifestations of viral pneumonia on chest imaging. Middle-aged and elderly patients with underlying comorbidities are susceptible to respiratory failure and may have a poorer prognosis. Abstract A B S T R A C T Viral diseases exhibit spatial and temporal variation, and there are many factors that can affect their occurrence. The identification of these factors is critical in the efforts to predict and lessen viral disease burden. Because viral infection is able to spread to humans from the environment, animals, and other humans, the One-Health framework can be used to investigate the critical pathways through which viruses are transported and transmitted. A holistic approach, incorporating publicly available clinical data for human, livestock, and wildlife disease occurrence, together with environmental data reported in federal and state databases such as parameters related to land use, environmental quality, and weather, can enhance the understanding of variations in disease patterns, leading to the design and implementation of surveillance systems. An example analysis approach is presented for Michigan, United States, which is a state with large urban centers as well as a sizeable rural and agricultural population. Analysis of publicly available data from 2017 indicates that gastrointestinal (GI) and influenza-associated illnesses in Michigan may have been related with agricultural land use to a higher extent than with developed land use during that year. Meanwhile, hepatitis A virus appears to be most closely related with developed land use in dense population areas. GI illnesses may be related to precipitation, and this relationship is strongest in the springtime, although GI illnesses are most common in the winter months. Integration of human-related clinical data, animal disease data, and environmental data can ultimately be used for prioritization of the most critical locations and times for viral outbreaks in both urban and rural environments. Abstract Bovine Respiratory Disease is the most costly disease that affects beef and dairy cattle industry. Its etiology is multifactorial, arising from predisposing environmental stress conditions as well as the action of several different respiratory pathogens. This situation has hindered the development of effective control strategies. Although different type of vaccines are available, many currently marketed vaccines are based on inactivated cultures of the main viral and bacterial agents involved in this pathology. The molecular composition of commercial veterinary vaccines is a critical issue. The present work aims to define at the proteomic level the most relevant valence of a line of commercial respiratory vaccines widely used in Central and South America. Since Mannheimia haemolytica is responsible for most of the disease associated morbid-mortality, we focused on the main proteins secreted by this pathogen, in particular Leukotoxin A, its main virulence factor. By Western blot analysis and mass spectrometry, Leukotoxin A was identified as a major component of (http://creativecommons.org/licenses/by-nc-nd/4.0/). M. haemolytica culture supernatants. We also identified other ten M. haemolytica proteins, including outer membrane proteins, periplasmic transmembrane solute transporters and iron binding proteins, which are relevant to achieve protective immunity against the pathogen. This work allowed a detailed molecular characterization of this vaccine component, providing evidence of its quality and efficacy. Furthermore, our results contributed to the identification of several proteins of interest as subunit vaccine candidates. Abstract INTRODUCTION: Acute respiratory failure has been one of the most important causes of death in intensive care units, and certain aspects of its pulmonary pathology are currently unknown.The objective was to describe the demographic data, etiology, and pulmonary histopathological findings of different diseases in the autopsies of patients with acute respiratory failure.METHOD: Autopsies of 4,710 patients with acute respiratory failure from 1990 to 2008 were reviewed, and the following data were obtained: age, sex, and major associated diseases. The pulmonary histopathology was categorized as diffuse alveolar damage, pulmonary edema, alveolar hemorrhage, and lymphoplasmacytic interstitial pneumonia. The odds ratio of the concordance between the major associated diseases and specific autopsy findings was calculated using logistic regression.RESULTS: Bacterial bronchopneumonia was present in 33.9% of the cases and cancer in 28.1%. The pulmonary histopathology showed diffuse alveolar damage in 40.7% (1,917) of the cases. A multivariate analysis showed a significant and powerful association between diffuse alveolar damage and bronchopneumonia, HIV/AIDS, sepsis, and septic shock, between liver cirrhosis and pulmonary embolism, between pulmonary edema and acute myocardial infarction, between dilated cardiomyopathy and cancer, between alveolar hemorrhage and bronchopneumonia and pulmonary embolism, and between lymphoplasmacytic interstitial pneumonia and HIV/ AIDS and liver cirrhosis.CONCLUSIONS: Bronchopneumonia was the most common diagnosis in these cases. The most prevalent pulmonary histopathological pattern was diffuse alveolar damage, which was associated with different inflammatory conditions. Further studies are necessary to elucidate the complete pathophysiological mechanisms involved with each disease and the development of acute respiratory failure. Abstract Peptidases are enzymes that hydrolyse peptide bonds in proteins and peptides. Peptidases are important in pathological conditions such as Alzheimer's disease, tumour and parasite invasion, and for processing viral polyproteins. The MEROPS database is an Internet resource containing information on peptidases, their substrates and inhibitors. The database now includes details of cleavage positions in substrates, both physiological and non-physiological, natural and synthetic. There are 39 118 cleavages in the collection; including 34 606 from a total of 10 513 different proteins and 2677 cleavages in synthetic substrates. The number of cleavages designated as 'physiological' is 13 307. The data are derived from 6095 publications. At least one substrate cleavage is known for 45% of the 2415 different peptidases recognized in the MEROPS database. The website now has three new displays: two showing peptidase specificity as a logo and a frequency matrix, the third showing a dynamically generated alignment between each protein substrate and its most closely related homologues. Many of the proteins described in the literature as peptidase substrates have been studied only in vitro. On the assumption that a physiologically relevant cleavage site would be conserved between species, the conservation of every site in terms of peptidase preference has been examined and a number have been identified that are not conserved. There are a number of cogent reasons why a site might not be conserved. Each poorly conserved site has been examined and a reason postulated. Some sites are identified that are very poorly conserved where cleavage is more likely to be fortuitous than of physiological relevance. This data-set is freely available via the Internet and is a useful training set for algorithms to predict substrates for peptidases and cleavage positions within those substrates. The data may also be useful for the design of inhibitors and for engineering novel specificities into peptidases.Database URL: http://merops.sanger.ac.uk . Abstract A B S T R A C T Viral diseases exhibit spatial and temporal variation, and there are many factors that can affect their occurrence. The identification of these factors is critical in the efforts to predict and lessen viral disease burden. Because viral infection is able to spread to humans from the environment, animals, and other humans, the One-Health framework can be used to investigate the critical pathways through which viruses are transported and transmitted. A holistic approach, incorporating publicly available clinical data for human, livestock, and wildlife disease occurrence, together with environmental data reported in federal and state databases such as parameters related to land use, environmental quality, and weather, can enhance the understanding of variations in disease patterns, leading to the design and implementation of surveillance systems. An example analysis approach is presented for Michigan, United States, which is a state with large urban centers as well as a sizeable rural and agricultural population. Analysis of publicly available data from 2017 indicates that gastrointestinal (GI) and influenza-associated illnesses in Michigan may have been related with agricultural land use to a higher extent than with developed land use during that year. Meanwhile, hepatitis A virus appears to be most closely related with developed land use in dense population areas. GI illnesses may be related to precipitation, and this relationship is strongest in the springtime, although GI illnesses are most common in the winter months. Integration of human-related clinical data, animal disease data, and environmental data can ultimately be used for prioritization of the most critical locations and times for viral outbreaks in both urban and rural environments. Abstract The enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH) is responsible for the synthesis of N-glycolylneuraminic acid (Neu5Gc), a sialic acid present on the cell surface proteins of most deuterostomes. The CMAH gene is thought to be present in most deuterostomes, but it has been inactivated in a number of lineages, including humans. The inability of humans to synthesize Neu5Gc has had several evolutionary and biomedical implications. Remarkably, Neu5Gc is a xenoantigen for humans, and consumption of Neu5Gc-containing foods, such as red meats, may promote inflammation, arthritis, and cancer. Likewise, xenotransplantation of organs producing Neu5Gc can result in inflammation and organ rejection. Therefore, knowing what animal species contain a functional CMAH gene, and are thus capable of endogenous Neu5Gc synthesis, has potentially far-reaching implications. In addition to humans, other lineages are known, or suspected, to have lost CMAH; however, to date reports of absent and pseudogenic CMAH genes are restricted to a handful of species. Here, we analyze all available genomic data for nondeuterostomes, and 322 deuterostome genomes, to ascertain the phylogenetic distribution of CMAH. Among nondeuterostomes, we found CMAH homologs in two green algae and a few prokaryotes. Within deuterostomes, putatively functional CMAH homologs are present in 184 of the studied genomes, and a total of 31 independent gene losses/pseudogenization events were inferred. Our work produces a list of animals inferred to be free from endogenous Neu5Gc based on the absence of CMAH homologs and are thus potential candidates for human consumption, xenotransplantation research, and model organisms for investigation of human diseases. Abstract New epidemics of infectious diseases often involve health care workers. In this short communication we present a case report of a health care professional who became the fi rst case of infl uenza H1N1 virus to be notifi ed in the United Arab Emirates. There are several issues related to workplace considerations and general public health, including preventive measures, the need for isolation of the patient, dealing with contacts, return to work, and communication with the workforce. Abstract The diagnostics of respiratory viral infections has improved markedly during the last 15 years with the development of PCR techniques. Since 1997, several new respiratory viruses and their subgroups have been discovered: influenza A viruses H5N1 and H1N1, human metapneumovirus, coronaviruses SARS, NL63 and HKU1, human bocavirus, human rhinoviruses C and D and potential respiratory pathogens, the KI and WU polyomaviruses and the torque teno virus. The detection of previously known viruses has also improved. Currently, a viral cause of respiratory illness is almost exclusively identifiable in children, but in the elderly, the detection rates of a viral etiology are below 40%, and this holds also true for exacerbations of chronic respiratory illnesses. The new viruses cause respiratory symptoms like the common cold, cough, bronchitis, bronchiolitis, exacerbations of asthma and chronic obstructive pulmonary disease and pneumonia. Acute respiratory failure may occur. These viruses are distributed throughout the globe and affect people of all ages. Data regarding these viruses and the elderly are scarce. This review introduces these new viruses and reviews their clinical significance, especially with regard to the elderly population. Abstract Angiotensin converting enzyme inhibitors (ACEi) are standard of care for treatment of congestive heart failure, but aldosterone breakthrough occurs when ACEi are used in both dogs and humans. Entresto®, a combinational angiotensin-receptor blocker/neprilysin inhibitor, showed superiority in reducing mortality in human patients with heart failure compared to enalapril. Pharmacodynamics of Entresto® have been evaluated in healthy dogs, showing efficacy in altering the renin-angiotensin-aldosterone system (RAAS) without causing adverse effects. The aim of this prospective, blinded, randomized, placebo-controlled study was to compare the pharmacodynamic effects of Entresto® to placebo in dogs with preclinical myxomatous mitral valve disease (MMVD) and to evaluate the safety profile of Entresto® in dogs with cardiac disease. Client-owned dogs weighing 4-15 kg with ACVIM Stage B2 MMVD were enrolled. Dogs with clinically significant pulmonary hypertension or systemic disease were excluded, as were dogs on any medication(s) known to alter the RAAS. All dogs received pimobendan throughout the study period. Each patient was evaluated at three time points (Day 0, Day 7, and Day 30). Echocardiography, thoracic radiographs (CXR), Doppler systemic arterial pressure (SAP), complete blood count, serum biochemical profile, plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration, and urinary aldosterone to creatinine ratio (UAldo:C) were evaluated on Day 0. On Day 0, each dog was randomly allocated to a treatment group (Entresto®; 20 mg/kg BID) or a placebo group by the attending pharmacist. Investigators, veterinary technicians, and owners were blinded to treatment. Serum renal enzyme and electrolyte concentrations and SAP were rechecked on Day 7. Echocardiography, CXR, SAP, renal panel, plasma NT-proBNP concentration, and UAldo:C were rechecked on Day 30. Thirteen dogs were recruited: Entresto® (n = 7) and placebo (n = 6). The median percent increase in UAldo:C between Day 0 and Day 30 was significantly lower in the Entresto® group (12%; P = 0.032) as compared to the placebo group (195%). The median percent decrease of NT-proBNP concentration from Day 0 to Day 30 was not statistically different between groups (P = 0.68). No statistical differences were seen in echocardiographic, CXR, SAP, or biochemical profiles measured at any time point between groups. No adverse events were noted by the owners for dogs in either group. This study suggests that Entresto® efficiently inhibits RAAS in dogs with cardiomegaly secondary to MMVD in comparison to placebo. Entresto® is safe in regards to SAP and renal enzyme and electrolyte concentrations. No adverse effects were noted in any dog taking Entresto®. Future studies comparing Entresto® to ACEi, evaluating the concomitant use of diuretics and Entresto®, and evaluating longterm (> 30 days) effects of Entresto® are warranted. Trimethylamine N-oxide (TMAO), the oxidized product of trimethylamine, is produced by gastrointestinal microbiota from certain dietary nutrients including choline and L-carnitine. Elevated plasma concentrations of TMAO, choline, and L-carnitine have been shown to be associated with the presence and severity of cardiovascular disease in people and are independent predictors of adverse cardiac events and mortality. This prospective study investigated whether there are differences in concentrations of circulating TMAO, choline, or L-carnitine in dogs with degenerative mitral valve disease (DMVD) compared to healthy controls (Controls).Thirty client-owned dogs were recruited, including 10 dogs with CHF secondary to DMVD, 10 dogs with asymptomatic DMVD, and 10 Controls. Echocardiography was performed, and fasting plasma concentrations of TMAO, choline, and carnitine fractions were measured. Data were compared among the three groups using ANOVA or Kruskal-Wallis tests.Trimethylamine N-oxide (P = 0.034), total L-carnitine (P = 0.034), carnitine esters, and carnitine esters to free carnitine ratio (E/F ratio) were significantly higher in dogs with CHF compared to asymptomatic DMVD. Trimethylamine N-oxide (P = 0.022), choline (P = 0.011), total L-carnitine (P = 0.011), carnitine esters, free carnitine, and E/F ratio were significantly higher in dogs with CHF compared to Controls. No differences were detected between asymptomatic DMVD dogs and Controls.Dogs with DMVD and CHF had higher concentrations of TMAO compared to both asymptomatic DMVD dogs and Controls. Prospective studies are warranted to determine if TMAO concentrations can be altered with dietary or gut microflora modification, and whether altering TMAO concentrations could impact disease progression.Loop diuretics provide symptomatic relief for congestive heart failure, yet stimulate the renin-angiotensin-aldosterone system (RAAS). We hypothesized that the two potent loop diuretics, torsemide and furosemide, at approximately equipotent dosages (similar diuresis), would have comparable effects on the circulating RAAS.Six, healthy, middle-aged, male beagles were randomized to receive torsemide (0.1mg/kg PO q12h), furosemide (2.0mg/kg PO q12h), or placebo for 10 days during 3 separate experiments, in a crossover design with a 10-day washout period between experiments. Blood was collected on days 1, 5, and 9 and 24-hour urine collection ended on days 2, 6, and 10. After repeated measures analysis and Bonferonni correction, variables with an adjusted P < 0.05 were investigated further, using Tukey';s method.Twenty-four hour urine production differed significantly between the diuretics only on day 10, with torsemide causing a 38% greater diuresis than furosemide. There was, however, no significant difference in average 3-day diuresis. There were no significant differences between diuretics in the 24-hour urinary excretion of Na + , Cl -, or K + , though furosemide caused less kaliuresis than torsemide. Serum renin, angiotensin II, and aldosterone and the urine aldosterone-to-creatinine ratio were significantly increased in the diuretic groups, as compared to placebo on days 5/6 and 9/10. There were no significant differences in these parameters between diuretics. Creatinine and BUN concentrations rose comparably in the 2 diuretic groups, remaining within reference intervals in all dogs.At approximately equipotent dosages (20:1), torsemide and furosemide produced comparable RAAS activation. This, and torsemide';s greater kaliuretic effect decrease support for torsemide';s hypothesized mineralocorticoid-receptor blocking capability.Transvenous pacemaker implantation is the definitive treatment for pathologic bradyarrhythmias, though reported complication rates have historically been high. The purpose of this study was to determine survival times and complication rates for dogs who underwent transve-Atenolol is a β 1 -receptor antagonist commonly prescribed in cats affected with hypertrophic obstructive cardiomyopathy. The traditional oral dose is 6.25 to 12.5 mg per cat q12-24h. The oral tablet is highly bioavailable (90 ± 9%) with peak atenolol concentration occurring 1-2 hours post administration in cats. However results from several studies indicate owner compliance may be poor with long-term oral treatment, and feline disposition can present specific challenges to owners with chronic oral medication administration. Previous Abstract Viral 2=,5=-phosphodiesterases (2=,5=-PDEs) help disparate RNA viruses evade the antiviral activity of interferon (IFN) by degrading 2=,5=-oligoadenylate (2-5A) activators of RNase L. A kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) to localize and organize cyclic AMP (cAMP) signaling during diverse physiological processes. Among more than 43 AKAP isoforms, AKAP7 appears to be unique in its homology to viral 2=,5=-PDEs. Here we show that mouse AKAP7 rapidly degrades 2-5A with kinetics similar to that of murine coronavirus (mouse hepatitis virus [MHV]) strain A59 ns2 and human rotavirus strain WA VP3 proteins. To determine whether AKAP7 could substitute for a viral 2=,5=-PDE, we inserted AKAP7 cDNA into an MHV genome with an inactivated ns2 gene. The AKAP7 PDE domain or N-terminally truncated AKAP7 (both lacking a nuclear localization motif), but not full-length AKAP7 or a mutant, AKAP7 H185R , PDE domain restored the infectivity of ns2 mutant MHV in bone marrow macrophages and in livers of infected mice. Interestingly, the AKAP7 PDE domain and N-terminally deleted AKAP7 were present in the cytoplasm (the site of MHV replication), whereas full-length AKAP7 was observed only in nuclei. We suggest the possibility that viral acquisition of the host AKAP7 PDE domain might have occurred during evolution, allowing diverse RNA viruses to antagonize the RNase L pathway.We also reported that the rotavirus VP3 C-terminal domain (VP3-CTD) cleaves 2-5A and that it may rescue ns2 mutant MHV. Here we report that a cellular protein, AKAP7, has an analogous 2=,5=-phosphodiesterase (2=,5=-PDE) domain that is able to restore the growth of chimeric MHV expressing inactive ns2. The proviral effect requires cytoplasmic localization of the AKAP7 PDE domain. We speculate that AKAP7 is the ancestral precursor of viral proteins, such as ns2 and VP3, that degrade 2-5A to evade the antiviral activity of RNase L. RH. 2014 . Murine AKAP7 has a 2=,5=-phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene. mBio 5(4):e01312-14. Abstract Rationale: Cardiac stem cell-derived exosomes have been demonstrated to promote cardiac regeneration following myocardial infarction in preclinical studies. Recent studies have used intramyocardial injection in order to concentrate exosomes in the infarct. Though effective in a research setting, this method is not clinically appealing due to its invasive nature. We propose the use of a targeting peptide, cardiac homing peptide (CHP), to target intravenously-infused exosomes to the infarcted heart. Methods: Exosomes were conjugated with CHP through a DOPE-NHS linker. Ex vivo targeting was analyzed by incubating organ sections with the CHP exosomes and analyzing with fluorescence microscopy. In vitro assays were performed on neonatal rat cardiomyocytes and H9C2 cells. For the animal study, we utilized an ischemia/reperfusion rat model. Animals were treated with either saline, scramble peptide exosomes, or CHP exosomes 24 h after surgery. Echocardiography was performed 4 h after surgery and 21 d after surgery. At 21 d, animals were sacrificed, and organs were collected for analysis. Results: By conjugating the exosomes with CHP, we demonstrate increased retention of the exosomes within heart sections ex vivo and in vitro with neonatal rat cardiomyocytes. In vitro studies showed improved viability, reduced apoptosis and increased exosome uptake when using CHP-XOs. Using an animal model of ischemia/reperfusion injury, we measured the heart function, infarct size, cellular proliferation, and angiogenesis, with improved outcomes with the CHP exosomes. Conclusions: Our results demonstrate a novel method for increasing delivery of for treatment of myocardial infarction. By targeting exosomes to the infarcted heart, there was a significant improvement in outcomes with reduced fibrosis and scar size, and increased cellular proliferation and angiogenesis. Abstract The cancer burden is a global problem, and oncology nurses should be accountable for delivering safe and effective cancer care and providing the best possible experience for patients. The development and application of evidence-based practice in cancer care is an effective strategy in achieving this goal; however, the journey in which such practice involves may encounter various challenges. In this article, the author discusses her own experience, successful and unsuccessful of such a journey. Both challenges and opportunities are identified, and suggestions put forward for making collaborative efforts. Abstract Despite consistent efforts to protect public health there is still a heavy burden of viral disease, both in the United States and abroad. In addition to conventional medical treatment, there is a need for a holistic approach for early detection and prevention of viral outbreaks at a population level. One-Health is a relatively new integrative approach to the solving of global health challenges. A key component to the One-Health approach is the notion that human health, animal health, and environmental health are all innately interrelated. One-Health interventions, initiated by veterinary doctors, have proven to be effective in controlling outbreaks, but thus far the applications focus on zoonotic viruses transmitted from animals to humans. Environmental engineers and environmental scientists hold a critical role in the further development of One-Health approaches that include water-related transport and transmission of human, animal, and zoonotic viruses. In addition to waterborne viruses, the proposed approach is applicable to a wide range of viruses that are found in human excrement since contaminated water-based surveillance systems may be used for early detection of viral disease. This paper proposes a greater One-Health based framework that involves water-related pathways. The first step in the proposed framework is the identification of critical exposure pathways of viruses in the water environment. Identification of critical pathways informs the second and third steps, which include water-based surveillance systems for early detection at a population level and implementation of intervention approaches to block the critical pathways of exposure. Abstract To estimate the pharyngeal carriage rate of Neisseria meningitidis (N. meningitidis ), Streptococcus pneumoniae (S. pneumoniae ) and Staphylococcus aureus (S. aureus ) among Australian Hajj pilgrims.In 2014, surveillance was conducted in two phases among Australian Hajj pilgrims: The first phase during Hajj in Mina, and the second phase soon after returning home to Australia. Nasopharyngeal or oropharyngeal swabs were taken from participants then tested, firstly by nucleic acid testing, and also by standard culture.Of 183 participants recruited in the first phase, 26 (14.2%) tested positive for S. pneumoniae ; 4 had received pneumococcal conjugate vaccine (PCV13). Only one tested positive for N. meningitidis (W). Of 93 2 nd phase samples cultured, 17 (18.3%) grew S. aureus , all methicillin sensitive, 2 (2.2%) grew N. meningitidis (on subculture; one serotype B, one negative), and 1 (1%), from an unvaccinated pilgrim, grew S. pneumoniae .Relatively high carriage of S. pneumoniae and little meningococcal carriage was found. This indicates the importance of a larger study for improved infection surveillance and possible vaccine evaluation. Abstract The difference noted in Rotavirus vaccine efficiency between high and low income countries correlates with the lack of universal access to clean water and higher standards of hygiene. Overcoming these obstacles will require great investment and also time, therefore more effective vaccines should be developed to meet the needs of those who would benefit the most from them. Increasing our current knowledge of mucosal immunity, response to Rotavirus infection and its modulation by circadian rhythms could point at actionable pathways to improve vaccination efficacy, especially in the case of individuals affected by environmental enteropathy. Also, a better understanding and validation of Rotavirus entry factors as well as the systematic monitoring of dominant strains could assist in tailoring vaccines to individual's needs. Another aspect that could improve vaccine efficiency is targeting to M cells, for which new ligands could potentially be sought. Finally, alternative mucosal adjuvants and vaccine expression, storage and delivery systems could have a positive impact in the outcome of Rotavirus vaccination.ARTICLE HISTORY Abstract Background: Osteonecrosis (ON) is known to be one of the most disabling complications following corticosteroid (CS) medications. However, evidence regarding risk of asymptomatic prevalence of ON among different diseases and the impact of variable steroid regimens are conflicting. We aimed to determine the prevalence of ON of femoral head in asymptomatic patients with systemic rheumatic diseases who received high-dose CS and also clarify its relationship with different dosages and regimens.Methods: In this cross-sectional study, 50 consecutive patients receiving high-dose CS for rheumatic diseases who have no pelvic pain were recruited. MRI of both hips was performed on all patients using a 1.5 Tesla to diagnose ON.Results: Of 50 subjects, 18 (36%) developed ON of the femoral head. Groups with and without ON were comparable in terms of sex, age and mean starting CS dose. There was no statistical difference in the type of CS regimen including daily dose, peak dose and cumulative dose between the two groups. However, silent ON was associated with both the cumulative CS dose and the duration of CS therapy.Conclusion: According to high prevalence of ON in our selected patients with no other identifiable risk factor for ON, monitoring of high risk patients with periodic hip MRI would help diagnose necrosis in early stage.Kianmehr N, Bidari A, Mofidi M, Bahar N. Silent osteonecrosis of the femoral head following high-dose corticosteroids in patients with systemic rheumatic diseases s. Abstract The Middle East respiratory syndrome coronavirus (MERS-CoV) has been recognized as a highly pathogenic virus that infects the human respiratory tract and has high morbidity and mortality. The MERS-CoV is a huge burden on Saudi Arabian health-care facilities, causing approximately 40% mortality. The transmission mechanism of the virus is still not well understood. Therefore, the prevention of any route of transmission is the best measure to arrest the spread of this disease. Using the real time polymerase chain reaction (RT-PCR), MERS-CoV was detected in the nasal secretions of a human cadaver. Full precautions should be applied and carefully followed to prevent the transmission of the virus, especially among health care workers. Abstract Hand washing plays a key role in preventing respiratory infection in many clinical settings. However, its effectiveness in preventing acute respiratory illness (ARI) during field training in military training facilities has been not studied.A quasi-interventional study was performed to evaluate the prevalence of ARIs over 4 weeks in a Korean army training center in South Korea from January 2009 to February 2009. A total of 1291 recruits participating in military training for 4 weeks were randomly distributed to 2 battalions (one with 631 and the other with 660). After noticing there is a difference between the 2 battalions in terms of the development of ARIs at the end of 2 weeks of training, we conducted interviews with the battle commanders to determine factors that may be related to one battalion having a higher incidence of ARI. Thereafter, we performed an intervention, which consists of instructing the battalion having a higher incidence of ARI to implement field hand washing from the third week. Following the intervention, we compared the cumulative rate of ARI during 4 weeks of training.The interviews revealed that there were no major differences between the 2 battalions in terms of the training schedules, living environments, or indoor hand washing methods. However, there was difference in terms of hand washing during field training for the first 2 weeks; whereas one battalion (the early hand washing group) implemented hand washing during field training starting in the first week, the other battalion did not implement hand washing for the first 2 weeks but instead began in the third week (the late hand washing group). The cumulative incidence rate of ARI during 4 weeks of training was significantly lower in the early hand washing group (13.0%, 95% confidence interval [CI]: 10.6%-15.9%) than in the late hand washing group (28.0%, 95% CI, 24.7%-31.5%).Our study suggests that outdoor hand washing during field training may be an effective precaution for reducing ARI incidence among recruits participating in military training.Abbreviations: ARI = acute respiratory illness, CI = confidence interval. Abstract It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release. Abstract and Blautia (P = 0.008) significantly decreased on day 20 and C. hiranonis (p = 0.001) on day 60. Blautia (P = 0.017) remained decreased on day 60. E.coli remained unchanged on day 20 (P = 0.999) but decreased on day 60 (P = 0.0001). Faecalibacterium remained unchanged on day 20 (P = 0.711) but increased on day 60 (P = 0.0002). In group 3, no statistically significant differences were identified.Administration of amoxicillin/clavulanic acid or doxycycline had profound effects on certain bacterial groups potentially associated with dysbiosis in these kittens.Several studies have reported intestinal microbial dysbiosis in dogs with chronic enteropathy. Limited data is available about the microbiota gene function in this pathology in dogs. Determining the functional attributes of the microbiome is essential for understanding their role on host metabolism and disease. The aim of this study was to compare the functional roles of the fecal microbiota in healthy dogs and dogs with CE by fecal DNA shotgun sequencing.Fecal samples were collected from 14 healthy dogs and 20 dogs with chronic enteropathy (CE). Fecal DNA was extracted using a commercial kit (PowerSoil, QIAGEN). Functional characterization of the shotgun sequence reads in the KEGG database was performed using next generation sequencing, in order to identify the relative abundance of specific metabolic pathways. A Wilcoxon test was used for comparison of the gene abundance between groups. Significance was set at q < 0.05.At phylum level, low abundance of Bacteroidetes was observed in dog with CE, compared to healthy control dogs (48.5 vs 1.6%; q = 0.0006).Fusobacteria was also significantly increased in healthy controls (0.25 vs 0.04%; q = 0.0111). The pathway enrichment analysis of the bacterial metagenomes showed that 130 of 360 (36.1%) total metabolic modules were differentially abundant between studied groups. Genes for carbohydrate metabolism, biosynthesis of amino acids (lysine, threonine, histidine, isoleucine, tryptophan, leucine and serine) and vitamins (ascorbate, thiamine and riboflavin) were decreased in dogs with CE, while genes involved in transport of molecules and homeostasis maintenance during oxidative stress (glutathione biosynthesis) were increased in CE.Our data presents, as previous reported, an intestinal microbial dysbiosis in dogs with CE. As new finding, our results show an altered microbial metabolism in dogs with CE compared to healthy dogs, characterized by reduction of amino acid biosynthesis and carbohydrate metabolism. Further studies including transcriptomic analysis are warranted to define the consequences of this microbiota dysfunction on dogs with CE.'Protein-losing-enteropathy' (PLE) is a syndrome caused by various diseases including idiopathic inflammatory bowel disease, primary lymphangiectasia, lymphoma, and severe acute gastroenteritis. Certain breed predispositions, such as the soft-coated Wheaten Terrier, are well known. Our clinical experience suggests that pugs with PLE respond poorly to treatment, but this is not described in the literature.The aim of the current study was to assess whether the pug breed demonstrates a worse response to treatment for PLE than other breeds of dog.This was a retrospective study comparing the response to treatment in all pugs diagnosed with PLE between 2009 and 2018 in five referral centres in the United Kingdom. Approval for the study was granted by the University of Liverpool Research Ethics Committee. A group of non-pug dogs, also diagnosed with PLE within the same period, was selected for comparison. PLE was defined as any gastrointestinal disease resulting in serum albumin below the laboratory reference interval that could not be explained by another cause. Factors associated with survival were assessed using simple statistics (Mann-Whitney tests and chi-square test as appropriate) and Cox's proportional hazards regression. Initially, factors were tested individually using simple regression; a multiple regression model was then created, subsequently refined by backwards stepwise elimination until the best model was found. A total of 35 pugs were diagnosed with a PLE between 2009 and 2018 and were compared with 113 dogs from other breeds. On simple regression analysis, factors associated with survival (at P < 0.1) were pug breed (P = 0.002), diet used for treatment (P = 0.022), receiving immunosuppressive therapy (P = 0.089), and treatment with cobalamin (P = 0.005). However, the only factors that remained in the final model were the pug breed and the diet used for treatment. In this respect, dogs of the pug breed were associated with a greater hazard of dying (compared with non-pugs: hazards ratio [HR] 2.67 (CI 1.60-4.47; P < 0.001), whilst being fed a hydrolysed diet was associated with a lesser hazard risk of death than when fed other diets (compared with low-fat diets: HR 0.50, 95%-CI: 0.26-0.97; P = 0.042; compared with other diets including highly-digestible diets: HR 0.35, 95%-CI: 0.17-0.71; P = 0.004).In conclusion, this study demonstrated an association between the pug breed and a poorer response to treatment for PLE compared to other breeds. Further research should be undertaken as to the underlying cause.ABSTRACTSDisclosures to report.AJG is an employee of the University of Liverpool, but his post is financially supported by Royal Canin, which is also owned by MarsPetcare. AJG has also received financial remuneration for providing educational material, speaking at conferences, and consultancy work for Mars Petcare; all such remuneration has been for projects unrelated to the work reported in this manuscript. All other authors declare no conflict of interest. ESCG-O-5 Is measuring serum folate pointless? Retrospective analysis of prevalence and clinical significance of hypo-or hyperfolataemia in dogs with chronic enteropathies A. Petrelli, S. Salavati Royal (Dick) School of Veterinary Studies and the Roslin Institute, Edinburgh, United Kingdom Assessment of serum folate (SF) is routinely performed in dogs with chronic enteropathies (CE), most often in conjunction with serum cobalamin. Traditionally, their combination has been used to differentiate intestinal malabsorption from dysbiosis. Despite the fact that the diagnostic and prognostic value of serum cobalamin is well documented, the prevalence of hypo-and hyperfolataemia and the clinical and prognostic value of its assessment has not been scrutinised in dogs with CE. The aims of this study were to determine the prevalence of SF abnormalities in dogs with CE, as well as its relationship to other laboratory parameters and outcome. Files of dogs presented for chronic gastrointestinal (GI) signs (> 3 weeks duration) between 2014 and 2017 were retrospectively evaluated. Exclusion criteria were lack of SF assessment, and supplementation of folate or cobalamin beforehand. 321 dogs (100 FN, 109 MN, 34 FE, 78 ME; median age of 65 m, range 2-171) were included. Hypofolataemia was present in 97/321 (30%), hyperfolataemia in 86/321 (27%), with the remaining 138/321 dogs (43%) having normal SF values. Initially, dogs were divided into groups according to the final diagnosis: CE (n = 215), other GI disease (n = 76), non-GI disease (n = 30). SF values were not significantly different across those groups (Kruskal-Wallis, P = 0.83). When dividing CE dogs into different subgroups of food-(FRE), antibiotic-(ARE), steroid-responsive (SRE) or protein-losing enteropathy (PLE), no difference in SF values was observed (ANOVA, P = 0.92). When all dogs were grouped by their SF status (low, normal, high), significant differences in serum cobalamin (P = 0.001), alkaline phosphatase (p = 0.01), cholesterol (p = 0.03) and total calcium (p = 0.01) were identified (ANOVA, Dunn's post hoc test). Multivariate analysis confirmed the correlation between SF and cobalamin (P = 0.009), as well as cholesterol (P = 0.028) and total calcium (p = 0.038). However, none of these correlations were linear (Spearman, all P > 0.05). Kaplan Maier analysis of follow-up and survival times by SF status showed no significant differences. In conclusion, SF was not associated with GI disease or subgroup of CE. Improvement of clinical signs or survival was not associated with SF status. In this study, no diagnostic or prognostic benefit of assessing SF in dogs with chronic GI signs could be detected. Future prospective studies should assess if folate supplementation in the 30% of CE dogs with hypofolataemia can accelerate clinical improvement or influence outcome/ prognosis.Acute haemorrhagic diarrhoea syndrome (AHDS) in dogs is often treated with antibiotics due to the potential risk of bacterial translocation from the gastrointestinal tract to the blood stream. However, recent studies indicate that antibiotics are not always necessary. According to the Danish antibiotic use guidelines for companion animals 2012, antibiotics are not recommended for routine treatment of AHDS but only indicated in hospitalized dogs with severely affected overall condition and signs of systemic inflammation (SIRS)/sepsis. The aim of this study was to evaluate severity of disease and outcome in hospitalized dogs with acute haemorrhagic diarrhoea that did not receive antibiotics. The study was performed as a retrospective, observational study based on information from medical records for dogs with acute haemorrhagic diarrhoea of unknown aetiology, hospitalized at the University Hospital for Companion Animals during the period 25/2-2014 to 9/10-2018. Signalments, concurrent diseases, clinical disease at the time of hospitalization and during each consecutive day, treatment prior to and during hospitalization, days of hospitalization/euthanasia and laboratory results were registered for each patient. Clinical disease was scored according to the AHDS-scoring system from 0-18 and the number of SIRS criteria (tachycardia (HR > 120), tachypnea (RR > 40), hyper or hypothermia (T > 39.0 C or < 37.5 C), leucocytosis (WBC > 18*10 9 / L), leukopenia (WBC < 5*10 9 /L, band neutrophilia and/or hypoglycaemia [glucose <4 mmol/L]) were recorded. One-hundred and seventy-two dogs were excluded from the analysis due to suspected drug induced disease (vaccination(s) (N = 4), anaesthetics (N = 5), corticosteroids or NSAIDs (N = 81)), alimentary foreign body (N = 5) or treatment with antibiotics during hospitalization (N = 128). Of the 128 dogs, where an obvious cause for the diarrhoea were not found and that only received supportive treatment (intravenous fluid therapy N = 128; antiemetics N = 98; gastroprotectants N = 107), 98% survived to discharge (125/128 dogs). Two dogs were euthanized due to financial constraints and reluctance from the owner to proceed with further treatment due to advanced age and one brachycephalic dog suffered a respiratory crisis with respiratory arrest nonresponsive to resuscitation. The surviving 125 dogs were hospitalized for an average of 1.7 days (range 1-4 days) with a mean AHDS-score of 12 at hospitalization (range 4-16). The mean AHDS-score after 24 hours of hospitalization was 5.5 (range 0-14). 29% (37/128) of the ABSTRACTS 341 dogs met ≥2 SIRS criteria during hospitalization. None of the 128 dogs had a degenerative left-shift. These results suggest that antimicrobial therapy in dogs with acute haemorrhagic diarrhoea may not always be necessary in dogs even when 2 or more SIRS-criteria are met. Recent studies have shown alterations in faecal bile acid (BA) profiles in dogs with chronic enteropathy, potentially contributing to clinical signs. The study aim was to assess faecal BA concentrations in dogs with acute haemorrhagic diarrhoea syndrome (AHDS-D) over time and to compare it to that of healthy control dogs (HC-D). Twenty-five AHDS-D and 53 HC-D were enrolled. Faecal BA concentrations were measured by gas chromatography-mass spectrometry on days 0, 2, 7, and 14 in AHDS-D, and on day 0 in HC-D. Statistical analysis was performed with Friedman test for comparison of BA over time in AHDS-D, and unpaired t-test or Mann-Whitney-test for comparison between AHDS-D and HC-D (P < 0.05). On day 0, concentration of lithocholic acid was significantly lower in AHDS-D than in HC-D (p < 0.001). On day 2, total faecal BA were significantly increased in AHDS-D (p < 0.001) compared to HC-D. More precisely, primary BA were significantly increased in AHDS-D on day 2 (P = 0.034) with significantly higher concentration of cholic acid (p = 0.004), while secondary BA were significantly decreased (p < 0.001) with significantly lower concentrations of deoxycholic acid (p < 0.001) and ursodeoxycholic acid (UDCA) (p < 0.001). Dogs in AHDS-D showed a significant decrease in total primary BA concentrations (P = 0.021) with significantly lower concentrations of cholic acid (P = 0.027) and deoxycholic acid (p = 0.021) on day 14 compared to day 2. In conclusion, AHDS-D show alterations in faecal BA profiles compared to HC-D. However, faecal BA profiles normalize rapidly suggesting that BA dysmetabolism does not seem to play a major role in the pathophysiology of AHDS. Pimobendan is frequently used off-label for treatment of cats with congestive heart failure (CHF) secondary hypertrophy cardiomyopathy (HCM). Concerns exist regarding the safety of pimobendan in the subset of cats with HCM and dynamic outflow tract obstruction (HOCM). The purpose of this study was to evaluate safety and tolerability of pimobendan in cats with CHF secondary to HOCM compared with nonobstructive HCM.Medical records from 94 cats with CHF (47 with HOCM, 47 with nonobstructive HCM) at two tertiary referral hospitals were reviewed.Demographic, clinicopathologic, echocardiographic, and treatment data were collected and compared between groups, including information regarding possible adverse effects of pimobendan.Average age of cats (9 +/− 4 years) did not differ between HOCM and HCM (P = 0.12). Compared to cats with HCM, cats with HOCM were more likely to manifest CHF as pulmonary edema (44/47 versus 32/47; P = 0.003) and less likely to have pleural effusion (13/47 versus 25/47; P = 0.02). Other than a higher incidence of heart murmurs in cats with HOCM (P < 0.001), clinical variables did not differ between groups. Pimobendan was typically initiated on the date of CHF diagnosis (median time from diagnosis of CHF to initiation of pimobendan was 0 days). Initial dose of pimobendan was 0.25 +/− 0.07 mg/kg every 12 hours; dose (or frequency) was escalated at some point during CHF management in 31/94 (33%) of cases, with no difference between HOCM and HCM cats. Clinical signs that could potentially represent adverse effects of pimobendan (vomiting, diarrhea, anorexia, lethargy, new-onset arrhythmias) were noted in 13/47 (28%) HCM cats and 9/47 (19%) HOCM cats (P = 0.34). Based on patterns of timing and resolution, these signs were generally ascribed to recurrence of CHF rather than pimobendan administration. Pimobendan was discontinued due to adverse effects in only 1 cat with nonobstructive HCM that experienced lethargy and nausea 2-3 hours following pimobendan administration (resolved when pimobendan discontinued). Pimobendan was discontinued in 7 additional cats, either because owners were unable to administer the medication (n = 2) or because CHF had resolved (n = 5 cases where CHF was precipitated by an acute external event, such as fluid overload or injectable glucocorticoid administration). No cats experienced acute adverse hemodynamic effects (hypotension, cardiovascular collapse) following pimobendan administration. Results of this study suggest that pimobendan is well-tolerated in cats with cardiomyopathy and CHF, regardless of presence of dynamic outflow tract obstruction. Systolic heart murmurs (SM) are commonly diagnosed in healthy cats. The aim of this study was to determine whether the aorto-septal angle (AoSA) is associated with SM in cats. More, we hypothesised that SM are related to the presence of septal bulge, systolic anterior motion of the mitral valve (SAM) and increased aortic flow velocity (AoV). Between November 2014 and February 2018, 316 client owned cats referred for a cardiology evaluation were prospectively examined. Regardless the presence of a systolic murmur, cats with a ABSTRACTSPathways that disrupt the cardiovascular-renal axis in dogs are incompletely defined. The renal endothelin (ET-1) system may play a key role because it regulates blood pressure and sodium homeostasis in the kidney, but also mediates vascular dysfunction and pro-fibrotic/ inflammatory changes that increase cardiovascular risk in people. Urinary ET-1 (UET-1) is a marker of renal vascular and tubular ET-1 activity, and so could provide insight into changes in renal ET-1 signalling that contribute to cardiovascular-renal interactions in dogs. We hypothesised that renal ET-1 activity increases in advanced canine chronic cardiac and renal diseases.In this pilot study, we compared UET-1 and cystatin C (a marker of renal injury/dysfunction) concentrations in surplus urine from four ABSTRACTS Complete and balanced diets, one test and one control, were evaluated in this prospective, randomized, double-blind, multicenter study for effect on clinical, biochemical and echocardiographic parameters in forty-four client-owned cats with asymptomatic hypertrophic cardiomyopathy (aHCM).Cats with diastolic interventricular septum (IVSd) and/or left ventricular wall (LVWd) thickness ≥ 6 mm were included after informed ownerconsent. Examination of non-sedated, fasted cats before and after 6 and 12 months of test or control diet included auscultation, bodyweight (BW), body condition score (BCS) and echocardiography. WallClinical environment can be stressful for cats, thereby affecting blood pressure (BP) recordings.The aim was to investigate how different clinical settings, and the order (sequence) in which settings are performed, affect BP recordings in healthy cats.Ninety-six healthy cats were prospectively included. The health examination included physical examination, echocardiography, hematology, and biochemistry. Blood pressure was measured with a high-definition oscillometric (HDO) device, with cuff on tail in three clinical settings;(1) cat taken out of its carrier and placed on examination table with veterinarian present, (2) cat in its own carrier with veterinarian present, or(3) cat in its own carrier without veterinarian present. The owner was ABSTRACTS 347 Abstract Public health officials are increasingly recognizing the need to develop disease-forecasting systems to respond to epidemic and pandemic outbreaks. For instance, simple epidemic models relying on a small number of parameters can play an important role in characterizing epidemic growth and generating short-term epidemic forecasts. In the absence of reliable information about transmission mechanisms of emerging infectious diseases, phenomenological models are useful to characterize epidemic growth patterns without the need to explicitly model transmission mechanisms and the natural history of the disease. In this article, our goal is to discuss and illustrate the role of regularization methods for estimating parameters and generating disease forecasts using the generalized Richards model in the context of the 2014e15 Ebola epidemic in West Africa. Abstract Field parasitological studies consistently demonstrate the reality of polyparasitism in natural systems. However, only recently, studies from ecological and evolutionary fields have emphasised a broad spectrum of potential multiple infections-related impacts. The main goal of our review is to reunify the different approaches on the impacts of polyparasitism, not only from laboratory or human medical studies but also from field or theoretical studies. We put forward that ecological and epidemiological determinants to explain the level of polyparasitism, which regularly affects not only host body condition, survival or reproduction but also host metabolism, genetics or immune investment. Despite inherent limitations of all these studies, multiple infections should be considered more systematically in wildlife to better appreciate the importance of parasite diversity in wildlife, cumulative effects of parasitism on the ecology and evolution of their hosts. Abstract Graphical Abstract Highlights d A Powassan virus LNP-mRNA vaccine induces potently neutralizing antibodies in mice d One dose of the mRNA vaccine protects against lethal Powassan virus challenge d The antibody response to the vaccine neutralizes other tickborne flaviviruses d The vaccine cross-protects against disease following challenge with Langat virus Abstract Nowadays, the metagenomic approach has been a very important tool in the discovery of new viruses in environmental and biological samples. Here we discuss how these discoveries may help to elucidate the etiology of diseases and the criteria necessary to establish a causal association between a virus and a disease. Abstract Twenty years ago (1992), a landmark Institute of Medicine report entitled "Emerging Infections: Microbial Threats to Health in the United States" underscored the important but often underappreciated concept of emerging infectious diseases (EIDs). A review of the progress made and setbacks experienced over the past 2 decades suggests that even though many new diseases have emerged, such as SARS (severe acute respiratory syndrome) and the 2009 pandemic influenza, significant advances have occurred in EID control, prevention, and treatment. Among many elements of the increase in the capacity to control EIDs are genomics-associated advances in microbial detection and treatment, improved disease surveillance, and greater awareness of EIDs and the complicated variables that underlie emergence. In looking back over the past 20 years, it is apparent that we are in a time of great change in which both the challenge of EIDs and our responses to them are being transformed. Recent advances support guarded optimism that further breakthroughs lie ahead. Abstract Feline endometrial adenocarcinomas are uncommon malignant neoplasms that have been poorly characterized to date. In this study, we describe a uterine adenocarcinoma in a Persian cat with feline leukemia virus infection. At the time of presentation, the cat, a female Persian chinchilla, was 2 years old. The cat underwent surgical ovariohystectomy. A cross-section of the uterine wall revealed a thickened uterine horn. The cat tested positive for feline leukemia virus as detected by polymerase chain reaction. Histopathological examination revealed uterine adenocarcinoma that had metastasized to the omentum, resulting in thickening and the formation of inflammatory lesions. Based on the histopathological findings, this case was diagnosed as a uterine adenocarcinoma with abdominal metastasis. To the best of our knowledge, this is the first report of a uterine adenocarcinoma with feline leukemia virus infection. Abstract The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) infection in 2012 resulted in an increased concern of the spread of the infection globally. MERS-CoV infection had previously caused multiple health-care-associated outbreaks and resulted in transmission of the virus within families. Community onset MERS-CoV cases continue to occur. Dromedary camels are currently the most likely animal to be linked to human MERS-CoV cases. Serologic tests showed significant infection in adult camels compared to juvenile camels. The control of MERS-CoV infection relies on prompt identification of cases within health care facilities, with institutions applying appropriate infection control measures. In addition, determining the exact route of transmission from camels to humans would further add to the control measures of MERS-CoV infection. Abstract A cat was referred because of diffuse parenchymal lung disease. Close examinations revealed a swollen abdominal lymph node and multiple nodules of the liver. Mycobacterium avium subspecies hominissuis infection was confirmed by culture and single nucleotide polymorphism analysis of samples recovered from the liver and bronchoalveolar lavage. After administration of combination antibiotics for 6 months, culture results were negative. Though atonic seizures were observed during the treatment, it disappeared after isoniazid discontinuation and pyridoxal phosphate administration. On day 771 of illness, no clinical signs, lung diseases, or obvious swelling of lymph nodes was observed. This is the first report to confirm Mycobacterium avium subspecies hominissuis infection in cats through gene analysis and to completely cure it with combination antibiotics.Mycobacterium avium subspecies hominissuis (MAH), a species of nontuberculous Mycobacterium (NTM), is a slow-growing bacteria that is widely detected in the environment. In human medicine, NTM produce pulmonary infiltrates and disseminated diseases, mainly in immunocompromised hosts [5, 7] . Incidence of NTM infections is reportedly increasing worldwide, and the prevalence of NTM species varies between countries. In Japan, MAH is the most common pathogen of NTM diseases [14, 24, 25] . A previous study has revealed that combination antibiotics following guidelines for the treatment of pulmonary NTM disease achieves positive clinical results [16] .In dogs and cats, MAH infection occurs via ingestion of infected meat or water, or contact with polluted soils [6] . Despite the widespread distribution of MAH, infection in dogs and cats is uncommon due to their innate immunity [13] . Little is therefore known about the clinical implications, treatment, and prognosis of MAH infection in cats. We report here a case of disseminated MAH infection in a young male Somali cat who recovered due to combination antibiotics and describe a side effect observed during treatment.A 4-year-old neutered male Somali cat was kept completely indoors, and received a medical check-up every year. Diffuse parenchymal lung disease had been detected without any symptoms or blood test abnormalities in a medical check-up 3 months previously. Antibiotics and prednisolone (0.55-1.1 mg/kg every 24 hr) had been administered for the previous 2 months due to symptoms such as a poor appetite, moderate tachypnea, and a mild fever. The antibiotics clindamycin (5.5 mg/kg every 12 hr), doxycycline (11 mg/kg every 12 hr), and enrofloxacin (5.6 mg/kg every 24 hr) were administered separately. Despite the drug administration, his symptoms and lung disease were not improved. He was therefore referred to the Veterinary Medical Center of Osaka Prefecture University.On physical examination, a body weight of 4.4 kg, body temperature of 39.0°C, heart rate of 180/min, and rhonchi lung sounds (respiratory rate: 30/min) were observed. A complete blood cell count revealed neutropenia (Table 1) . The general chemical profile Abstract Introduction: Healthcare-associated respiratory viral infections (HARVIs) result in significant harm to infants in the neonatal intensive care unit (NICU). Healthcare workers and visitors can serve as transmission vectors to patients. We hypothesized that improved family and visitor hand hygiene (FVHH) and visitor screening would reduce HARVIs by at least 25%. Methods: This quality improvement project took place in a large tertiary NICU to reduce HARVIs. Interventions primarily focused on improving FVHH and reducing visitation by symptomatic family members and visitors. We defined correct FVHH as hand hygiene performed immediately before touching their child. Hand hygiene observations were performed by direct observation by NICU staff using a standardized tool. Interventions to improve FVHH included education of staff and visitors, reminder signs, and immediate reminders to families to prevent lapses in hand hygiene. Staff screened family and visitors before NICU entry. Symptomatic individuals were asked to defer visitation until symptoms resolved. HARVIs were identified during prospective surveillance by infection preventionists using standard definitions. Results: Baseline FVHH was 27% in 2015. After May 2017, the average FVHH remained at 85%. When reminded, family members and visitors performed hand hygiene 99% of the time. Staff screened ~129,000 people for FVHH. Between January 2013 and March 2019, there were 74 HARVIs; 80% were rhinovirus/enterovirus. After the implementation of improved FVHH, the HARVI rate decreased from 0.67 to 0.23/1,000 patient days. Conclusions: Adding interventions to improve FVHH and visitor management to existing healthcare worker prevention efforts can help reduce Abstract We report group B Betacoronavirus infection in little Japanese horseshoe bats in Iwate prefecture. We then used reversetranscription PCR to look for the coronavirus RNA-dependent RNA polymerase gene in fecal samples collected from 27 little Japanese horseshoe bats and found eight were provisionally positive. We had a success in the nucleotide sequencing of six of the eight positive samples and compared them with those of authentic coronaviruses. We found that these six samples were positive in coronavirus infection, and they belonged to the group B Betacornavirus by phylogenetic analysis. Virus isolation using the Vero cell culture was unsuccessful. Pathogenic trait of these bat coronaviruses remained unexplored. Abstract Background: Middle East Respiratory Syndrome (MERS) is a respiratory disease caused by a novel coronavirus that was identified in 2012 in Saudi Arabia. It is associated with significant mortality and morbidity. We identified factors associated with the Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) infection among suspected cases presented with sign and symptoms of upper respiratory infection or exposure to the virus. We also looked at the impact of medication history on virus transmission. Method: We included subjects with suspected MERS-CoV infection and confirmed cases of MERS infection. Subjects were excluded if there were any missing data that prevent the final analysis. Descriptive statistics were used to report demographic data. Percentages and frequencies were used to summarize the categorical variables, while means and standard deviations were calculated for continuous variables. Logistic regression was used to assess the risk of MERS-CoV infection among the suspected cases. A value of p < 0.05 was considered statistically significant. Results: A total of 16,189 suspected cases were identified, complete data were analyzed for 3154 to assess factors that are independently associated with MERS-CoV infection. MERS-CoV infection was associated with age (adjusted odds ratio [AOR] = 1.06; 95% CI [1.02-1.098], P-value = 0.004), male gender (AOR = 1.617; 95% CI [1.365-1.77], P-value < 0.001) and diabetes (AOR = 1.68; 95% CI [1.346-1.848], P-value = 0.002. There was no significant association with the other comorbidities. Medication history was not associated with an increase or decrease the likelihood of the infection. Conclusions: MERS-Cov infection is more common in male, advanced age and diabetes. No medications were associated with an increase or decrease the likelihood of the infection. This is important to focus on screening and detection to this patient population. Abstract The objectives are to introduce a provincial level surveillance system, which has been initiated in response to the MERS-CoV outbreak of South Korea, and describe findings from systematic investigation of individual admissions attributed to acute febrile illness for the first year. Abstract Mis-secreted glycoproteins (LGI1, reelin) are emerging causes of epilepsy. LMAN2L belongs to a glycoprotein secretion chaperone family. One recessive LMAN2L missense mutation predicted to impair the chaperone's interaction with glycoproteins was reported in a family with intellectual disability (ID) and remitting epilepsy. We describe four members of a family with autosomal dominant inheritance of a similar phenotype. We show that they segregate a NM_001142292.1:c.1073delT mutation that eliminates LMAN2L's endoplasmic reticulum retention signal and mislocalizes the protein from that compartment to the plasma membrane. LMAN2L mislocalization, like impaired glycoprotein interaction, disturbs brain development, including generation of developmentally restricted epilepsy. Abstract In the era of the "sickest first" policy, patients with very high model for end-stage liver disease (MELD) scores have been increasingly admitted to the intensive care unit with the expectation that they will receive a liver transplant (LT) in the absence of improvement on supportive therapies. Such patients are often admitted in a context of acute-on-chronic liver failure with extrahepatic failures. Sequential assessment of scores or classification based on organ failures within the first days after admission help to stratify the risk of mortality in this population. Although the prognosis of severely ill cirrhotic patients has recently improved, transplant-free mortality remains high. LT is still the only curative treatment in this population. Yet, the increased relative scarcity of graft resource must be considered alongside the increased risk of losing a graft in the initial postoperative period when performing LT in "too sick to transplant" patients. Variables associated with poor immediate post-LT outcomes have been identified in large studies. Despite this, the performance of scores based on these variables is still insufficient. Consideration of a patient's comorbidities and frailty is an appealing predictive approach in this population that has proven of great value in many other diseases. So far, local expertise remains the last safeguard to LT. Using this expertise, data are accumulating on favourable post-LT outcomes in very high MELD populations, particularly when LT is performed in a situation of stabilization/improvement of organ failures in selected candidates. The absence of "definitive" contraindications and the control of "dynamic" contraindications allow a "transplantation window" to be defined. This window must be identified swiftly after admission given the poor short-term survival of patients with very high MELD scores. In the absence of any prospect of LT, withdrawal of care could be discussed to ensure respect of patient life, dignity and wishes. Abstract This year, the Editorial Board has given much attention to convergence. By systematically combining diverse fields of study, efforts have been made to overcome the limitations of existing interpretations. The convergent status of rehabilitation exercises and the need to overcome medical and societal problems through convergence, which was emphasized during the MERS (Middle East respiratory syndrome) outbreak, were introduced. Now, in the last issue of the year, we reconsider the concept of penile rehabilitation, which has until now been recognized as safe. We suggest an urgent need to conduct high-quality prospective studies with regard to the biochemical recurrence of cancer, specifically with regard to the mechanism by which phosphodiesterase type 5 inhibitors (PDE5I) may affect the course of prostate cancer when taken after radical prostatectomy.Drugs like Viagra and Cialis work by deactivating the enzyme PDE5-a chemical that takes away an erection after sex by limiting blood supply.However, scientists now think that malignant melanoma is fueled by a faulty gene (a family of serine-threonine protein kinases, BRAF) which suppresses the enzyme PDE5, suggesting that PDE5 plays an important role in preventing the spread of cancer cells, including prostate cancer cells. It is feared that Viagra, and drugs like it, could be mimicking the effect of the mutated BRAF gene.The early administration of PDE5I after radical prostatectomy is helpful in the patient's fast recovery of erectile function, and it is often used clinically as a means of penile rehabilitation. However, according to one recent retrospective analysis of patients who received bilateral nerve-sparing radical prostatectomy, it was suggested that taking PDE5I might be associated with the biochemical recurrence of prostate cancer (Loeb et al., 2015; Michl et al., 2015) . Hence, the relationship between PDE5I medication and the biochemical recurrence of prostate cancer needs to be reviewed, based on reports of the impact of PDE5I on prostate can- Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV-exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedariesapart from conventional antibodies-have relatively unique, heavy chain-only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventionalVHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs-composed of the camel VHH linked to a human Fc domain lacking the CH1 exon-had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens. Abstract Kawasaki disease (KD) is an acute self-limiting inflammatory disorder, associated with vasculitis, affecting predominantly medium-sized arteries, particularly the coronary arteries. In developed countries KD is the commonest cause of acquired heart disease in childhood. The aetiology of KD remains unknown, and it is currently believed that one or more as yet unidentified infectious agents induce an intense inflammatory host response in genetically susceptible individuals. Genetic studies have identified several susceptibility genes for KD and its sequelae in different ethnic populations, including FCGR2A, CD40, ITPKC, FAM167A-BLK and CASP3, as well as genes influencing response to intravenous immunoglobulin (IVIG) and aneurysm formation such as FCGR3B, and transforming growth factor (TGF) β pathway genes. IVIG and aspirin are effective therapeutically, but recent clinical trials and meta-analyses have demonstrated that the addition of corticosteroids to IVIG is beneficial for the prevention of coronary artery aneurysms (CAA) in severe cases with highest risk of IVIG resistance. Outside of Japan, however, clinical scores to predict IVIG resistance perform suboptimally. Furthermore, the evidence base does not provide clear guidance on which corticosteroid regimen is most effective. Other therapies, including anti-TNFα, could also have a role for IVIG-resistant KD. Irrespective of these caveats, it is clear that therapy that reduces inflammation in acute KD, improves outcome. This paper summarises recent advances in the understanding of KD pathogenesis and therapeutics, and provides an approach for managing KD patients in the UK in the light of these advances. Abstract Canine infectious respiratory disease complex (CIRDC) is associated with multiple factors. The possible transmission source can be via community-acquired infection (CAI) or hospital-associated infection (HAI), but the variable factors within these two routes are not well described. This study aimed to (i) investigate a crosssectional incidence of canine respiratory viruses, including influenza (CIV), parainfluenza, distemper (CDV), respiratory coronavirus (CRCoV), adenovirus-2, and herpesvirus, in respiratory-diseased dogs, and (ii) analyze the possibly related risk factors. In total 209 dogs with respiratory illness, consisting of 133 CAI and 76 HAI dogs, were studied. Both nasal and oropharyngeal swabs were sampled from each dog and subjected for CIRDC virus detection using multiplex PCRs. Common six viruses associated with CIRDC were detected in both groups with CIV and CRCoV being predominantly found. Only CDV was significantly more prevalent in CAI than HAI dogs. Multiple virus detections were found in 81.2% and 78.9% of CAI and HAI dogs, respectively. Co-detection of CIV and CRCoV was represented the highest proportion and most often found with other CIRD viruses. Moreover, the clinical severity level was notably related to the age of infected dogs, but not to the vaccination status, sex and transmission route. Since healthy or control dogs were not included in this study, the prevalence of the CIRD virus infections could not be assessed. Abstract Connexins, the proteins that form gap junction channels, are polytopic plasma membrane (PM) proteins that traverse the plasma membrane bilayer four times. The insertion of five different connexins into the membrane of the ER was studied by synthesizing connexins in translation-competent cell lysates supplemented with pancreatic ER-derived microsomes, and by expressing connexins in vivo in several eucaryotic cell types. In addition, the subcellular distribution of the connexins was determined. In vitro-synthesis in the presence of microsomes resulted in the signal recognition particle-dependent membrane insertion of the connexins. The membrane insertion of all connexins was accompanied by an efficient proteolytic processing that was dependent on the microsome concentration. Endogenous unprocessed connexins were detectable in the microsomes used, indicating Abstract Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1-ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem-loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce 11 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the 11 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5 0 of the shift site and to polyamine levels. Abstract Coccidiosis and necrotic enteritis (NE) are among the most significant diseases affecting the poultry industry. These diseases have become more prominent in the wake of policies to reduce the use of antibiotics in animal production. This has led to more research focused on better understanding the immune system and its responses to pathogen challenge, and thus developing informed strategies to exploit immune responses that can support enhanced disease resistance and growth performance. Some chicken breeds and lines show greater resistance or susceptibility to various diseases, and thus these birds maybe able to shed light on immune processes or pathways that contribute to the more resistant/susceptible state. This review attempts to identify potentially important genes that show some consistency in (relative) up or downregulation in key tissues between the resistant and susceptible chickens. For coccidiosis and NE, relative downregulation of IL-10 and (slightly less consistently) upregulation of IFNγ appear to be features of more resistant birds. Data for IFN-α, IL-12, and IL-17D are currently less consistent. Gene expression data from NE studies have identified some potentially interesting, perhaps less well understood, immune-related genes (e.g., TCF12, BCL2, IRF2, TRAF3, TAB3, etc.,) that maybe associated with the resistant and/or susceptible phenotype. Salmonella and Campylobacter are important foodborne pathogens harbored by the chicken intestinal tract, while infectious bursal disease and infectious bronchitis are also important viral diseases of poultry. We, therefore, consider whether there are consistent features from resistant/susceptible disease models with these pathogens that relate to findings from the coccidiosis and NE studies. It is not anticipated that ideal immune responses to these pathogens will be identical but rather that consistent elements maybe identified that could help inform breeding or alternative strategies to support general disease resistance and enhanced (and efficient) flock productivity. Abstract The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and characteristics of live, recombinant viral vector vaccines. A recent publication by the V3SWG described live, attenuated, recombinant vesicular stomatitis virus (rVSV) as a chimeric virus vaccine for HIV-1 (Clarke et al., 2016). The rVSV vector system is being explored as a platform for development of multiple vaccines. This paper reviews the molecular and biological features of the rVSV vector system, followed by a template with details on the safety and characteristics of a rVSV vaccine against Zaire ebolavirus (ZEBOV). The rVSV-ZEBOV vaccine is a live, replication competent vector in which the VSV glycoprotein (G) gene is replaced with the glycoprotein (GP) gene of ZEBOV. Multiple copies of GP are expressed and assembled into the viral envelope responsible for inducing protective immunity. The vaccine (designated V920) was originally constructed by the National Microbiology Laboratory, Public Health Agency of Canada, further developed by NewLink Genetics Corp. and Merck & Co., and is now in final stages of registration by Merck. The vaccine is attenuated by deletion of the principal virulence factor of VSV (the G protein), which also removes the primary target for anti-vector immunity. The V920 vaccine caused no toxicities after intramuscular (IM) or intracranial injection of nonhuman primates and no reproductive or developmental toxicity in a rat model. In multiple studies, cynomolgus macaques immunized IM with a wide range of virus doses rapidly developed ZEBOV-specific antibodies measured in IgG ELISA and neutralization assays and were fully protected against lethal challenge with ZEBOV virus. Over 20,000 people have received the vaccine in clinical trials; the vaccine has proven to be safe and well tolerated. During the first few days after vaccination, many vaccinees experience a mild acute-phase reaction with fever, headache, myalgia, and arthralgia of short duration; this period is associated with a low-level viremia, activation of anti-viral genes, and increased levels of chemokines and cytokines. Oligoarthritis and rash appearing in the second week occur at a low incidence, and are typically mild-moderate in severity and self-limited. V920 vaccine was used in a Phase III efficacy trial during the West African Ebola epidemic in 2015, showing 100% protection against Ebola Virus Disease, and it has subsequently been deployed for emergency control of Ebola outbreaks in central Africa. The template Abstract Because primary care is the cornerstone of an effective health care system, many developed countries have striven to establish and strengthen their primary care systems. However, the primary care system in South Korea is not well established, and primary care research is still in its infancy. This study aimed to show the benefits of regular doctors as primary care providers in South Korea by analyzing the effect of regular doctor visits on emergency room (ER) visits. Methods: We analyzed cross-sectional data on 11,293 adults aged 18 years and over collected from the 2013 Korea Health Panel Survey (beta version 1.0). We classified those participants with and without regular doctors into the treatment and control groups, respectively, and estimated the average treatment effect (ATE) of having a regular doctor on ER visits. We used counterfactual framework and propensity score analysis to adjust for unevenly distributed confounding covariates between treatments and control groups. Results: The estimated conditional ATE of a regular doctor on ER visits was statistically insignificant in the general population (-0.4%; 95% confidence interval [CI], -2.0 to 1.2) and in the subgroup of patients with hypertension (-1.8%; 95% CI, -4.5 to 0.9). However, in patients with diabetes mellitus (DM), the estimated ATE was statistically significant (-5.0; 95% CI, -9.2 to -0.7). Conclusion: In the total study population, having a regular doctor did not result in a significant difference in ER visits. However, there was a decrease in ER visits in patients with DM in South Korea. Abstract The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology Abstract Coronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of doublemembrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6.the ability of these viruses to cause severe pathology and fatality. Insight into the molecular biology of how coronaviruses take over the host cell is critical for a full understanding of any known and possible future outbreaks caused by these viruses. Additionally, since membrane rearrangement is a tactic used by all known positive-sense single-stranded RNA viruses, this work adds to that body of knowledge and may prove beneficial in the development of future therapies not only for human coronavirus infections but for other pathogens as well.Citation Angelini MM, Akhlaghpour M, Neuman BW, Buchmeier MJ. 2013. Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce doublemembrane vesicles. mBio 4(4):e00524-13. Abstract Purpose We aimed to evaluate psychometric properties of the Korean version of the European Organization for Research and Treatment of Cancer (EORTC) QLQ-NMIBC24 when applied to Korean non-muscle invasive bladder cancer (NMIBC) patients.A total of 249 patients who underwent curative transurethral resection of bladder tumor (TURBT) for primary or recurrent NMIBC were asked to complete the Korean version of EORTC QLQ-C30 and -NMIBC24 questionnaires three times (preoperative, post-TURBT 3 months and 6 months). Linguistic validation and psychometric evaluation of the questionnaire was conducted.Multitrait scaling analysis confirmed satisfactory construct validity in five scales except the malaise scale. Internal consistency was good (Cronbach's alpha ! 0.70) for the five scales except the malaise scale at the all three time points. Known-group comparison analyses showed better quality-of-life (QOL) scores in patients with higher performance status as expected, and better sexual function in men than women (p < 0.05). Most of the scales had low correlations (< 0.40) with the scales in QLQ-C30 showing divergent validity, except for malaise scale which showed higher correlations (0.42 to 0.60). Responsiveness to change was consistent with clinical implications over time after TURBT.The Korean version of the EORTC QLQ-NMIBC24 has good reliability and cross-cultural validity for measuring various QOL aspects that can be self-administered to Korean NMIBC patients undergoing TURBT. Abstract Background: Given that YKL-40 is a known marker of inflammation, we sought to determine its association with urinary tract infection (UTI) in febrile children.Methods: In total, 44 children aged 0 to 24 months with febrile UTI and 35 age-and sexmatched controls with fever from other causes were enrolled in the study. ELISA was performed to determine the level of YKL-40 in urine collected from each child.The ratio of urinary YKL-40 to creatinine (Cr) was higher in the children with a UTI than in the controls (P < 0.001). The area under the ROC curve for detecting UTI was 0.88 for the urinary YKL-40/Cr ratio, 0.86 for pyuria, and 0.71 for positive nitrite on urinalysis. We applied a cut-off value of 125.23 pg/mg to urinary YKL-40/Cr for detecting UTI. Eight of nine children in the control group with pyuria had urinary YKL-40/Cr levels lower than 125.23 pg/mg, and the one child in the UTI group without pyuria or positive nitrite had a urinary YKL-40/Cr level greater than 125.23 pg/mg.Determining the levels of urinary YKL-40/Cr may help identify true cases of UTI in febrile young children, especially when they have pyuria but not nitrite, or have neither pyuria nor nitrite in the urine. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection. Abstract We previously reported widespread differential expression of long non-protein-coding RNAs (ncRNAs) in response to virus infection. Here, we expanded the study through small RNA transcriptome sequencing analysis of the host response to both severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza virus infections across four founder mouse strains of the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits. We observed differential expression of over 200 smallThe small RNAs identified also included many non-miRNA small RNAs, such as small nucleolar RNAs (snoRNAs), in addition to nonannotated small RNAs. An integrative sequencing analysis of both small RNAs and long transcripts from the same samples showed that the results revealing differential expression of miRNAs during infection were largely due to transcriptional regulation and that the predicted miRNA-mRNAIMPORTANCE Most studies examining the host transcriptional response to infection focus only on protein-coding genes. However, mammalian genomes transcribe many short and long non-protein-coding RNAs (ncRNAs). With the advent of deepsequencing technologies, systematic transcriptome analysis of the host response, including analysis of ncRNAs of different sizes, is now possible. Using this approach, we recently discovered widespread differential expression of host long (>200 nucleotide [nt]) ncRNAs in response to virus infection. Here, the samples described in the previous report were again used, but we sequenced another fraction of the transcriptome to study very short (about 20 to 30 nt) ncRNAs. We demonstrated that virus infection also altered expression of many short ncRNAs of diverse classes. Putting the results of the two studies together, we show that small RNAs may also play an important role in regulating the host response to virus infection.Citation Peng X, et al. 2011. Integrative deep sequencing of the mouse lung transcriptome reveals differential expression of diverse classes of small RNAs in response to respiratory virus infection. mBio 2(6):e00198-11. Abstract The seventh novel human infecting Betacoronavirus that causes pneumonia (2019 novel coronavirus, 2019-nCoV) originated in Wuhan, China. The evolutionary relationship between 2019-nCoV and the other human respiratory illness-causing coronavirus is not closely related. We sought to characterize the relationship of the translated proteins of 2019-nCoV with other species of Orthocoronavirinae. A phylogenetic tree was constructed from the genome sequences. A cluster tree was developed from the profiles retrieved from the presence and absence of homologs of ten 2019-nCoV proteins. The combined data were used to characterize the relationship of the translated proteins of 2019-nCoV to other species of Orthocoronavirinae. Our analysis reliably suggests that 2019-nCoV is most closely related to BatCoV RaTG13 and belongs to subgenus Sarbecovirus of Betacoronavirus, together with SARS coronavirus and Bat-SARS-like coronavirus. The phylogenetic profiling cluster of homolog proteins of one annotated 2019-nCoV protein against other genome sequences revealed two clades of ten 2019-nCoV proteins. Clade 1 consisted of a group of conserved proteins in Orthocoronavirinae comprising Orf1ab polyprotein, Nucleocapsid protein, Spike glycoprotein, and Membrane protein. Clade 2 comprised six proteins exclusive to Sarbecovirus and Hibecovirus. Two of six Clade 2 nonstructural proteins, NS7b and NS8, were exclusively conserved among 2019-nCoV, BetaCoV_RaTG, and BatSARS-like Cov. NS7b and NS8 have previously been shown to affect immune response signaling in the SARS-CoV experimental model. Thus, we speculated that knowledge of the functional changes in the NS7b and NS8 proteins during evolution may provide important information to explore the human infective property of 2019-nCoV. Abstract Background. Molecular detection methods allow for the simultaneous detection of several infectious agents. This study assesses whether co-infection with 2 viruses as compared with 1 is associated with increased hospitalization in those with acute respiratory infections.Methods. We prospectively enrolled a cohort of pediatric and adult participants with influenza-like illness during 2010-2014 in Mexico. Clinical information and respiratory samples were collected at enrollment. Respiratory viruses were detected with multiplex polymerase chain reaction (PCR) and influenza-specific reverse transcription PCR assays. Participants were followed for 14 and 28 days after inclusion. Severity of disease, as measured by hospitalization with acute respiratory infections, was compared between single and dual viral infections.Results. Among 5662 participants in the study, either 1 (n = 3285) or 2 (n = 641) viruses were detected in 3926 participants. Rhinovirus (n = 1433), influenza (n = 888), and coronaviruses (n = 703) were the most frequently detected viruses (either alone or in co-infection). Bocavirus, respiratory syncytial virus (RSV), metapneumovirus, and rhinovirus cases were hospitalized more often than other viruses. Bocavirus+rhinovirus cases were hospitalized more often than those with rhinovirus alone (but not bocavirus alone). RSV cases were more likely to be hospitalized than cases with co-infections of RSV and parainfluenza virus or coronavirus. Metapneumovirus cases were hospitalized more often than those co-infected with metapneumovirus+coronavirus.Conclusions. In this study, detection of 2 viruses did not significantly increase hospitalizations compared with single virus infections. Larger studies will allow for distinguishing between sequential and simultaneous infection and for a better understanding of the role of each virus during the evolution of acute respiratory episodes. Abstract The devastating Ebola virus (EBOV) outbreak in West Africa in 2013-2016 has flagged the need for the timely development of vaccines for high-threat pathogens. To be better prepared for new epidemics, the WHO has compiled a list of priority pathogens that are likely to cause future outbreaks and for which R&D efforts are, therefore, paramount (R&D Blueprint: https://www.who.int/blueprint/priority-diseases/en/). To this end, the detailed characterization of vaccine platforms is needed. The vesicular stomatitis virus (VSV) has been established as a robust vaccine vector backbone for infectious diseases for well over a decade. The recent clinical trials testing the vaccine candidate VSV-EBOV against EBOV disease now have added a substantial amount of clinical data and suggest VSV to be an ideal vaccine vector candidate for outbreak pathogens. In this review, we discuss insights gained from the clinical VSV-EBOV vaccine trials as well as from animal studies investigating vaccine candidates for Blueprint pathogens.ARTICLE HISTORY Abstract Background: It is crucial to understand the current status of clinical laboratory practices for the largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infections in the Republic of Korea to be well prepared for future emerging infectious diseases.We conducted a survey of 49 clinical laboratories in medical institutions and referral medical laboratories. A short questionnaire to survey clinical laboratory practices relating to MERS-CoV diagnostic testing was sent by email to the directors and clinical pathologists in charge of the clinical laboratories performing MERS-CoV testing. The survey focused on testing volume, reporting of results, resources, and laboratory safety.Results: A total of 40 clinical laboratories responded to the survey. A total of 27,009 MERS-CoV real-time reverse transcription PCR (rRT-PCR) tests were performed. Most of the specimens were sputum (73.5%). The median turnaround time (TAT) was 5.29 hr (first and third quartile, 4.11 and 7.48 hr) in 26 medical institutions. The median TAT of more than a half of the laboratories (57.7%) was less than 6 hr. Many laboratories were able to perform tests throughout the whole week. Laboratory biosafety preparedness included class II biosafety cabinets (100%); separated pre-PCR, PCR, and post-PCR rooms (88.6%); negative pressure pretreatment rooms (48.6%); and negative pressure sputum collection rooms (20.0%).Clinical laboratories were able to quickly expand their diagnostic capacity in response to the 2015 MERS-CoV outbreak. Our results show that clinical laboratories play an important role in the maintenance and enhancement of laboratory response in preparation for future emerging infections. Abstract Objectives: Complement activation product C5a plays a critical role in systemic inflammatory response syndrome induced by viruses, bacteria, and toxic agents including paraquat poisoning. This study is to explore the efficiency of anti-C5a-based intervention on systemic inflammatory responses induced by paraquat poisoning. Design: Study of cynomolgus macaque model and plasma from paraquat-poisoning patients. Setting: Laboratory investigation.Subjects: Cynomolgus macaque (n = 12) and samples of plasma from patients (n = 16). Interventions: The neutralizing antihuman C5a antibody (IFX-1) was administered to investigate the new treatment strategy for paraquat-induced systemic inflammatory responses in cynomolgus macaque model. In addition, C5a activation in plasma of paraquat patients was blocked by IFX-1 to investigate the blockade role of anti-C5a antibody in activation of inflammatory cells. Measurements and Main Results: Dysregulated complement activation and the subsequent cytokine storm were found in patients with acute lung injury and in a primate model of paraquat poisoning. Targeted inhibition of C5a by IFX-1 led to marked alleviation of systemic inflammatory responses and multiple organ damage in the primate model. In addition, blockade of C5a activity in plasma from patients completely inhibited activation of CD11b on blood granulocytes from normal donors, suggesting that IFX-1 may alleviate the excessive activation of inflammatory responses and have clinical utility for patients with acute lung injury. Conclusions: Anti-C5a antibodies such as IFX-1 may be used as effective therapeutics for treatment of those suffering from systemic inflammatory responses induced by chemical poisoning like paraquat. (Crit Care Med 2018; 46:e419-e425) Abstract A new human betacoronavirus in lineage c, tentatively called HCoV-EMC, was isolated from a patient from the Kingdom of Saudi Arabia who died from acute severe pneumonia and renal failure. The viral RNA has been detected in eight additional cases. Sequencing and bioinformatic analysis of the viral genomic RNA showed that it is a novel virus not previously detected in any other species and that its closest relatives are two Asian bat coronaviruses. HCoV-EMC may represent a sporadic spillover to humans from an unknown animal reservoir. In a recent article, van Boheemen et al. demonstrated how state-of-theart sequencing technology and bioinformatic analysis can quickly provide critical insight into the viral genome sequence, phylogeny, replication strategy, and potential drug and vaccine targets and generate tools to evaluate the possible epidemic risk associated with this novel human virus.Citation Holmes KV, Dominguez SR. 2013. The new age of virus discovery: genomic analysis of a novel human betacoronavirus isolated from a fatal case of pneumonia. mBio 4(1):e00548-12. Abstract Background. Registration of interventional trials of Food and Drug Administration-regulated drug and biological products and devices became a legal requirement in 2007; the vast majority of these trials are registered in ClinicalTrials.gov. An analysis of ClinicalTrials.gov offers an opportunity to define the clinical research landscape; here we analyze 10 years of infectious disease (ID) clinical trial research.Methods. Beginning with 166 415 interventional trials registered in ClinicalTrials.gov from 2007-2017, ID trials were selected by study conditions and interventions. Relevance to ID was confirmed through manual review, resulting in 13 707 ID trials and 152 708 non-ID trials.Results. ID-related trials represented 6.9%-9.9% of all trials with no significant trend over time. ID trials tended to be more focused on treatment and prevention, with a focus on testing drugs, biologics, and vaccines. ID trials tended to be large, randomized, and nonblinded with a greater degree of international enrollment. Industry was the primary funding source for 45.2% of ID trials. Compared with the global burden of disease, human immunodeficiency virus/AIDS and hepatitis C trials were overrepresented, and lower respiratory tract infection trials were underrepresented. Hepatitis C trials fluctuated, keeping with a wave of new drug development. Influenza vaccine trials peaked during the 2009 H1N1 swine influenza outbreak.Conclusions. This study presents the most comprehensive characterization of ID clinical trials over the past decade. These results help define how clinical research aligns with clinical need. Temporal trends reflect changes in disease epidemiology and the impact of scientific discovery and market forces. Periodic review of ID clinical trials can help identify gaps and serve as a mechanism to realign resources. Abstract Purpose: Despite the availability of molecular methods, identification of the causative virus in children with acute respiratory infections (ARIs) has proven difficult as the same viruses are often detected in asymptomatic children. Methods: Multiplex reverse transcription polymerase chain reaction assays were performed to detect 15 common respiratory viruses in children under 15 years of age who were hospitalized with ARI between January 2013 and December 2015. Viral epidemiology and clinical profiles of single virus infections were evaluated. Results: Of 3,505 patients, viruses were identified in 2,424 (69.1%), with the assay revealing a single virus in 1,747 cases (49.8%). While major pathogens in single virus-positive cases differed according to age, human rhinovirus (hRV) was common in patients of all ages. Respiratory syncytial virus (RSV), influenza virus (IF), and human metapneumovirus (hMPV) were found to be seasonal pathogens, appearing from fall through winter and spring, whereas hRV and adenovirus (AdV) were detected in every season. Patients with ARIs caused by RSV and hRV were frequently afebrile and more commonly had wheezing compared with patients with other viral ARIs. Neutrophil-dominant inflammation was observed in ARIs caused by IF, AdV, and hRV, whereas lymphocyte-dominant inflammation was observed with RSV A, parainfluenza virus, and hMPV. Monocytosis was common with RSV and AdV, whereas eosinophilia was observed with hRV. Conclusion: In combination with viral identification, recognition of virus-specific clinical and laboratory patterns will expand our understanding of the epidemiology of viral ARIs and help us to establish more efficient therapeutic and preventive strategies. Abstract Recent advances in the development and discovery of pharmacological interventions within the ubiquitin-proteasome system (UPS) have uncovered an enormous potential for possible novel treatments of neurodegenerative disease, cancer, immunological disorder and microbial infection. Interference with proteasome activity, although initially considered unlikely to be exploitable clinically, has already proved to be very effective against haematological malignancies, and more specific derivatives that target subsets of proteasomes are emerging. Recent small-molecule screens have revealed inhibitors against ubiquitin-conjugating and -deconjugating enzymes, many of which have been evaluated for their potential use as therapeutics, either as single agents or in synergy with other drugs. Here, we discuss recent advances in the characterisation of novel UPS modulators (in particular, inhibitors of ubiquitin-conjugating and -deconjugating enzymes) and how they pave the way towards new therapeutic approaches for the treatment of proteotoxic disease, cancer and microbial infection. Abstract Motivation: An important aspect of infectious disease research involves understanding the differences and commonalities in the infection mechanisms underlying various diseases. Systems biology-based approaches study infectious diseases by analyzing the interactions between the host species and the pathogen organisms. This work aims to combine the knowledge from experimental studies of hostpathogen interactions in several diseases to build stronger predictive models. Our approach is based on a formalism from machine learning called 'multitask learning', which considers the problem of building models across tasks that are related to each other. A 'task' in our scenario is the set of host-pathogen protein interactions involved in one disease. To integrate interactions from several tasks (i.e. diseases), our method exploits the similarity in the infection process across the diseases. In particular, we use the biological hypothesis that similar pathogens target the same critical biological processes in the host, in defining a common structure across the tasks. Results: Our current work on host-pathogen protein interaction prediction focuses on human as the host, and four bacterial species as pathogens. The multitask learning technique we develop uses a taskbased regularization approach. We find that the resulting optimization problem is a difference of convex (DC) functions. To optimize, we implement a Convex-Concave procedure-based algorithm. We compare our integrative approach to baseline methods that build models on a single host-pathogen protein interaction dataset. Our results show that our approach outperforms the baselines on the training data. We further analyze the protein interaction predictions generated by the models, and find some interesting insights. Abstract Health professionals' knowledge and awareness of the disease surveillance is essential for reporting diseases to health departments. This study aimed to assess the knowledge and attitudes of Jordanian physicians toward public health surveillance of communicable disease. A cross-sectional study was conducted among resident doctors who were working in 4 main Ministry of Health hospitals and 2 teaching hospitals in Jordan in September 2017. A self-administered paper-based questionnaire was used to collect the data. The questionnaire collected information about sociodemographic and practicerelated characteristics of physicians and included items to assess their knowledge of surveillance and reporting practices. This study included 223 physicians (152 males and 71 females). About 60.1% of the residents were graduates from medical schools in Jordan and the remaining (39.9%) were graduates from medical schools in other countries. Approximately two thirds of residents (62.3%) were doing their residency in Ministry of Health hospitals and the rest (37.7%) in 2 teaching hospitals. Only 44.8% of physicians had defined surveillance correctly. Only 27.4% of physicians had been educated or trained on surveillance. About 39.5% of physicians had filled at least one report form during their practice. The main reasons for not reporting mandatory diseases were high workload (49.8%) and being not trained on reporting diseases (46.6%). A relatively high percentage of physicians have insufficient knowledge of surveillance and reporting of notifiable communicable diseases. Training of physicians on surveillance and diseases notification is highly needed. The practice of disease notification should be enforced in Jordanian hospitals.Incomplete and untimely reporting of notifiable communicable diseases are one of the main challenges facing the effective implementation of the disease surveillance systems. How does your research contribute to the field? This research helps to understand the physicians' knowledge, awareness, and compliance of disease surveillance and notification in Jordan. Abstract Despite of the role of domestic dogs as reservoirs for threatening viral diseases for wild carnivores, few studies have focused to identify circulation of viruses among dogs living in human/wildlife interfaces. To identify canine parvovirus (CPV) types circulating in dogs living in an Atlantic forest biome, faecal samples (n = 100) were collected at the same period (one week) corresponding to each of four areas, during 2014 to 2016 and corresponded to 100 different individuals. CPV was isolated in cell culture from 67 out 100 (67%) samples from healthy dogs.Cytopathic effects were characterized by total or partial cell culture lysis. Genome sequences of CPV-2a (10%), CPV-2b (7%) and CPV-2c (50%) were concomitantly detected by PCR and nucleotide sequencing. The current study addresses the importance of monitoring CPV circulation among dogs presenting potential contact with wildlife species. Abstract Over the past decade, the RNA revolution has revealed thousands of non-coding RNAs that are essential for cellular regulation and are misregulated in disease. While the development of methods and tools to study these RNAs has been challenging, the power and promise of small molecule chemical probes is increasingly recognized. To harness existing knowledge, we compiled a list of 116 ligands with reported activity against RNA targets in biological systems (R-BIND). In this survey, we examine the RNA targets, design and discovery strategies, and chemical probe characterization techniques of these ligands. We discuss the applicability of current tools to identify and evaluate RNA-targeted chemical probes, suggest criteria to assess the quality of RNA chemical probes and targets, and propose areas where new tools are particularly needed. We anticipate that this knowledge will expedite the discovery of RNA-targeted ligands and the next phase of the RNA revolution. Abstract Calf diarrhea caused by infectious agents is associated with economic losses in the cattle industry. The purpose of this study was to identify the causative agents and epidemiological characteristics of diarrhea in Korean native calves (KNC). In total, 207 diarrheal KNC aged less than 7 months were investigated. Fecal samples collected from the rectum were examined for causative agents using polymerase chain reaction (PCR) or real-time PCR and the number of oocysts were counted. Fourteen causative agents were detected from 164 of the 207 diarrheal KNC. Rotavirus was the most common agent (34.8%), followed by Eimeria spp. (31.7%), Escherichia coli (22.0%), Giardia spp. (14.0%), Clostridium difficile (9.8%), bovine viral diarrhea virus (8.5%), coronavirus (7.9%), Cryptosporidium spp. (7.3%), torovirus (6.7%), parvovirus (5.5%), norovirus (4.9%), kobuvirus (1.8%), adenovirus (1.2%), and Salmonella spp. (0.6%). About 95 (57.9%) of 164 calves were infected with a single causative agent and 42.1% were infected by multiple agents. No significant difference was observed in mortality between calves infected with a single agent and multiple agents. The occurrence of diarrhea caused by rotavirus, Eimeria spp., kobuvirus, and Giardia spp. was significantly different based on onset age, and the prevalence of diarrhea caused by rotavirus or C. difficile was significantly different between seasons. This study help the understanding of KNC diarrhea for the development of an effective strategy for disease prevention and control, especially in Eastern provinces of South Korea. Abstract Cryo-electron microscopy (cryo-EM) plays an increasingly more important role in structural biology. With the construction of an arm of the Chinese National Protein Science Facility at Tsinghua University, biological cryo-EM has entered a phase of rapid development in China. This article briefly reviews the history of biological cryo-EM in China, describes its current status, comments on its impact on the various biological research fields, and presents future outlook. Abstract Consultation-liaison psychiatry (CLP) was first established in China after liberation in 1949. It has developed more rapidly over the last two decades but, despite major regional differences in the level of CLP, the overall practice of CLP in the country remains quite basic, largely limited to case-based consultation with other medical departments. There is little ongoing collaboration between departments of psychiatry and other departments, and medical students and nonpsychiatric clinicians rarely get training in CLP. Abstract RNA dependent DNA-polymerases, reverse transcriptases, are key enzymes for retroviruses and retroelements. Their fidelity, including indel generation, is significant for their use as reagents including for deep sequencing. Here, we report that certain RNA template structures and G-rich sequences, ahead of diverse reverse transcriptases can be strong stimulators for slippage at slippageprone template motif sequence 3 of such 'slippagestimulatory' structures. Where slippage is stimulated, the resulting products have one or more additional base(s) compared to the corresponding template motif. Such structures also inhibit slippagemediated base omission which can be more frequent in the absence of a relevant stem-loop. Slippage directionality, base insertion and omission, is sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5 adjacent base. The retrotransposon-derived enzyme TGIRT exhibits more slippage in vitro than the retroviral enzymes tested including that from HIV. Structure-mediated slippage may be exhibited by other polymerases and enrich gene expression. A cassette from Drosophila retrotransposon Dme1 chrX 2630566, a candidate for utilizing slippage for its GagPol synthesis, exhibits strong slippage in vitro. Given the widespread occurrence and importance of retrotransposons, systematic studies to reveal the extent of their functional utilization of RT slippage are merited. Abstract In December 2019, several patients with pneumonia of an unknown cause were detected in Wuhan, China. On 7 January 2020, the causal organism was identified as a new coronavirus, later named as the 2019 novel coronavirus (2019-nCoV). Genome sequencing found the genetic sequence of 2019-nCoV homologous to that of severe acute respiratory syndrome-associated coronavirus. As of 29 January 2020, the virus had been diagnosed in more than 7000 patients in China and 77 patients in other countries. It is reported that both symptomatic and asymptomatic patients with 2019-nCoV can play a role in disease transmission via airborne and contact. This finding has caused a great concern about the prevention of illness spread. The clinical features of the infection are not specific and are often indistinguishable from those of other respiratory infections, making it difficult to diagnose. Given that the virus has a strong ability to spread between individuals, it is of top priority to identify potential or suspected patients as soon as possible-or the virus may cause a serious pandemic. Therefore, a precision medicine approach to managing this disease is urgently needed for detecting and controlling the spread of the virus. In this article, we present such an approach to managing 2019-nCoV-related pneumonia based on the unique traits of the virus recently revealed and on our experience with coronaviruses at West China Hospital in Chengdu, China. Abstract All cellular functions, ranging from regular cell maintenance and homeostasis, specialized functions specific to cellular types, or generating responses due to external stimulus, are mediated by proteins within the cell. Regulation of these proteins allows the cell to alter its behavior under different circumstances. A major mechanism of protein regulation is utilizing protein kinases and phosphatases; enzymes that catalyze the transfer of phosphates between substrates [1]. Proteins involved in phosphate signaling are well studied and include kinases and phosphatases that catalyze opposing reactions regulating both structure and function of the cell. Kinomics is the study of kinases, phosphatases and their targets, and has been used to study the functional changes in numerous diseases and infectious diseases with aims to delineate the cellular functions affected. Identifying the phosphate signaling pathways changed by certain diseases or infections can lead to novel therapeutic targets. However, a daunting 518 putative protein kinase genes have been identified [2], indicating that this protein family is very large and complex. Identifying which enzymes are specific to a particular disease can be a laborious task. In this review, we will provide information on large-scale systems biology methodologies that allow global screening of the kinome to more efficiently identify which kinase pathways are pertinent for further study. Abstract Zoonoses are frequently reported, and outbreaks of the highly pathogenic influenza virus, severe acute respiratory syndrome, and Middle East respiratory syndrome have occurred recently, in Africa, the Middle East, and Southeast Asia. Sterilization using a chemical reactor with plasma assisted catalytic technology (PACT) was investigated. Tests were carried out on the feline calicivirus (FCV) vaccine strain F9, which is a surrogate of airborne pathogen human norovirus. Results showed that the PACT device could inactivate FCV, which passed through the plasma chamber. Sterilization rate may be more than 99.99% (below the detection limit). These results indicate that PACT may be an effective mean to inactivate many viruses, including human norovirus, and potentially other airborne, infectious microorganisms. Abstract Previous studies have examined different strategies for siRNa delivery with varying degrees of success. These include use of viral vectors, cationic liposomes, and polymers. several copolymers were designed and synthesized based on blocks of poly(ethylene glycol) PeG, poly(propylene glycol) PPG, and poly(l-lysine). These were designated as P1, P2, and P3. We studied the copolymer self-assembly, siRNa binding, particle size, surface potential, architecture of the complexes, and siRNa delivery. silencing of GFP using copolymer P3 to deliver GFP-specific siRNa to Neuro-2a cells expressing GFP was almost as effective as using Lipofectamine 2000, with minimal cytotoxicity. Thus, we have provided a new copolymer platform for siRNa delivery that we can continue to modify for improved delivery of siRNa in vitro and eventually in vivo. Figure 9 . Gene silencing of GFP protein in Neuro 2a/GFP cells at a final concentration of 80 nM siRNa. cells were treated with complexes of copolymer P1, P2, and P3 with GFP siRNa at N/P ratio of 6, 12, and 24. cells were also treated with naked siRNa or Lipofectamine-complexed siRNas as additional controls. Abstract Background: Infection by multidrug-resistant (MDR) pathogens leads to poor patient outcomes in intensive care units (ICUs). Contact precautions are necessary to reduce the transmission of MDR pathogens. However, the importance of the surrounding environment is not well known. We studied the effects of ICU relocation on MDR respiratory pathogen detection rates and patient outcomes. Methods: Patients admitted to the ICU before and after the relocation were retrospectively analyzed. Baseline patient characteristics, types of respiratory pathogens detected, antibiotics used, and patient outcomes were measured. Results: A total of 463 adult patients admitted to the ICU, 4 months before and after the relocation, were included. Of them, 234 were admitted to the ICU before the relocation and 229 afterward. Baseline characteristics, including age, sex, and underlying comorbidities, did not differ between the two groups. After the relocation, the incidence rate of MDR respiratory pathogen detection decreased from 90.0 to 68.8 cases per 1,000 patient-days, but that difference was statistically insignificant. The use of colistin was significantly reduced from 53.5 days (95% confidence interval [CI], 20.3 to 86.7 days) to 18.7 days (95% CI, 5.6 to 31.7 days). Furthermore, the duration of hospital stay was significantly reduced from a median of 29 days (interquartile range [IQR], 14 to 50 days) to 21 days (IQR, 11 to 39 days). Conclusions: Incidence rates of MDR respiratory pathogen detection were not significantly different before and after ICU relocation. However, ICU relocation could be helpful in reducing the use of antibiotics against MDR pathogens and improving patient outcomes. Abstract Consensus Statements of the American College of Veterinary Internal Medicine (ACVIM) provide the veterinary community with up-to-date information on the pathophysiology, diagnosis, and treatment of clinically important animal diseases. The ACVIM Board of Regents oversees selection of relevant topics, identification of panel members with the expertise to draft the statements, and other aspects of assuring the integrity of the process. The statements are derived from evidence-based medicine whenever possible and the panel offers interpretive comments when such evidence is inadequate or contradictory. A draft is prepared by the panel, followed by solicitation of input by the ACVIM membership which may be incorporated into the statement. It is then submitted to the Journal of Veterinary Internal Medicine, where it is edited prior to publication. The authors are solely responsible for the content of the statements.Immune-mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune-mediated erythrocyte destruction, and adverse consequences of long-term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence-based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for Abstract Previously developed Asn-Gly-Arg (NGR) peptide-modified multifunctional poly(ethyleneimine)-poly(ethylene glycol) (PEI-PEG)-based nanoparticles (TPIC) have been considered to be promising carriers for the co-delivery of DNA and doxorubicin (DOX). As a continued effort, the aim of the present study was to further evaluate the interaction between TPIC and human umbilical vein endothelial cells (HUVEC) to better understand the cellular entry mechanism. In the present investigation, experiments relevant to co-localization, endocytosis inhibitors and factors influencing the internalization were performed. Without any treatment, there was no co-localization between aminopeptidase N/CD13 (APN/CD13) and caveolin 1 (CAV1). However, co-localization between CD13 and CAV1 was observed when cells were incubated with an anti-CD13 antibody or TPIC. As compared with antibody treatment, TPIC accelerated the speed and enhanced the degree of co-localization. TPIC entered HUVEC not only together with CD13 but also together with CAV1. However, this internalization was not dependent on the enzyme activity of CD13 but could be inhibited by methyl-β-eyclodextfin (MβCD), further identifying the involvement of caveolae-mediated endocytosis (CvME). This conclusion was also verified by endocytosis inhibitor experiments. Abstract Background: Plasma citrulline (CIT) concentration is considered to be a reliable marker of functional enterocyte mass, primarily in humans. However, information about CIT levels along with related metabolites, arginine (ARG), nitric oxide (NO), and ammonia in neonatal calves are lacking.Objectives: To compare plasma CIT, ARG, NO, and whole blood ammonia concentrations in neonatal calves with acute diarrhea with those in healthy calves and to assess their possible relationships with diarrhea-related criteria.Animals: Seventy neonatal calves (60 with acute diarrhea and 10 healthy).: Observational case-control study. Diarrheic calves were classified into subgroups on the basis of etiology, severity of diarrhea, degree of dehydration, and systemic inflammatory response syndrome (SIRS) status. Plasma CIT and ARG concentrations were measured by liquid chromatography/tandem mass spectrometry. Results: Plasma CIT (median [range]: 67.5 [61.9-75.4] vs 30.1 [15.0-56.1] μmol/L) and ARG (170.7 [148.5-219.5] vs 106.1 [54.4-190.7] μmol/L) were lower and plasma NO (4.42 [3.29-5.58] vs 6.78 [5.29-8.92] μM) and blood ammonia concentrations (28.7 [26.1-36.9] vs 59.8 [34.6-99.5] μmol/L) were higher in the neonatal calves with diarrhea (P < .001). Plasma CIT (β = −0.29, P = .02), ARG (β = −0.33, P = .01), NO (β = 0.55, P < .001), and blood ammonia (β = 0.63, P <.001) were affected by SIRS status. Except for ammonia (0.52), the effects sizes for severity of diarrhea and degree of dehydration were small (ηp2 ≤ 0.45) for CIT, ARG, and NO. Conclusions and Clinical Importance: The changes in these variables might have diagnostic, prognostic, and therapeutic value in diarrheic neonatal calves. K E Y W O R D S amino acids, dehydration, etiology, infectious diseases, systemic inflammatory response, urea cycle Abstract The devastation caused by the Ebola virus disease (EVD) outbreak in West Africa has brought to the fore a number of important ethical debates about how best to respond to a health crisis. These debates include issues related to prevention and containment, management of the health care workforce, clinical care, and research design, all of which are situated within the overarching moral problem of severe transnational disadvantage, which has very real and specific impacts upon the ability of citizens of EVD-affected countries to respond to a disease outbreak. Ethical issues related to prevention and containment include the appropriateness and scope of quarantine and isolation within and outside affected countries. The possibility of infection in health care workers impelled consideration of whether there is an obligation to provide health services where personal protection equipment is inadequate, alongside the issue of whether the health care workforce should have special access to experimental treatment and care interventions under development. In clinical care, ethical issues include the standards of care owed to people who comply with quarantine and isolation restrictions. Ethical issues in research include appropriate study design related to experimental vaccines and treatment interventions, and the sharing of data and biospecimens between research groups. The compassionate use of experimental drugs intersects both with research ethics and clinical care. The role of developed countries also came under scrutiny, and we concluded that developed countries have an obligation to contribute to the containment of EVD infection by contributing to the strengthening of local health care systems and infrastructure in an effort to provide fair benefits to communities engaged in research, ensuring that affected countries have ready and affordable access to any therapeutic or preventative interventions developed, and supporting affected countries on their way to recovery from the impact of EVD on their social and economic lives. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) is continuously spreading and causing severe and fatal acute respiratory disease in humans. Prophylactic and therapeutic strategies are therefore urgently needed to control MERS-CoV infection. Here, we generated a humanized monoclonal antibody (mAb), designated hMS-1, which targeted the MERS-CoV receptor-binding domain (RBD) with high affinity. hMS-1 significantly blocked MERS-CoV RBD binding to its viral receptor, human dipeptidyl peptidase 4 (hDPP4), potently neutralized infection by a prototype MERS-CoV, and effectively cross-neutralized evolved MERS-CoV isolates through recognizing highly conserved RBD epitopes. Notably, single-dose treatment with hMS-1 completely protected hDPP4 transgenic (hDPP4-Tg) mice from lethal infection with MERS-CoV. Taken together, our data suggest that hMS-1 might be developed as an effective immunotherapeutic agent to treat patients infected with MERS-CoV, particularly in emergent cases. Abstract Acute respiratory tract infection (ARTI) is the most common causes of outpatient visit and hospital admission for children. The study aimed to report epidemiological data on respiratory viruses in a university-affiliated children's hospital.The study was a retrospective study conducted in a university affiliated children's hospital from 2016 May to 2017 April. The results of all nasopharyngeal swab and sputum samples sent for the test for respiratory viruses (adenovirus, influenza A, influenza B, and respiratory syncytial virus) were extracted from the electronic healthcare records. Clinical characteristics were compared between groups with positive versus negative results for respiratory viruses. Multivariable regression models were employed by including age, gender, type of sample (swab vs sputum), source (emergency department vs others), and season to explore the independent factors associated with positive results for respiratory viruses.A total of 34,961 samples were identified during the study period. A total of 3102 (8.9%) samples were positive for adenovirus, 2811 (8.0%) were positive for influenza A, 3460 (9.9%) were positive for influenza B, and 4527 (13.0%) were positive for respiratory syncytial virus. The positive rate of adenovirus was highest in April (50.8%), and lowest in November (3%). The absolute number of positive samples for adenovirus was highest in June (n = 587) and April (n = 544). For the test of influenza A, age was independently associated with positive result. With 1 year increase in age, the odds of positive result increased by 12% (odds ratio [OR]: 1.12; 95% confidence interval [CI]: 1.11-1.13; P < .001). As compared with the autumn, the summer showed significantly lower rate of positive for RSV (OR: 0.49; 95% CI: 0.38-0.62; P < .001), whereas the winter had higher risk of positive result (OR: 3.88; 95% CI: 3.37-4.50; P < .001).The study reported epidemiological data on the prevalence of respiratory viruses in a large tertiary care children's hospital. Age, gender, type of sample, source, and season were associated with the positive rates for respiratory viruses.Abbreviations: ARDS = acute respiratory distress syndrome, ARTI = acute respiratory tract infection, ER = emergency room, HER = electronic healthcare record, RSV = respiratory syncytial virus. Abstract In this study, we report that human plantar fascia consists of two distinct tissues with differential structural properties. These tissues also contain stem/progenitor cells with differential biological properties. The mechanobiological responses of these two plantar fascia stem cells also differ in terms of expression of collagen I and IV, non-ligament-related genes, and proinflammatory genes. The production of inflammatory agents (prostaglandin E 2 , interleukin-6) and matrix degradative enzymes (matrix metalloproteinase-1, matrix metalloproteinase-2) are also different between the two types of plantar fascia stem cells. Based on the findings from this study, we suggest that plantar fasciitis results from the aberrant mechanobiological responses of the stem cells from plantar fascia sheath and core tissues. Our findings may also be used to devise tissue engineering approaches to treat plantar fascia injury effectively. Abstract Viruses are replication competent genomes which are relatively gene-poor. Even the largest viruses (i.e. Herpesviruses) encode only slightly .200 open reading frames (ORFs). However, because viruses replicate obligatorily inside cells, and considering that evolution may be driven by a principle of economy of scale, it is reasonable to surmise that many viruses have evolved the ability to co-opt cell-encoded proteins to provide needed surrogate functions. An in silico survey of viral sequence databases reveals that most positive-strand and double-stranded RNA viruses have ORFs for RNA helicases. On the other hand, the genomes of retroviruses are devoid of virally-encoded helicase. Here, we review in brief the notion that the human immunodeficiency virus (HIV-1) has adopted the ability to use one or more cellular RNA helicases for its replicative life cycle. Abstract Background: Chronic Hepatitis B (CHB) remains a major problem for global public health. Viral persistence and immune defects are the two major reasons for CHB, and it was hypothesized that based on a transient clearance of serum viral DNA and HBsAg "window stage", active immunization against hepatitis B virus (HBV) might initiate effective host immune responses versus HBV to achieve functional cure of CHB. Methods: Two experimental mouse models that mice hydrodynamic injected HBV DNA or infected with recombinant AAV/HBV were used. The "sandwich" therapeutic effect by using a potent human anti-HBsAg neutralizing monoclonal antibody (G12) in combination with antiviral drug tenofovir disoproxil fumarate (TDF), followed by active immunization with HBsAg-HBsAb (mYIC) was evaluated. Findings: A single G12 injection rapidly cleared serum HBsAg in HDI-HBV carrier mice, with a synergistic effect in decreasing viral DNA load when TDF was given orally. When both serum viral DNA and HBsAg load became low or undetectable, mYIC was administered. A more effective clearance of viral DNA and HBsAg was observed and serum HBsAb was developed only in these "sandwich"-treated mice. Efficient intrahepatic anti-HBV immune responses were also observed in these mice, including the formation of aggregates of myeloid cells with CD8 + T cells and increased TNF-α, granzyme B production.The "sandwich" combination therapy not only efficiently decreased HBsAg and HBV DNA levels but also induced effective cellular and humoral immunity, which may result in functional cure of CHB.Research in context sectionPersistence of HBV cccDNA and various host immune defects were shown as the two main obstacles for HBV clearance. Blocking a single target will not be sufficient to achieve functional cure of CHB. A very small percentage of CHB patients could attain "functional cure" by antiviral treatment fol- * Corresponding authors. .cn (Z. Yuan). lowed by interferon therapy. Therapeutic vaccination for CHB showed substantial efficacy. A humanized HBsAb G12 could efficiently block HBV infection and decrease HBsAg level in mice model.This research showed that when both serum viral DNA and HB-sAg load became low or undetectable by antiviral and HBsAb treatment, therapeutic vaccine (mYIC) add on induced effective, host humoral and intrahepatic cellular anti-HBV immune responses, with more rapid and efficient clearance of HBV and HBsAg. Abstract A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration. Abstract The possible occurrence of a highly pathogenic influenza strain is of concern to health authorities worldwide. It is believed that developing countries may experience a higher illness attack rate compared to developed countries. Intervention strategies shown by modelling studies to be highly effective for developed countries give limited guidance as to the impact which an influenza pandemic may have on low-income countries such as Papua New Guinea (PNG), given different demographics and resource constraints. To address this, an individual-based model of a PNG community was created, used to simulate the spread of a novel influenza strain and the results compared to those obtained from a comparable Australian model.Design A modelling study.Setting The towns of Madang in PNG (population ~35,000) and Albany (population ~30,000) in Australia.Daily and cumulative illness attack rates in both models following introduction of a novel influenza strain into a naive population, for an unmitigated scenario and two social distancing intervention scenarios.The unmitigated scenario indicated a ~50% higher attack rate in PNG compared to the Australian model. The two social distancing-based interventions strategies were 60% -70% less effective in a PNG setting compared to an Australian setting. Abstract Steroid-associated osteonecrosis (SAON) is a common orthopaedic problem caused by administration of corticosteroids prescribed for many nonorthopaedic medical conditions. We summarised different pathophysiologies of SAON which have adverse effects on multiple systems such as bone marrow stem cells (BMSCs) pool, bone matrix, cell apoptosis, lipid metabolism, and angiogenesis. Different animal models were introduced to mimic the pathophysiology of SAON and for testing the efficacy of both prevention and treatment effects of various chemical drugs, biological, and physical therapies. According to the classification of SAON, several prevention and treatment methods are applied at the different stages of SAON. For the current period, Chinese herbs may also have the potential to prevent the occurrence of SAON. In the future, genetic analysis might also be helpful to effectively predict the development of ON and provide information for personalised prevention and treatment of patients with SAON. Abstract Coronavirus disease 2019 (COVID-2019) has been recognized as a global threat, and several studies are being conducted using various mathematical models to predict the probable evolution of this epidemic. These mathematical models based on various factors and analyses are subject to potential bias. Here, we propose a simple econometric model that could be useful to predict the spread of COVID-2019. We performed Auto Regressive Integrated Moving Average (ARIMA) model prediction on the Johns Hopkins epidemiological data to predict the epidemiological trend of the prevalence and incidence of COVID-2019. For further comparison or for future perspective, case definition and data collection have to be maintained in real time. Abstract This paper provides an introduction to Geographical Information Systems (GIS) and how they can be used. It reviews the current state of GIS use in health care before identifying the barriers to more pervasive use of GIS in health. Finally, it makes recommendations for the direction of health GIS research over the next decade and concludes with a call to action to health informatics researchers to stop ignoring a tool and methodology that has such immense potential for improving the health of our communities. Abstract There are many challenges to quantifying and evaluating the media impact on the control of emerging infectious diseases. We modeled such media impacts using a piecewise smooth function depending on both the case number and its rate of change. The proposed model was then converted into a switching system, with the switching surface determined by a functional relationship between susceptible populations and different subgroups of infectives. By parameterizing the proposed model with the 2009 A/H1N1 influenza outbreak data in the Shaanxi province of China, we observed that media impact switched off almost as the epidemic peaked. Our analysis implies that media coverage significantly delayed the epidemic's peak and decreased the severity of the outbreak. Moreover, media impacts are not always effective in lowering the disease transmission during the entire outbreak, but switch on and off in a highly nonlinear fashion with the greatest effect during the early stage of the outbreak. The finding draws the attention to the important role of informing the public about 'the rate of change of case numbers' rather than 'the absolute number of cases' to alter behavioral changes, through a self-adaptive media impact switching on and off, for better control of disease transmission. E merging infectious diseases such as the 2003 outbreak of Severe Acute Respiratory Syndrome (SARS) 1,2 and the 2009 novel influenza A(H1N1) pandemic 3,4 have significant societal impacts not only through diseaseinduced morbidity and mortality, but also through their interference with socio-economic activities and population movement. Global public health systems of surveillance and response have been substantially improved in order to curb an emerging disease by containing it at source 5-7 or by slowing down its spread from the source 8,9 . Effective public health information processing is at the core of any global surveillance and response system. This is because at the start of an emerging epidemic, massive news coverage and fast information flow can generate profound psychological impacts on the public, and hence greatly alter individuals' behaviour and influence the implementation of public intervention and control policies 10 . How long and how effective media impact remains is therefore an issue of great importance for future epidemics control, and quantifying this impact through a mathematical modeling framework falls within the scope of this study.Recently, several mathematical models have been proposed to investigate media impacts. Existing approaches to modeling the impact of media coverage have focused on how this coverage depends on the number of infected individuals 11-14 , where prototype decreasing functions such as e 2mI , e {a1E{a2I{a3H (with H denoting hospitalized individuals, I infectives, E exposed individuals and nonnegative constants m, a i , i 5 1, 2, 3) and c 1 2 c 2 f(I) (with constants c 1 , c 2 ) have been embedded into the incidence rate. However, individuals may also change their behaviour due to their awareness and interpretation of the rate of change of the case numbers. Here we propose a novel mathematical model to represent the behavioral changes or implementation of interventions which are dependent on both the case number and its rate of change. We hope to examine how long media impacts last and how effective they are for different types of behavioral changes. In practice, media impact affects disease control (or spread) in conjunction with other visible measures, including non-pharmaceutical interventions (NPIs) (e.g., quarantine and isolation following contact tracing) and pharmaceutical interventions (PIs) (e.g., vaccines, treatment). We also incorporate these interventions into our model in order to identify the most effective strategy (or combination of strategies) including media impacted-behavioral changes to mitigate the epidemic during the entire outbreak.The model with media impact. We investigate a general SIR(susceptible-infective-recovery)-type epidemiological model, which incorporates media impacts and other interventions such as quarantine, isolation, vaccination and OPEN SUBJECT AREAS: APPLIED MATHEMATICS INFECTIOUS DISEASES Abstract Highlights d 5 early innate markers correlate with antibody response to Ebola vaccine rVSV-ZEBOV d IP-10 on day 3 after vaccination is an independent correlate of antibody induction d RNA-seq analysis identifies early gene expression signature linked to IP-10 Abstract Background: Suicide is a grave public health issue that is responsible for a high mortality rate among individuals aged 15-44 years. Attitudes toward suicide among medical staff members have been associated with appropriate therapeutic responses to suicidal individuals. The aim of this study was to examine the effects of parental rearing on attitudes toward suicide among Japanese medical college students. Methods: We examined the association between parental bonding and attitudes toward suicide in 160 medical college students in Japan. The Parental Bonding Instrument was used to assess the attitudes and behaviors of parents. The attitudes toward suicide were evaluated using the Japanese version of the Attitudes Toward Suicide questionnaire. Results: The mean age of the subjects was 25.2±4.0 years old. The majority of the participants in our study agreed that anyone could commit suicide (88.8%) and that suicide is preventable (86.3%). After adjusting for age and sex, multivariate regression analysis revealed that maternal care approached a statistically significant association with the "right to suicide" attitude. Under the same conditions, maternal care was shown to be significantly associated with the "common occurrence" attitude. No other significant relationships were observed between parental bonding and attitudes toward suicide.This study suggests that a higher level of maternal care ensures that children think that suicide occurs less commonly. The promotion of best practices for suicide prevention among medical students is needed. Child rearing support might be associated with suicide prevention. Abstract Pulmonary complications, including acute respiratory distress syndrome (ARDS), are well described in P. falciparum (PF) and to a lesser extent in other malaria species. In non-endemic areas, malaria diagnosis may be overlooked; if a thorough travel history is not obtained on all patients with acute febrile illness. Three patients with malaria associated respiratory distress were admitted to our intensive care unit. The diagnosis was delayed; however, all patients received artesunate and intensive therapy with a satisfactory outcome. One patient presented with respiratory disease while the others developed ARDS during or following appropriate therapy. Similarly, level of parasitemia was variable ranging from undetectable to over 5%. Variability in timing and severity of illness is exciting and gives emphasis to the different pathological processes contemplated in this complication. Abstract IFN-l4 is a novel type-III interferon with strong clinical significance in humans. Only a subset of individuals-up to 10% of Asians, 50% of Europeans, and 90% of Africans-carry the DG allele of a genetic variant rs368234815-TT/DG and are genetically able to produce IFN-l4 protein. Carriers of the DG allele have impaired ability to clear infection with hepatitis C virus (HCV). IFN-l4 is also predicted to exist and be functionally important in several nonhuman mammals. In this study, we present the first comparative analysis of 12 mammalian IFN-l4 orthologs in a human hepatic cell line, HepG2, which supports signaling of the human IFN-l4. We show that despite differences in protein sequences, functional properties of the recombinant human and nonhuman IFN-l4 proteins are comparable-they are all expressed as predominantly cytoplasmic proteins that are biologically active for induction of interferon signaling. We show that several IFN-l4 orthologs can be detected by Western blotting, flow cytometry, and confocal imaging using a monoclonal antibody developed for the human IFN-l4. Studies of IFN-l4 in animals should help improve our understanding of the biology of this novel clinically important interferon in normal and disease conditions. Abstract The 1977-1978 influenza epidemic was probably not a natural event, as the genetic sequence of the virus was nearly identical to the sequences of decades-old strains. While there are several hypotheses that could explain its origin, the possibility that the 1977 epidemic resulted from a laboratory accident has recently gained popularity in discussions about the biosafety risks of gain-of-function (GOF) influenza virus research, as an argument for why this research should not be performed. There is now a moratorium in the United States on funding GOF research while the benefits and risks, including the potential for accident, are analyzed. Given the importance of this historical epidemic to ongoing policy debates, we revisit the evidence that the 1977 epidemic was not natural and examine three potential origins: a laboratory accident, a live-vaccine trial escape, or deliberate release as a biological weapon. Based on available evidence, the 1977 strain was indeed too closely matched to decades-old strains to likely be a natural occurrence. While the origin of the outbreak cannot be conclusively determined without additional evidence, there are very plausible alternatives to the laboratory accident hypothesis, diminishing the relevance of the 1977 experience to the modern GOF debate.Citation Rozo M, Gronvall GK. 2015. The reemergent 1977 H1N1 strain and the gain-offunction debate. mBio 6(4):e01013-15. Abstract In one more years, we will 'celebrate' an exact centenary of the Spanish flu pandemic. With the rapid evolution of the influenza virus, the possibility of novel pandemic remains ever a concern. This review covers our current knowledge of the influenza A virus: on the role of RNA in translation, replication, what is known of the expressed proteins and the protein products generated from alternative splicing, and on the role of base pairing in RNA structure. We highlight the main events associated with viral entry into the cell, the transcription and replication process, an export of the viral genetic material from the nucleus and the final release of the virus. We discuss the observed potential roles of RNA secondary structure (the RNA base-pairing arrangement) and RNA/RNA interactions in this scheme. Abstract Interest in bat-borne diseases and parasites has grown in the past decade over concerns for human health. However, the drivers of parasite diversity among bat host species are understudied as are the links between parasite richness and emerging risks. Thus, we aimed at exploring factors that explain macro and microparasite species richness in bats from Southeast Asia, a hotspot of emerging infectious diseases. First, we identified bat species that need increased sampling effort for pathogen discovery. Our approach highlights pathogen investigation disparities among species within the same genus, such as Rhinolophus and Pteropus. Secondly, comparative analysis using independent contrasts method allowed the identification of likely factors explaining parasite and viral diversity of bats. Our results showed a key role of bat distribution shape, an index of the fragmentation of bat distribution, on parasite diversity, linked to a decrease for both viral and endoparasite species richness. We discuss how our study may contribute to a better understanding of the link between parasite species richness and emergence. Abstract Gaining insight in likely disease emergence scenarios is critical to preventing such events from happening. Recent focus has been on emerging zoonoses and on identifying common patterns and drivers of emerging diseases. However, no overarching framework exists to integrate knowledge on all emerging infectious disease events. Here, we propose such a conceptual framework based on changes in the interplay of pathogens, hosts and environment that lead to the formation of novel disease patterns and pathogen genetic adjustment. We categorize infectious disease emergence events into three groups: (i) pathogens showing up in a novel host, ranging from spill-over, including zoonoses, to complete species jumps; (ii) mutant pathogens displaying novel traits in the same host, including an increase in virulence, antimicrobial resistance and host immune escape; and (iii) disease complexes emerging in a new geographic area, either through range expansion or through long distance jumps. Each of these categories is characterized by a typical set of drivers of emergence, matching pathogen trait profiles, disease ecology and transmission dynamics. Our framework may assist in disentangling and structuring the rapidly growing amount of available information on infectious diseases. Moreover, it may contribute to a better understanding of how human action changes disease landscapes globally. Abstract The induction of ATF4 expression has previously been shown to increase the replication of HIV-1. However, the detailed mechanism underlying this effect and the factors involved in the regulation of ATF4 function are still unknown. Here, we demonstrate first that knocking out ATF4 using siRNA shows a strong negative effect on HIV-1 production, indicating that ATF4 is a functional positive cellular factor in HIV-1 production. To determine the mechanism by which ATF4 regulates the HIV-1 life cycle, we assessed the effect of the overexpression of wild type ATF4 and its various derivatives on HIV-1 LTR-mediated transcriptional activation and the production of HIV-1 particles. This effect was studied through co-transfection experiments with either reporter vectors or proviral DNA. We found that the N-terminal domains of ATF4 are involved in HIV-1 LTR-mediated transcriptional activation, and thus in HIV-1 production. [BMB Reports 2018; 51(8): 388-393] BMB Rep. 2018; 51(8): 388-393 www.bmbreports.org Abstract There are many age-associated changes in the respiratory and pulmonary immune system. These changes include decreases in the volume of the thoracic cavity, reduced lung volumes, and alterations in the muscles that aid respiration. Muscle function on a cellular level in the aging population is less efficient. The elderly population has less pulmonary reserve, and cough strength is decreased in the elderly population due to anatomic changes and muscle atrophy. Clearance of particles from the lung through the mucociliary elevator is decreased and associated with ciliary dysfunction. Many complex changes in immunity with aging contribute to increased susceptibility to infections including a less robust immune response from both the innate and adaptive immune systems. Considering all of these age-related changes to the lungs, pulmonary disease has significant consequences for the aging population. Chronic lower respiratory tract disease is the third leading cause of death in people aged 65 years and older. With a large and growing aging population, it is critical to understand how the body changes with age and how this impacts the entire respiratory system. Understanding the aging process in the lung is necessary in order to provide optimal care to our aging population. This review focuses on the nonpathologic aging process in the lung, including structural changes, changes in muscle function, and pulmonary immunologic function, with special consideration of obstructive lung disease in the elderly. Abstract No specific antivirals are currently available for two emerging infectious diseases, Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS). A literature search covering pathogenesis, clinical features and therapeutics, clinically developed drugs for repurposing and novel drug targets was performed. This review presents current knowledge on the epidemiology, pathogenesis and clinical features of the SARS and MERS coronaviruses. The rationale for and outcomes with treatments used for SARS and MERS is discussed. The main focus of the review is on drug development and the potential that drugs approved for other indications provide for repurposing. The drugs we discuss belong to a wide range of different drug classes, such as cancer therapeutics, antipsychotics, and antimalarials. In addition to their activity against MERS and SARS coronaviruses, many of these approved drugs have broad-spectrum potential and have already been in clinical use for treating other viral infections. A wealth of knowledge is available for these drugs. However, the information in this review is not meant to guide clinical decisions, and any therapeutic described here should only be used in context of a clinical trial. Potential targets for novel antivirals and antibodies are discussed as well as lessons This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract The effect of long-distance transport on cattle health has not frequently been studied in Bangladesh. The current study investigated the health conditions, and the extent and pattern of cattle injuries, along with haematobiochemical and hormonal changes, before and after long-distance transportation (≈648 km) from the market of origin to the market of destination. A total of 100 adult cattle were selected at the Benapole live cattle market, Abstract in one case, whereas Pneumocystis jiroveci was identified as a co-pathogen in one cMV-infected patient. Severe combined immunodeficiency is a pediatric emergency condition and given the significant impact of pulmonary CMV infection in SCID children, establishing an accurate etiological diagnosis is of essential importance in instituting the specific treatment and improving the outcome. Abstract Acute lung injury and acute respiratory distress syndrome (ARDS) represent a heterogenous group of lung disease in critically ill patients that continues to have high mortality. Despite the increased understanding of the molecular pathogenesis of ARDS, specific targeted treatments for ARDS have yet to be developed. ARDS represents an unmet medical need with an urgency to develop effective pharmacotherapies. Multiple promising targets have been identified that could lead to the development of potential therapies for ARDS; however, they have been limited because of difficulty with the mode of delivery, especially in critically ill patients. Nanobiotechnology is the basis of innovative techniques to deliver drugs targeted to the site of inflamed organs, such as the lungs. Nanoscale drug delivery systems have the ability to Abstract Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease. Abstract Background: Chronic cough in children is a diagnostic challenge. Objective: To discover the utility of nasal dipsticks and polymerase chain reaction (PCR)-DNA analysis in differentiating bacterial sinusitis from other causes of chronic cough and identifying pathogens from the nasal cavity. Method: We recruited 22 patients under 15 years of age with cough lasting longer than 4 weeks (group 1), 7 controls with allergic rhinitis (group 2), and 10 controls without respiratory symptoms (group 3). Based on symptoms, the results of nasal secretion assays, and nasal endoscopy, a diagnosis of clinical bacterial sinusitis was made. We identified potential pathogens by quantitative PCR of nasal secretions. Results: Group 1A (cough with clinical bacterial sinusitis n ¼ 10): Eight (80%) patients had bacterial sinusitis associated with dominant potential pathogenic bacteria (PPB): Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Group 1B (cough without clinical bacterial sinusitis n ¼ 12): None had dominant PPB. Group 2 (allergic rhinitis n ¼ 7): None had dominant PPB. Group 3 (asymptomatic n ¼ 10): None had dominant PPB. Twenty to 57% of all groups were colonized with Staphylococcus aureus. Fifty to 70% were colonized with Staphylococcus epidermidis, Corynebacterium pseudodiphtheriticum, and Dolosigranulum pigrum. Conclusion: In children with chronic cough, clinicians can utilize a simple and inexpensive nasal secretion dipstick assay for rapid diagnosis of sinusitis and identify PPB by DNA-PCR test for specific antibiotic treatment. Abstract Background: Researchers increasingly use social contact data to inform models for infectious disease spread with the aim of guiding effective policies about disease prevention and control. In this article, we undertake a systematic review of the study design, statistical analyses, and outcomes of the many social contact surveys that have been published. Methods: We systematically searched PubMed and Web of Science for articles regarding social contact surveys. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines as closely as possible. Results: In total, we identified 64 social contact surveys, with more than 80% of the surveys conducted in high-income countries. Study settings included general population (58%), schools or universities (37%), and health care/conference/research institutes (5%). The largest number of studies did not focus on a specific age group (38%), whereas others focused on adults (32%) or children (19%). Retrospective (45%) and prospective (41%) designs were used most often with 6% using both for comparison purposes. The definition of a contact varied among surveys, e.g., a nonphysical contact may require conversation, close proximity, or both. We identified age, time schedule (e.g., weekday/weekend), and household size as relevant determinants of contact patterns across a large number of studies. Conclusions: We found that the overall features of the contact patterns were remarkably robust across several countries, and irrespective of the study details. By considering the most common approach in each aspect of design (e.g., sampling schemes, data collection, definition of contact), we could identify recommendations for future contact data surveys that may be used to facilitate comparison between studies. Abstract Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and the necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralization assays. In this study we generated an equine influenza pseudotyped lentivirus for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8 subtype equine influenza virus strain, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. In order to produce infective pseudotype particles it was necessary to additionally co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave the HA. High titre pseudotype virus (PV) was then used in PV antibody neutralization assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% correlation between the results of the two assays, with the PVNA assay appearing slightly more sensitive. Future work will extend the testing of the PVNA with a larger number of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre. Abstract Phalaenopsis equestris remain largely unknown, which led to deficiency of effective measures to control disease of P. equestris caused by infecting viruses. In this study, for the first time, we characterized viral small interfering RNAs (vsiRNAs) profiles in P. equestris co-infected with CymMV and ORSV through small RNA sequencing technology. CymMV and ORSV small interfering RNAs (siRNAs) demonstrated several general and specific/new characteristics. vsiRNAs, with A/ U bias at the first nucleotide, were predominantly 21-nt long and they were derived predominantly (90%) from viral positive-strand RNA. 21-nt siRNA duplexes with 0-nt overhangs were the most abundant 21-nt duplexes, followed by 2-nt overhangs and then 1-nt overhangs 21-nt duplexes in infected P. equestris. Continuous but heterogeneous distribution and secondary structures prediction implied that vsiRNAs originate predominantly by direct Dicer-like enzymes cleavage of imperfect duplexes in the most folded regions of the positive strand of both viruses RNA molecular. Furthermore, we totally predicted 54 target genes by vsiRNAs with psRNATarget server, including disease/stress responserelated genes, RNA interference core components, cytoskeleton-related genes, photosynthesis or energy supply related genes. Gene Ontology classification showed that a majority of the predicted targets were related to cellular components and cellular processes and performed a certain function. All target genes were down-regulated with different degree by vsiRNAs as shown by realtime reverse transcription polymerase chain reaction. Taken together, CymMV and ORSV siRNAs played important roles in interplay with P. equestris by down modulating the expression levels of endogenous genes in host plant. Abstract For occupational safety, healthcare workers must select and wear appropriate personal protective equipment (PPE), protective clothing, and masks as countermeasures against exposure to infectious body fluids and blood splash. It is important for healthcare workers to ensure the protective performance of each PPE against penetration of pathogens. The International Standards Organization (ISO) 22609 test evaluates the effectiveness of medical facemasks to protect against penetration of splashed synthetic blood. However, in this method, the protective performance is determined only visually, without quantification of leaked liquid volume. Therefore, in this study, we modified the ISO 22609 test method to quantify the volume of leaked liquid and obtain a more accurate assessment of the protection performance. We tested non-woven and woven materials used for masks or protective clothing, and the performance of each material was classified using this new method. We found that the quantity of leaked synthetic blood was dependent on the structural characteristics of each material. These findings will allow healthcare workers to select the most appropriate PPE for a given situation or task. Abstract Genotyping of avian infectious bronchitis virus (IBV) was performed on trachea and kidney samples of six chickens obtained from a single farm in Japan. Using two primer sets targeting the spike (S) protein gene, the S1 and S2 regions of DNA fragments were amplified. Sequences of amplified S1 fragments extracted from both organs were identical among the six chickens, showing a JP-I genotype. Sequences of amplified S2 fragments differed between trachea and kidney samples. The kidney profile showed a group IV genotype, whereas the trachea profile showed an unclassified group. This result showed that two different IBVs infected the six chickens. The first IBV infection induced poor protective immunity in this farm, permitting a second IBV infection to occur. Abstract Very late antigens VLA-1, VLA 2, VLA-3, and VLA6, belonging to the 01 subfamily of integrins, have been identified as receptors for different binding domains of laminin (LM). We have detected VLA-6, but not VLA-1 and VLA-2 on a subset (50-70%) of fresh peripheral blood CD3 -, CD16+, CD56+ human natural killer (NK) cells by immunofluorimetric and biochemical analysis . Binding assays performed on LM-coated plates showed that 10-15% of NK cells spontaneously adhere to LM, and this adhesion is mediated by VLA-6 . Activation of NK cells through CD16 triggering or by phorbol ester results in a rapid increase of adhesion to LM, which is still mediated by VLA-6 . The enhanced adhesiveness is not associated with changes in (31 LM receptor expression, while it correlates with changes in the phosphorylation status of cx6 subunit . The expression of VLA6 on NK cells and the modulation of its avidity by activating stimuli may be relevant for NK cell migration and tissue location during inflammation or immune response. Abstract Using a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4 + CD25 + FOXP3 + regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-b1 and TGF-b2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments. Abstract Three-dimensional (3D) structure is now known for a large fraction of all protein families. Thus, it has become rather likely that one will find a homolog with known 3D structure when searching a sequence database with an arbitrary query sequence. Depending on the extent of similarity, such neighbor relationships may allow one to infer biological function and to identify functional sites such as binding motifs or catalytic centers. Entrez's 3D-structure database, the Molecular Modeling Database (MMDB), provides easy access to the richness of 3D structure data and its large potential for functional annotation. Entrez's search engine offers several tools to assist biologist users: (i) links between databases, such as between protein sequences and structures, (ii) precomputed sequence and structure neighbors, (iii) visualization of structure and sequence/structure alignment. Here, we describe an annotation service that combines some of these tools automatically, Entrez's 'Related Structure' links. For all proteins in Entrez, similar sequences with known 3D structure are detected by BLAST and alignments are recorded. The 'Related Structure' service summarizes this information and presents 3D views mapping sequence residues onto all 3D structures available in MMDB (http:// www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=structure).CONTENTThe molecular modeling database (MMDB) is Entrez's 'Structure' database (1). Querying MMDB with text terms, e.g. one may identify structures of interest based on a protein name. Links between databases provide other search mechanisms. A query of Entrez PubMed database, e.g. will identify articles citing a particular protein name. Links from this set of articles to 'Structure' may identify structures not found by direct query, since PubMed abstracts contain additional descriptive terms. Currently, MMDB and its visualization services handle 25 000 user queries per day. Abstract A B S T R A C T Macrophages (Mφ) are central players in mediating proinflammatory and immunomodulatory functions. Unchecked Mφ activities contribute to pathology across many diseases, including those caused by infectious pathogens and metabolic disorders. A fine balance of Mφ responses is crucial, which may be achieved by enforcing appropriate bioenergetics pathways. Metabolism serves as the provider of energy, substrates, and byproducts that support differential Mφ characteristics. The metabolic properties that control the polarization and response of Mφ remain to be fully uncovered for use in managing infectious diseases. Here, we review the various metabolic states in Mφ and how they influence the cell function. Abstract The interferon (IFN)-induced transmembrane (IFITM) proteins are critical mediators of the host antiviral response. Here, we expand the role of IFITM proteins to host defense against intracellular bacterial infection by demonstrating that they restrict Mycobacterium tuberculosis (MTb) intracellular growth. Simultaneous knockdown of IFITM1, IFITM2, and IFITM3 by RNAi significantly enhances MTb growth in human monocytic and alveolar/epithelial cells, whereas individual overexpression of each IFITM impairs MTb growth in these cell types. Furthermore, MTb infection, Toll-like receptor 2 and 4 ligands, and several proinflammatory cytokines induce IFITM1-3 gene expression in human myeloid cells. We find that IFITM3 co-localizes with early and, in particular, late MTb phagosomes, and overexpression of IFITM3 enhances endosomal acidification in MTb-infected monocytic cells. These findings provide evidence that the antiviral IFITMs participate in the restriction of mycobacterial growth, and they implicate IFITM-mediated endosomal maturation in its antimycobacterial activity.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Abstract Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2-4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2-4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2-4) by fusing the variable region of mouse mAb 2-4 to the constant region of feline antibody. The chimeric mAb 2-4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2-4 and chimeric mAb 2-4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2-4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2-4 was reduced. In contrast, in cats treated with chimeric mAb 2-4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2-4-treated cats. Abstract The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in September 2012 in Saudi Arabia had attracted the attention of the global health community. In 2017 the Saudi Ministry of Health released a visual triage system with scoring to alert healthcare workers in emergency departments (EDs) and haemodialysis units for the possibility of occurrence of MERS-CoV infection. We performed a retrospective analysis of this visual score to determine its sensitivity and specificity. The study included all cases from 2014 to 2017 in a MERS-CoV referral centre in Riyadh, Saudi Arabia. During the study period there were a total of 2435 suspected MERS cases. Of these, 1823 (75%) tested negative and the remaining 25% tested positive for MERS-CoV by PCR assay. The application of the visual triage score found a similar percentage of MERS-CoV and non-MERS-CoV patients, with each score from 0 to 11. The percentage of patients with a cutoff score of 4 was 75% in patients with MERS-CoV infection and 85% in patients without MERS-CoV infection (p 0.0001). The sensitivity and specificity of this cutoff score for MERS-CoV infection were 74.1% and 18.6%, respectively. The sensitivity and specificity of the scoring system were low, and further refinement of the score is needed for better prediction of MERS-CoV infection. Abstract Synthetic nanoparticles play an increasingly significant role in vaccine design and development as many nanoparticle vaccines show improved safety and efficacy over conventional formulations. These nanoformulations are structurally similar to viruses, which are nanoscale pathogenic organisms that have served as a key selective pressure driving the evolution of our immune system. As a result, mechanisms behind the benefits of nanoparticle vaccines can often find analogue to the interaction dynamics between the immune system and viruses. This review covers the advances in vaccine nanotechnology with a perspective on the advantages of virus mimicry towards immune potentiation. It provides an overview to the different types of nanomaterials utilized for nanoparticle vaccine development, including functionalization strategies that bestow nanoparticles with virus-like features. As understanding of human immunity and vaccine mechanisms continue to evolve, recognizing the fundamental semblance between synthetic nanoparticles and viruses may offer an explanation for the superiority of nanoparticle vaccines over conventional vaccines and may spur new design rationales for future vaccine research. These nanoformulations are poised to provide solutions towards pressing and emerging human diseases. Abstract An outbreak of COVID-19 developed aboard the Princess Cruises Ship during January-February 2020. Using mathematical modeling and time-series incidence data describing the trajectory of the outbreak among passengers and crew members, we characterize how the transmission potential varied over the course of the outbreak. Our estimate of the mean reproduction number in the confined setting reached values as high as ~11, which is higher than mean estimates reported from community-level transmission dynamics in China and Singapore (approximate range: 1.1-7). Our findings suggest that R t decreased substantially compared to values during the early phase after the Japanese government implemented an enhanced quarantine control. Most recent estimates of R t reached values largely below the epidemic threshold, indicating that a secondary outbreak of the novel coronavirus was unlikely to occur aboard the Diamond Princess Ship. Abstract: 73 Main: 2048 2 Abstract: An outbreak of COVID-19 developed aboard the Princess Cruises Ship during January-February 2020. Using mathematical modeling and time-series incidence data describing the trajectory of the outbreak among passengers and crew members, we characterize how the transmission potential varied over the course of the outbreak. Our estimate of the mean reproduction number in the confined setting reached values as high as ~11, which is higher than mean estimates reported from community-level transmission dynamics in China and Singapore (approximate range: 1.1-7). Our findings suggest that R t decreased substantially compared to values during the early phase after the Japanese government implemented an enhanced quarantine control. Most recent estimates of R t reached values largely below the epidemic threshold, indicating that a secondary outbreak of the novel coronavirus was unlikely to occur aboard the Diamond Princess Ship. Abstract Emergency Medical Countermeasures Enterprise (PHEMCE) has made great strides in strategic preparedness and response capabilities. There have been numerous advances in planning, biothreat countermeasure development, licensure, manufacturing, stockpiling and deployment. Increased biodefense surveillance capability has dramatically improved, while new tools and increased awareness have fostered rapid identification of new potential public health pathogens. Unfortunately, structural delays in vaccine design, development, manufacture, clinical testing and licensure processes remain significant obstacles to an effective national biodefense rapid response capability. This is particularly true for the very real threat of "novel pathogens" such as the avian-origin influenzas H7N9 and H5N1, and new coronaviruses such as hCoV-EMC. Conventional approaches to vaccine development, production, clinical testing and licensure are incompatible with the prompt deployment needed for an effective public health response. An alternative approach, proposed here, is to apply computational vaccine design tools and rapid production technologies that now make it possible to engineer vaccines for novel emerging pathogen and WMD biowarfare agent countermeasures in record time. These new tools have the potential to significantly reduce the time needed to design string-of-epitope vaccines for previously unknown Abstract N-myc oncogene amplification is associated but not present in all cases of high-risk neuroblastoma (NB). Since oncogene expression could often modulate sensitivity to oncolytic viruses, we wanted to examine if N-myc expression status would determine virotherapy efficacy to high-risk NB. We showed that induction of exogenous N-myc in a non-N-myc-amplified cell line background (TET-21N) increased susceptibility to oncolytic vesicular stomatitis virus (mutant VSVΔM51) and alleviated the type I IFN-induced antiviral state. Cells with basal N-myc, on the other hand, were less susceptible to virus-induced oncolysis and established a robust IFN-mediated antiviral state. The same effects were also observed in NB cell lines with and without N-myc amplification. Microarray analysis showed that N-myc overexpression in TET-21N cells downregulated IFN-stimulated genes (ISGs) with known antiviral functions. Furthermore, virus infection caused significant changes in global gene expression in TET-21N cells overexpressing N-myc. Such changes involved ISGs with various functions. Therefore, the present study showed that augmented susceptibility to VSVΔM51 by N-myc at least involves downregulation of ISGs with antiviral functions and alleviation of the IFNstimulated antiviral state. Our studies suggest the potential utility of N-myc amplification/overexpression as a predictive biomarker of virotherapy response for high-risk NB using IFN-sensitive oncolytic viruses. Abstract Objectives: To synthesize new transitional metal complexes derived from 3-aryl-azo-4-hydroxy coumarin analogues and to evaluate their antimicrobial activities.Methods: The syntheses of complexes of coumarin analogues were accomplished by mixing a hydro-alcoholic solution of 3-aryl-azo-4-hydroxy coumarin analogues with transition metal chlorides. The structural environment of the synthesized molecules was characterized using different instrumental methods. The antimicrobial activity of the compounds was determined by the agar well diffusion method.Results: The cobalt complexes of (E)-3-((4-chlorophenyl) diazenyl)-4-hydroxy-2H-chromen-2-one (HL 1 ): (4a) and (E)-3-((4-methoxyphenyl) diazenyl)-4-hydroxy-2H-chromen-2-one (HL 2 ): (4e) showed excellent antimicrobial activities compared with their ligands.The reports of the antimicrobial investigation showed that the cobalt complexes of 3-aryl-azo-4hydroxy coumarin analogues exhibited potential antimicrobial activity that was stronger than that of their ligands. Abstract The objectives of this study were to evaluate the effects of intravenous acetate Ringer's solution, with or without dextrose, on diarrheic calves with either experimentally induced or spontaneous diarrhea. In the experimental model, diarrhea was induced in nine healthy calves by administering cold milk (below 4°C) twice a day for 2 days. The calves were randomly assigned to the isotonic saline (ISS), acetated Ringer's (AR) or acetated Ringer's with 5% dextrose (ARD) groups, with three calves assigned to each group. The calves received 80 ml/kg of their designated solution, at a flow rate of 20 ml/kg/hr. Infusion of ISS, AR and ARD were all found to be safe and effective in increasing plasma volume. Intravenous (IV) infusion of ISS resulted in the acidification secondary to dilution, while AR and ARD infusion inhibited acidification. In addition, prevention of catabolism was observed only with IV infusion of ARD. Sixteen calves with spontaneous diarrhea were enrolled in the clinical study. The calves were randomly assigned to the AR or ARD groups, with eight calves being assigned to each group. The calves received 100 ml/kg of their designated solution, at a flow rate of 25 ml/kg/hr. Intravenous infusion of AR and ARD was found to be effective in increasing plasma volume and inhibiting acidification. Only infusion of ARD prevented catabolism, but it also led to hyperglycemia. Our results suggest that a solution containing dextrose may be beneficial for wasting diarrheic calves. Abstract Background: Acute respiratory infections (ARI) are the leading cause of death worldwide, especially among children. The majority of these infections in children are of viral etiology. In this study, we evaluated the incidence of viral ARI among children in Lebanon. Patients and Methods: Children presenting with symptoms of ARI were prospectively recruited between September 2009 to February 2012. Nasopharyngeal aspirates were obtained from patients and screened for 11 respiratory viruses using a multiplex Luminex-based PCR assay. Results: Two hundred twenty-one patients were recruited with a median age of 1 year (IQR: 0 -5). Out of 221 patients, 116 (52.5%) were positive for at least one virus, the majority (103/116; 88.8%) of which were in children under 6-year of age. Overall, 188 viruses were detected. Rhinovirus (RhV) was the most common virus detected in 81 (69.8%) patients followed by coxsackie virus and echovirus (CVEV) which were detected as one target in the panel in 45 (38.8%), and parainfluenza viruses (PIV types : 1, 2, 3, 4) in 24 (20.7%) patients. Coinfection with more than one virus was detected in 49 (42.9%) patients. RhV and CVEV were the most common viruses associated with co-infections and higher risk of rhinorrhea. Conclusions: Viral pathogens account for at least half of the ARIs in Lebanon, with a high frequency of co-infections being detected. Abstract The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor TM ) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutationcontaining DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine. Abstract Introduction: Fever of unknown origin (FUO) is a common initial presentation leading to a diagnostic challenge.Patient concerns: A 3-month history of moderate-to-high fever was reported in an otherwise healthy 54-year-old man. Enhanced computed tomography (CT) scans of his chest showed a remarkable progressive enlargement of bilateral cervical, supraclavicular, hilar, and mediastinal lymph nodes within 2 weeks. Bronchofibroscopy manifested obvious luminal stenosis with swelling, thick pale mucosa, and disappearing of structures of trachea cricoid cartilage, followed by a 18F-fluorodeoxyglucose positron-emission tomography-computed tomography (18F-FDG PET/CT) with intense symmetric FDG uptake in larynx, tracheobronchial tree, and hilar, mediastinal, and axillary lymph nodes being demonstrated.Diagnosis: A diagnosis of relapsing polychondritis (RP) was finally reached.Interventions: The patient received methylprednisolone 40 mg daily with a gradual tapering in a 4-month follow-up.Outcomes: The patient experienced no relapse of fever and lymph nodes enlargement in the 4-month follow-up.Lessons: Even though long-term fever with multiple lymphadenectasis usually lead to a diagnosis of lymphoma, the bronchoscopic features and evidence from 18F-FDG PET/CT in this case were much more approximate to RP, indicating an importance of a sensible differential diagnosis of RP in patients who present with nonspecific features such as FUO and lymph nodes enlargement. Keeping a high index of clinical suspicion in these patients can help recognize uncommon of RP and promote diagnosis and treatment. Our case highlights the significance of 18F-FDG PET/CT in helping reaching the diagnosis of RP in this condition. This report provides new data regarding the diagnostic difficulties of this rare type of autoimmune disease, and further investigations are needed as cases accumulate.Abbreviations: 18F-FDG PET/CT = 18F-fluorodeoxyglucose positron-emission tomography-computed tomography, FUO = fever of unknown origin, RP = relapsing pholychondritis. Abstract Porcine deltacoronavirus (PDCoV) has emerged in several pig-raising countries and has been a causative pathogen associated with diarrheal diseases in South Korea since 2014. In the present study, we were able to isolate and cultivate a Korean PDCoV strain (KNU16-07) in cell culture and investigate its pathogenicity. PDCoV-inoculated piglets showed watery diarrhea accompanied by acute enteritis in the natural host. Sequencing analysis demonstrated the genetic stability of KNU16-07 for at least thirty serial passages. Abstract Live attenuated vaccines are critical in the control of avian infectious bronchitis. It is necessary to know the protection conferred by commonly used commercial live vaccines. In this study, specific pathogen-free chicks were vaccinated with the commercial live vaccines H120, 4/91 and LDT3-A. Blood samples were collected at weekly intervals for the detection of IBV-specific antibodies and quantification of CD4 + and CD8 + T lymphocytes. At 21 days post-inoculation the vaccinated birds were challenged with the IBV prevalent local strains GX-YL5, GX-GL11079 and GX-NN09032, respectively. Trachea and kidney samples were collected at 5 days post-challenge for the detection of the virus. The results showed that the H120 group exhibited medium antibody levels, the lowest percentages of CD4 + , CD8 + T lymphocytes and the highest viral loads. The 4/91 group showed the lowest antibody levels, but the highest percentages of CD4 + , CD8 + T lymphocytes and the lowest viral loads. The LDT3-A group showed the highest antibody levels, the medium percentages of CD4 + , CD8 + T lymphocytes and the medium viral loads. The protection rates of H120, 4/91 and LDT3-A groups were 41.7-58.3%, 75.0-83.7% and 66.7-75.0%, respectively. The present study demonstrated that the vaccines H120, 4/91 and LDT3-A could stimulate the immunized chicks to produce different levels of humoral and cellular immunity to resist the infection of IBV, but couldn't provide complete protection against the prevalent local strains of IBV in southern China. Also, the vaccine 4/91 offered the best immune protection among the three vaccines.Avian infectious bronchitis (IB), an acute, highly contagious disease, is caused by avian coronavirus infectious bronchitis virus (IBV), which results in severe economic losses to the poultry industry worldwide. An increasing number of new serotypes or variants of IBV, which were caused by gene mutation and recombination, have been identified [4, 9, 16, 17, 19] . Vaccination is the main measure for the prevention and control of IB, but since there are multiple IBV serotypes and different serotypes of IBVs, vaccinations result in incomplete or no cross-protection against the various types of IBV. Hence, it is important to choose suitable vaccines for the control of IBV in the field.Live, attenuated and inactivated vaccines are commonly used to control IB at the present time. However, the effects of inactivated vaccines are uncertain and inactivated vaccines often fail to induce strong cellular immunity [23] . Although the live, attenuated vaccines can cause virus mutation and recombination, they can induce effective humoral and cellular immune responses as well as local immunity [3, 21] . In young birds, live, attenuated vaccines are especially used to achieve early protection and also to prime future layers and breeders, which will be boosted with the inactivated vaccines later [21] . Therefore, the live, attenuated vaccines are of importance in the prevention and control of IB in the field. Abstract As of February 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak started in China in December 2019 has been spreading in many countries in the world. With the numbers of confirmed cases are increasing, information on the epidemiologic investigation and clinical manifestation have been accumulated. However, data on viral load kinetics in confirmed cases are lacking. Here, we present the viral load kinetics of the first two confirmed patients with mild to moderate illnesses in Korea in whom distinct viral load kinetics are shown. This report suggests that viral load kinetics of SARS-CoV-2 may be different from that of previously reported other coronavirus infections such as SARS-CoV. Abstract Outbreaks of porcine epidemic diarrhea (PED) have resulted in significant economic losses in the swine industry, and another PED outbreak occurred in 2014 in Korea. Isolating and culturing PED virus (PEDV) allow investigations into its pathogenesis and the development of vaccines and diagnostic assays. In this study, we successfully isolated two PEDV isolates (QIAP1401 and QIAP1402) from naturally infected piglets at Jeju-do, Korea. Viral propagation was confirmed in Vero cells based on cytopathic effect, immunofluorescence assay, reverse transcription-polymerase chain reaction, and electron microscopic analyses. The QIAP401 isolate propagated well in Vero cells for 70 passages, with titers of 10 6.5 to 10 7.0 50% tissue culture infectious dose/mL, which increased gradually with passaging. The nucleotide and amino acid sequences of the QIAP1401 isolate were determined and compared with those of other PEDV isolates. The QIAP1401 isolate was determined to be closely related to the USA/Minnesota271/2014 strain (> 99.9% nucleotide similarity) that was isolated in the USA in 2014. Phylogenetic analysis based on several PEDV genes suggested that a new PEDV variant is circulating in the Korean swine industry, with 93.08% similarity to the SM98 strain isolated in 1998. In addition, the QIAP1401 strain showed strong virulence in 3-day-old piglets and 11-week-old growing pigs. Abstract China has exploded onto the world economy over the past few decades and is undergoing rapid transformation toward relatively more services. The health sector is an important part of this transition. This article provides a historical account of the development of health care in China since 1949. It also focuses on health insurance and macroeconomic structural adjustment to less saving and more consumption. In particular, the question of how health insurance impacts precautionary savings is considered. Multivariate analysis using data from 1990 to 2012 is employed. The household savings rate is the dependent variable in 3 models segmented for rural and urban populations. Independent variables include out-of-pocket health expenditures, health insurance payouts, housing expenditure, education expenditure, and consumption as a share of gross domestic product (GDP). Out-of-pocket health expenditures were positively correlated with household savings rates. But health insurance remains weak, and increased payouts by health insurers have not been associated with lower levels of household savings so far. Housing was positively correlated, whereas education had a negative association with savings rates. This latter finding was unexpected. Perhaps education is perceived as investment and a substitute for savings. China's shift toward a more service-oriented economy includes growing dependence on the health sector. Better health insurance is an important part of this evolution. The organization and finance of health care is integrally linked with macroeconomic policy in an environment constrained by prevailing institutional convention. Problems of agency relationships, professional hegemony, and special interest politics feature prominently, as they do elsewhere. China also has a dual approach to medicine relying heavily on providers of traditional Chinese medicine. Both of these segments will take part in China's evolution, adding another layer of complexity to policy. Abstract Hajj is a unique gathering with Mecca and Kaaba being spiritually important to many faiths across the globe, especially Muslims. This is because of the proclamation of the prophet's father, Ibrahaam, when he called all mankind to perform Hajj. That is why all Muslims on Earth feel that they have to visit Mecca and Kaaba on a specific date and time, and that is the reason this small location hosts one of the largest human gatherings in the world. Hajj is one of the five pillars of Islam that every financially and physically able Muslim must perform once in his/her lifetime. For 14 centuries countless millions of Muslim men and women from the four corners of the earth have undertaken pilgrimage to Mecca.In conclusion this review article confirm that Hajj is oldest and largest mass gathering in all mankind and there is some issues influence the health response such as size of gathering. diversity of population, climate and health facilities around hajj site, also we discuss the infectious and non infectious related illness in hajj and their prevention methods. Abstract South Korea was free of the Middle East Respiratory Syndrome (MERS) until 2015. The MERS outbreak in South Korea during 2015 was the largest outbreak of the Coronavirus outside the Middle East. The major characteristic of this outbreak is inter-or intra-hospital transmission. This recent MERS outbreak in South Korea is examined and assessed in this paper. The main objectives of the study is to characterize the pattern of the MERS outbreak in South Korea based on a basic reproductive ratio, the probability of ultimate extinction of the disease, and the spatio-temporal proximity of occurrence between patients. The survival function method and stochastic branching process model are adapted to calculate the basic reproductive ratio and the probability of ultimate extinction of the disease. We further investigate the occurrence pattern of the outbreak using a spatio-temporal autocorrelation function. Abstract While general medical practitioners (GPs) and veterinarians are often the first line responders in the face of a disease outbreak, pathways to improving the One Health efficacy of these clinicians remain unclear. A two-phase modified Delphi survey of professionals with known expertise in One Health ('expert panel') was used to 1) identify key knowledge, attitudes and practices (KAPs) of GPs and veterinarians that would be consistent with a One Health approach to zoonoses; and 2) determine priorities for future surveys with Australian GPs and veterinarians to identify important gaps that impede effective diagnosis and management of zoonoses. A list of 13 topics/sub-topics, as well as a list of 25 specific zoonotic diseases/agents emerged from the first phase of the survey. In the second phase the expert panel identified general knowledge of the clinical aspects and epidemiological aspects of zoonoses, as well as risk management practices, as the most important KAPs and research priorities for both GPs and veterinarians. In terms of diseases, the expert panel regarded knowledge of Hendra virus, Q fever, Australian bat lyssavirus (ABLV), anthrax and Brucella suis most important for veterinarians, whilst for GPs, Q fever, gastrointestinal/foodborne diseases, influenza, ABLV and local vector-borne diseases were found to be most important by the expert panel. Some differences were noted in terms of prioritization of topics/sub-topics and diseases/agents according to expert background (veterinary and non-veterinary). The Delphi survey technique enabled efficient collection of data from a diverse range of One Health 'experts'/specialists and provided clear priorities for proposed future research, and potentially for educational interventions to improve One Health efficacy of clinicians. Abstract Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARS-CoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us. Abstract There were severe panics caused by Severe Acute Respiratory Syndrome (SARS) and Middle-East Respiratory Syndrome-Coronavirus. Therefore, researches targeting these viruses have been required. Coronaviruses (CoVs) have been rising targets of some flavonoids. The antiviral activity of some flavonoids against CoVs is presumed directly caused by inhibiting 3C-like protease (3CLpro). Here, we applied a flavonoid library to systematically probe inhibitory compounds against SARS-CoV 3CLpro. Herbacetin, rhoifolin and pectolinarin were found to efficiently block the enzymatic activity of SARS-CoV 3CLpro. The interaction of the three flavonoids was confirmed using a tryptophan-based fluorescence method, too. An induced-fit docking analysis indicated that S1, S2 and S3 0 sites are involved in binding with flavonoids. The comparison with previous studies showed that Triton X-100 played a critical role in objecting false positive or overestimated inhibitory activity of flavonoids. With the systematic analysis, the three flavonoids are suggested to be templates to design functionally improved inhibitors.ARTICLE HISTORY Abstract Background: Burden of disease can be used to prioritize the healthcare budget allocation. We analyzed the research and development (R&D) budget of the Ministry of Health and Welfare (MOHW) in 2018 and compared the results with those of the 2015 Korean National Burden of Disease (KNBD) study. Methods: The 2018 MOHW R&D Project integrated implementation plan was used to analyze the R&D budget of the MOHW. The budget was allocated according to the KNBD disease group and according to the budget lines. The allocated budget was compared with the economic burden and the disability adjusted life years (DALYs) in 2015. Also, for budget targets for risk factors, DALYs of attributable risk factors were compared with corresponding budgets. Results: In 2018, the MOHW major R&D budget of USD 435.1 million accounted for 3% of the total government budget. Within the disease specific R&D budget, 35.9% was allocated to communicable disease groups, 64.1% to non-communicable diseases, and 0% to injury and violence. Among level 2 disease groups, neoplasm was ranked first. Among risk factors, climate change and behavioral risk were targeted for R&D. Conclusions: It would be difficult to say that current R&D allocations focus to minimize the burden of disease. A mismatch was observed between the R&D budget and the burden of disease in terms of economic burden and DALYs. There was a similar finding for risk factors R&D. A novel approach for allocating government R&D funding that is based on the goal of minimizing the disease burden in the Korean population should be considered. Abstract Neurotropic strains of mouse hepatitis viruses (MHV) such as MHV-A59 (A59) and MHV-4 (JHMV) cause acute and chronic encephalomyelitis and demyelination in susceptible strains of mice and rats. They are widely used as models of human demyelinating diseases such as multiple sclerosis (MS), in which immune mechanisms are thought to participate in the development of lesions in the central nervous system (CNS). The effects of MHV infection on target cell functions in the CNS are not well understood, but A59 has been shown to induce the expression of MHC class I molecules in glial cells after in vivo and in vitro infection. Changes in class I expression in infected cells may contribute to the immunopathogenesis of MHV infection in the CNS. In this communication, a large panel of MHV strains was tested for their ability to stimulate class I expression in primary astrocytes in vitro. The data show that the more hepatotropic strains, such as MHV-A59, MHV-1, MHV-2, MHV-3, MHV-D, MHV-K, and MHV-NuU, were potent inducers of class I expression in astrocytes during acute infection, measured by radioimmunoassay. The K b molecule was preferentially expressed over D b. By contrast, JHMV and several viral strains derived from it did not stimulate the expression of class I molecules. Assays of virus infectivity indicated that the class I-inducing activity did not correlate with the ability of the individual viral strain to replicate in astrocytes. However, exposure of the viruses or the supernatants from infected astrocytes to ultraviolet light abolished the class I-inducing activity, indicating that infectious virus is required for class I expression. These data also suggest that class I expression was induced directly by virus infection, and not by the secretion of a soluble substance into the medium by infected astrocytes. Finally, analyses of A59/JHMV recombinant viral strains suggest that class I-inducing activity resides in one of the A59 structural genes.J. Exp. Med. 9 The Rockefeller University Press Abstract Previously, we demonstrated that pyroptosis in alveolar macrophages (AMs) plays an essential role in lipopolysaccharide (LPS)-induced acute lung injury. However, the underlying mechanism remains largely unclear. Here, we show that the absence of interferon regulatory factor 1 (IRF-1) in genetic knock-out mice strongly abrogates pyroptosis in AMs and alleviates the LPS-induced lung injury and systemic inflammation. Our study demonstrates that IRF-1 contributes to caspase-1 activation and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain pyroptosome formation in AMs and leads to downstream inflammatory cytokine release, including that of IL-1b, IL-18, and HMGB1. The nuclear translocation of IRF-1 is linked to the presence of toll-like receptor 4 (TLR4). Our findings suggest that pyroptosis and the downstream inflammatory response in AMs induced by LPS is a process that is dependent on TLR4-mediated up-regulation of IRF-1. In summary, IRF-1 plays a key role in controlling caspase-1-dependent pyroptosis and inflammation.KEYWORDS-Acute lung injury, alveolar macrophage, caspase-1, interferon regulatory factor-1, pyroptosis ABBREVIATIONS-ALI-acute lung injury; AMs-alveolar macrophages; ARDS-acute respiratory distress syndrome; ASC-apoptosis-associated speck-like protein containing a caspase activation and recruitment domain; ATP-adenosine triphosphate; BALF-bronchoalveolar lavage fluid; HMGB1-high-mobility group box 1; IFN-interferon; IRF-1-interferon regulatory factor 1; KO-knockout; LDH-Lactate dehydrogenase; LPS-lipopolysaccharide; MyD88-myeloid differentiation factor 88; NLRP3-nod-like receptor protein 3; PAMP-pathogen-associated molecular pattern; qPCRquantitative PCR; TLR-4-toll-like receptor 4; WT-wild-type Address reprint requests to Pinhua Pan, PhD, Abstract Purpose: Severe ARDS is often associated with refractory hypoxemia, and early identification and treatment of hypoxemia is mandatory. For the management of severe ARDS ventilator settings, positioning therapy, infection control, and supportive measures are essential to improve survival.A precise definition of life-threating hypoxemia is not identified. Typical clinical determinations are: arterial partial pressure of oxygen < 60 mmHg and/or arterial oxygenation < 88 % and/or the ratio of PaO 2 /FIO 2 < 100. For mechanical ventilation specific settings are recommended: limitation of tidal volume (6 ml/ kg predicted body weight), adequate high PEEP (>12 cmH 2 O), a recruitment manoeuvre in special situations, and a 'balanced' respiratory rate (20-30/min). Individual bedside methods to guide PEEP/recruitment (e.g., transpulmonary pressure) are not (yet) available. Prone positioning [early (≤ 48 hrs after onset of severe ARDS) and prolonged (repetition of 16-hr-sessions)] improves survival. An advanced infection management/control includes early diagnosis of bacterial, atypical, viral and fungal specimen (blood culture, bronchoalveolar lavage), and of infection sources by CT scan, followed by administration of broad-spectrum anti-infectives. Neuromuscular blockage (Cisatracurium ≤ 48 hrs after onset of ARDS), as well as an adequate sedation strategy (score guided) is an important supportive therapy. A negative fluid balance is associated with improved lung function and the use of hemofiltration might be indicated for specific indications.A specific standard of care is required for the management of severe ARDS with refractory hypoxemia. Abstract Nucleoside and nucleotide analogs have played significant roles in antiviral therapies and are valued for their impressive potency and high barrier to resistance. They have been approved for treatment of herpes simplex virus-1, HIV, HBV, HCV, and influenza, and new drugs are being developed for the treatment of RSV, Ebola, coronavirus MERS, and other emerging viruses. However, this class of compounds has also experienced a high attrition rate in clinical trials due to toxicity. In this review, we discuss the utility of different biochemical and cell-based assays and provide recommendations for assessing toxicity liability before entering animal toxicity studies. Abstract In March 2013, a patient infected with a novel avian influenza A H7N9 virus was reported in China. Since then, there have been 458 confirmed infection cases and 177 deaths. The virus contains several human-adapted markers, indicating that H7N9 has pandemic potential. The outbreak of this new influenza virus highlighted the need for the development of universal influenza vaccines. Previously, we demonstrated that a tetrameric peptide vaccine based on the matrix protein 2 ectodomain (M2e) of the H5N1 virus (H5N1-M2e) could protect mice from lethal infection with different clades of H5N1 and 2009 pandemic H1N1 influenza viruses. In this study, we investigated the cross-protection of H5N1-M2e against lethal infection with the new H7N9 virus. Although five amino acid differences existed at positions 13, 14, 18, 20, and 21 between M2e of H5N1 and H7N9, H5N1-M2e vaccination with either Freund's adjuvant or the Sigma adjuvant system (SAS) induced a high level of anti-M2e antibody, which cross-reacted with H7N9-M2e peptide. A mouse-adapted H7N9 strain, A/Anhui/01/2013m, was used for lethal challenge in animal experiments. H5N1-M2e vaccination provided potent cross-protection against lethal challenge of the H7N9 virus. Reduced viral replication and histopathological damage of mouse lungs were also observed in the vaccinated mice. Our results suggest that the tetrameric H5N1-M2e peptide vaccine could protect against different subtypes of influenza virus infections. Therefore, this vaccine may be an ideal candidate for developing a universal vaccine to prevent the reemergence of avian influenza A H7N9 virus and the emergence of potential novel reassortants of influenza virus. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) was detected by monoclonal antibody-based nucleocapsid protein-capture enzyme-linked immunosorbent assay (ELISA), RNA detection, and viral culture from the nasal sample of a 1-month-old dromedary calf in Dubai with sudden death. Whole genome phylogeny showed that this MERS-CoV strain did not cluster with the other MERS-CoV strains from Dubai that we reported recently. Instead, it formed a unique branch more closely related to other MERS-CoV strains from patients in Qatar and Hafr-Al-Batin in Saudi Arabia, as well as the MERS-CoV strains from patients in the recent Korean outbreak, in which the index patient acquired the infection during travel in the eastern part of the Arabian Peninsula. Non-synonymous mutations, resulting in 11 unique amino acid differences, were observed between the MERS-CoV genome from the present study and all the other available MERS-CoV genomes. Among these 11 unique amino acid differences, four were found in ORF1ab, three were found in the S1 domain of the spike protein, and one each was found in the proteins encoded by ORF4b, ORF5, envelope gene, and ORF8. MERS-CoV detection for all other 254 dromedaries in this closed dairy herd was negative by nucleocapsid protein-capture ELISA and RNA detection. MERS-CoV IgG sero-positivity gradually increased in dromedary calves with increasing age, with positivity rates of 75% at zero to three months, 79% at four months, 89% at five to six months, and 90% at seven to twelve months. The development of a rapid antigen detection kit for instantaneous diagnosis is warranted. Abstract Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa protein of the lipocalin superfamily and its presence was initially observed in activated neutrophils. It has previously been demonstrated that the expression of NGAL is markedly increased in stimulated epithelia, and is important in the innate immunological response to various pathophysiological conditions, including infection, cancer, inflammation and kidney injury. The present study constructed a ventilator-associated lung injury model in mice. NGAL mRNA and protein expression levels in lung tissue were detected using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. In addition, NGAL protein levels in bronchoalveolar lavage fluid and serum were measured via western blotting. The results of the present study suggested that NGAL expression increased under all mechanical ventilation treatments. The increase was most prominent in the high peak inflation pressure and high-volume mechanical ventilation groups, where there was the greatest extent of lung injury. In addition, NGAL expression increased in a time-dependent manner under high-volume mechanical ventilation, consistent with the degree of lung injury. These findings suggested that NGAL may serve as a potential novel biomarker in ventilator-associated lung injury. Abstract Using a comprehensive set of discovery and optimization tools, antibodies were produced with the ability to neutralize SARS coronavirus (SARS-CoV) infection in Vero E6 cells and in animal models. These anti-SARS antibodies were discovered using a novel DNA display method, which can identify new antibodies within days. Once neutralizing antibodies were identified, a comprehensive and effective means of converting the mouse sequences to human frameworks was accomplished using HuFR TM (human framework reassembly) technology. The best variant (61G4) from this screen showed a 3.5 -4-fold improvement in neutralization of SARS-CoV infection in vitro. Finally, using a complete site-saturation mutagenesis methodology focused on the CDR (complementarity determining regions), a single point mutation (51E7) was identified that improved the 80% plaque reduction neutralization of the virus by greater than 8-fold. These discovery and evolution strategies can be applied to any emerging pathogen or toxin where a causative agent is known. Abstract Background: On May 19, 2017, the cluster of 6 acute respiratory infections due to adenovirus in the swimming department of a physical education school (School J) was reported to Korea Centers for Disease Control and Prevention. An epidemiological investigation was conducted to identify the transmission route of the infection and to control the outbreak. Materials and Methods: A retrospective cohort study (Study 1) was conducted on students and teachers of the athletic departments using the swimming pool, and a prospective surveillance (Study 2) was conducted on all students and teachers of the School J. A case was defined as any student and school personnel who developed more than two of the following symptoms from April 10 to July 2, 2017: fever, sore throat, cough, rhinorrhea, or headache. Relative risks (RRs) were calculated to compare the attack rates according to potential risk factors. Multivariable logistic regression was performed to identify the risk factors for infection in the outbreak. Results: 47 cases were identified: 33 (55.9%) cases occurred among 59 students and teachers in Study 1 and 14 (3.9%) among 362 students and school personnel in Study 2. There were 18 laboratory confirmed adenovirus infection cases. The common symptoms were headache (71.7%), fever (69.6%), rhinorrhea (63.0%), sputum (56.5%), and sore throat (54.3%). 23.9% of the cases were accompanied with diarrhea and 19.6% with eye congestion. None of the cases developed pneumonia. 32.6% of the cases were hospitalized. In Study 1, attack rate in the swimming department was higher than that in others (RR: 1.90; 95% confidence interval [CI]: 1.01-3.60). In Study 2, being a member of the shooting department (RR: 20.70; 95% CI: 4.90 -87.47) and being a first year high school student (RR: 10.95; 95% CI: 2.90 -41.33) were identified as risk factors for the infections. Genetic analyses of the adenoviruses showed 100% identical sequence in homology and confirmed the human adenovirus B55 (HAdV-B55). No adenovirus was detected at examining the water and environment of the swimming pool and dormitory. Conclusion: The outbreak is inferred to be occurred via propagated transmission among the students in the same athletic department, while the students with symptoms of respiratory infection continued performing school activities without any restrictions. Infection control measures such as early detection of symptoms of respiratory infection and restriction of group activity are necessary to prevent respiratory infection outbreak in the communal living setting. Abstract A scenario tree model was developed to propose efficient bovine viral diarrhea (BVD) control measures. The model used field data in eastern Hokkaido where the risk of BVDV infection in cattle has been reduced by an eradication program including mass vaccination, individual tests prior to communal pasture grazing, herd screening tests using bulk milk, and outbreak investigations of newly infected herds. These four activities were then used as hypothesized control measures in the simulation. In each simulation, the numbers of cattle infected persistently and transiently with BVDV detected by clinical manifestations and diagnosis tests and of missed by all of the diagnosis tests were calculated, and the numbers were used as indicators to be compared for the efficacy of the control measures. The model outputs indicated that the adoption of mass vaccination decreased the number of missed BVD cattle, although it did not increase the number of detected BVD cattle. Under implementation of mass vaccination, the efficacy of individual tests on selected 20% of the young and adult cattle was equal to that of the herd screening test performed in all the herds. When the virus prevalence or the number of sensitive animals becomes low, the efficacy of herd screening test was superior to one of individual tests. Considering the model outputs together, the scenario tree model developed in the present study was useful to compare the efficacy of the control measures for BVD.Bovine viral diarrhea (BVD) is a chronic disease with a global distribution caused by infection of bovine viral diarrhea virus (BVDV) in cattle [15] , and can result in economic losses within the livestock industry [5, 6, 11, 16, 17, 21, 23, 31] . Transient Abstract ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections. Abstract Israel. *Corresponding author (TT): tamirtul@post.tau.ac.ilRibo-seq reads mapping. Ribosome footprint sequences were obtained from 1 (GSE47509, induction 0-20min). We trimmed the poly-A adaptors from the reads using Cutadapt 2 (version 1.8.3), and utilized Bowtie 3 (version 1.1.1) to map them to the E. coli-lambda transcriptome. In the first phase, we discarded reads that mapped to rRNA and tRNA sequences with Bowtie parameters '-n 2 -seedlen 23 -k 1 --norc'. In the second phase, we mapped the remaining reads to the transcriptome with Bowtie parameters '-v 2 -a --strata --best -norc -m 200'. We attempted to extend alignments to their maximal length by comparing the polyA adaptor with the aligned transcript until reaching the maximal allowed error (2 mismatches across the read, with 3'-end mismatches avoided). We filtered out reads longer than 31-nt and shorter than 21-nt. Unique alignments were first assigned to the ribosome occupancy profiles. For multiple alignments, the best alignments in terms of number of mismatches were kept. Then, multiple aligned reads were distributed between locations according to the distribution of unique ribosomal reads in the respective surrounding regions. To this end, a 100-nt window was used to compute the read count density (total read counts in the window divided by length, based on unique reads) in vicinity of the M multiple aligned positions in the transcriptome, and the fraction of a read assigned to each position was . The location of the A-site was approximated by an 11nt shift from the 5' end of the aligned read. This shift maximized the correlation between MTDR (described below) and the observed read densities per E. coli gene.Ribosome profiling data normalization. We began the analysis by reconstructing ribosome profiles for E. coli and Bacteriophage Lambda expressed genes. The ribosome profiling method produces ribosome footprint counts that are proportional to the time spent in decoding each codon of all translated transcripts in a genome, at single nucleotide resolution. To avoid analyzing ribosomal profiles of genes with many missing read counts (RCs) that may result in a non-reliable estimation of the local ribosome density, genes profiles with fewer than 30 percent non-zero read counts were further filtered. Previous studies indicated an increase of RCs at the beginning of the ORF 4 and for some organisms at the end of ORF 4,5 ; therefore the first and last 20 codons were excluded when determining these thresholds or when calculating the average RCs per ORF. To enable comparison and analysis of RCs of codons of the same type originating from different genes, RCs of each Abstract China's public health surveillance system for HIV was established in late 1980s and has evolved significantly during the past three decades. With the gradually changing mode of HIV transmission from sharing of intravenous injecting equipment to sexual exposure and the rapid spread of HIV infection among Chinese homosexual men in recent years, an efficient and comprehensive population-level surveillance system for describing epidemics trends and risk behaviours associated with HIV acquisition are essential for effective public health interventions for HIV. The current review describes the overall strength of the Chinese HIV surveillance system and its structural weaknesses from a political and social perspective. The HIV surveillance system in China has undergone substantial revamping leading to a comprehensive, timely and efficient reporting system. However, large data gaps and lack of quality control and sharing of information obstruct the full performance of the system. This is largely due to fragmented authoritarianism brought about by the underlying political structure. Social stigma and discrimination in health institutes are also key barriers for further improvements of HIV diagnosis and surveillance in China. © Zhang et al.; Licensee Bentham Open. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/ 3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. Abstract The aim of this study was to evaluate the image quality of ultra-low-dose computed tomography (ULDCT) and its diagnostic performance in making a specific diagnosis of pneumonia in febrile neutropenic patients with hematological malignancy.ULDCT was performed prospectively in 207 febrile neutropenic patients with hematological malignancy. Three observers independently recorded the presence of lung parenchymal abnormality, and also indicated the cause of the lung parenchymal abnormality between infectious and noninfectious causes. If infectious pneumonia was considered the cause of lung abnormalities, they noted the two most appropriate diagnoses among four infectious conditions, including fungal, bacterial, viral, and Pneumocystis pneumonia. Sensitivity for correct diagnoses and receiver operating characteristic (ROC) curve analysis for evaluation of diagnostic accuracy were calculated. Interobserver agreements were determined using intraclass correlation coefficient.Of 207 patients, 139 (67%) had pneumonia, 12 had noninfectious lung disease, and 56 had no remarkable chest computed tomography (CT) (20 with extrathoracic fever focus and 36 with no specific disease). Mean radiation expose dose of ULDCT was 0.60±0.15 mSv. Each observer regarded low-dose CT scans as unacceptable in only four (1.9%), one (0.5%), and three (1.5%) cases of ULDCTs. Sensitivity and area under the ROC curve in making a specific pneumonia diagnosis were 63.0%, 0.65 for reader 1; 63.0%, 0.61 for reader 2; and 65.0%, 0.62 for reader 3; respectively. Conclusion ULDCT, with a sub-mSv radiation dose and acceptable image quality, provides ready and reasonably acceptable diagnostic information for pulmonary infection in febrile neutropenic patients with hematologic malignancy. Abstract Korean Red Ginseng (KRG) is a heat-processed ginseng developed by the repeated steaming and airdrying of fresh ginseng. Compared with fresh ginseng, KRG has been shown to possess greater pharmacological activities and stability because of changes that occur in its chemical constituents during the steaming process. In addition to anticancer, anti-inflammatory, and immune-modulatory activities, KRG and its purified components have also been shown to possess protective effects against microbial infections. Here, we summarize the current knowledge on the properties of KRG and its components on infections with human pathogenic viruses such as respiratory syncytial virus, rhinovirus, influenza virus, human immunodeficiency virus, human herpes virus, hepatitis virus, norovirus, rotavirus, enterovirus, and coxsackievirus. Additionally, the therapeutic potential of KRG as an antiviral and vaccine adjuvant is discussed. Abstract Pneumococcal infections are the leading cause of community-acquired pneumonia. Although the type 1 interferon-α (IFN-α) is a well-known antiviral cytokine, the role of IFN-α in antipneumococcal host defense and its therapeutic potential remain poorly understood. We have investigated these issues by using a murine transgene expression model. We found that in control animals, Streptococcus pneumoniae infection caused severe weight loss and excessive lung inflammation, associated with rapid bacterial outgrowth. In contrast, the animals that received a single dose of an adenoviral vector expressing IFN-α prior to pneumococcal infection demonstrated rapid and effective control of bacterial replication and lung inflammation and improved clinical outcome. Enhanced protection by IFN-α was due to increased activation of neutrophils and macrophages with increased release of reactive oxygen and nitrogen species and bacterial killing. Furthermore, we found that raised levels of IFN-α in the lung remained immune protective even when the gene transfer vector was given at a time postpneumococcal infection. Our study thus shows that the classically antiviral type 1 IFN can be exploited for enhancing immunity against pneumococcal infection via its activating effects on innate immune cells. Our findings hold implications for the therapeutic use of IFN-α gene transfer strategies to combat pneumococcal infections. Control of pneumococcal infection by IFN-α D Damjanovic et al. Abstract In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3 0 -adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the $150 nt 3 0 -adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem-loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem-loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3 0 RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis. Abstract a b s t r a c t A 3.5 year-old cat was admitted to the University of Melbourne Veterinary Teaching Hospital for chronic vomiting. Abdominal ultrasonography revealed a focal, circumferential thickening of the wall of the duodenum extending from the pylorus aborally for 3 cm, and an enlarged gastric lymph node. Cytology of fine-needle aspirates of the intestinal mass and lymph node revealed an eosinophilic inflammatory infiltrate and numerous extracellular septate acute angle branching fungal-type hyphae. Occasional hyphae had globose terminal ends, as well as round to oval blastospores and germ tubes. Candida albicans was cultured from a surgical biopsy of the duodenal mass. No underlying host immunodeficiencies were identified. Passage of an abrasive intestinal foreign body was suspected to have caused intestinal mucosal damage resulting in focal intestinal candidiasis. The cat was treated with a short course of oral itraconazole and all clinical signs resolved. Abstract The evaluation of the immunogenicity of Sabin strain based Inactivated Poliovirus Vaccines (sIPV) necessitates the use of wild strains in neutralization assays to assess the potential cross-reactivity of antibodies. The live virus strains including wild and Sabin strains must be handled in level 3 biocontainment laboratories. To develop an alternative assay without the use of a live virus, we constructed Mahoney, MEF-1, and Saukett pseudovirions by inserting luciferase reporter genes into intact capsid proteins. Afterward, we developed a pseudovirus-based neutralization test (pNT) and evaluated for the specificity and reproducibility. We tested serum samples from a clinical trial on sIPV vaccines by pNT and compared the results with those obtained from conventional neutralization tests (cNT). A strong correlation was observed between two methods, with the correlation coefficients of all three types of IPV vaccines being greater than 0.82 (p < 0.0001). The Geometric Mean Titer (GMT) values obtained by pNT were approximately four times higher than that by cNT, revealing the better sensitivity of pNT. In conclusion, pNT is a safe, rapid and sensitive quantitative assay with the potential of being an alternative for the evaluation of the potency of polio vaccines.ARTICLE HISTORY Abstract Although vaccines and antibiotics could kill or inhibit microbes, many infectious diseases remain difficult to treat because of acquired resistance and adverse side effects. Nano-carriers-based technology has made significant progress for a long time and is introducing a new paradigm in drug delivery. However, it still has some challenges like lack of specificity toward targeting the infectious site. Nanocarriers utilized targeting ligands on their surface called 'active target' provide the promising way to solve the problems like accelerating drug delivery to infectious areas and preventing toxicity or sideeffects. In this mini review, we demonstrate the recent studies using the active targeted strategy to kill or inhibit microbes. The four common nano-carriers (e.g. liposomes, nanoparticles, dendrimers and carbon nanotubes) delivering encapsulated drugs are introduced. Abstract Terminal sialic acids on cell surface glycoconjugates can carry 9-O -acetyl esters. For technical reasons, it has previously been difficult to determine their precise distribution on different cell types. Using a recombinant soluble form of the Influenza C virus hemagglutinin-esterase as a probe for 9-O -acetylated sialic acids, we demonstrate here their preferential expression on the CD4 T cell lineage in normal B10.A mouse lymphoid organs. Of total thymocytes, 8-10% carry 9-O -acetylation; the great majority of these are the more mature PNA Ϫ , HSA Ϫ , and TCR hi medullary cells. While low levels of 9-O -acetylation are seen on some CD4/CD8 double positive (DP) and CD8 single positive (SP) cells, high levels are present primarily on 80-85% of CD4 SP cells. Correlation with CD4 and CD8 levels suggests that 9-O -acetylation appears as an early differentiation marker as cells mature from the DP to the CD4 SP phenotype. This high degree of 9-O -acetylation is also present on 90-95% of peripheral spleen and lymph node CD4 T cells. In contrast, only a small minority of CD8 T cells and B cells show such levels of 9-O -acetylation. Among mature peripheral CD4 T lymphocytes, the highly O -acetylated cells are Mel 14 hi , CD44 lo , and CD45R(exon B) hi , features typical of naive cells. Digestions with trypsin and O -sialoglycoprotease (OSGPase) and ELISA studies of lipid extracts indicate that the 9-O -acetylated sialic acids on peripheral CD4 T cells are predominantly on O-linked mucintype glycoproteins and to a lesser degree, on sialylated glycolipids (gangliosides). In contrast, sialic acids on mucin type molecules of CD8 T cells are not O -acetylated; instead these molecules mask the recognition of O -acetylated gangliosides that seem to be present at similar levels as on CD4 cells. The 9-O -acetylated gangliosides on mouse T cells are not bound by CD60 antibodies, which recognize O -acetylated gangliosides in human T cells. Tethering 9-O -acetylated mucins with the Influenza C probe with or without secondary cross-linking did not cause activation of CD4 T cells. However, activation by other stimuli including TCR ligation is associated with a substantial decrease in surface 9-O -acetylation, primarily in the mucin glycoprotein component. Thus, 9-O -acetylation of sialic acids on cell surface mucins is a novel marker on CD4 T cells that appears on maturation and is modulated downwards upon activation. Abstract Influenza virus (IV) is a continuously evolving virus that widely spreads in humans and contributes to substantial morbidity and mortality. Re-emergence of human infection with avian influenza virus H5N1 poses extra challenge to IV control. Artificial microRNA (amiRNA)-mediated RNA interference has become a powerful antiviral approach due to its high specificity and rapid effect. Here, we designed several amiRNAs targeting the hemagglutinin gene of H5N1, a major determinant of pathogenicity. Expression and delivery efficiency were enhanced by presenting functional amiRNA with chimpanzee adenovirus serotype 68 (AdC68). One amiRNA, HA-1405, significantly limited H5N1 replication in vitro and inhibited 96.7% of clade 2.3.2 replication. AdC68-conjugated HA-1405 treatment remarkably decreased different clades of H5N1 plaque formation in Madin-Darby canine kidney cells. Moreover, prophylactic administration with rAd(HA-1405) markedly alleviated clinical symptoms and reduced ~3to 40-folds of lung viral RNA copies against four clades of H5N1 in Institute of Cancer Research (ICR) mice. Our results further showed that rAd(HA-1405) conferred 70 and 40% immediate protection against lethal clade 2.3.2 and clade 2.3.4 H5N1 challenge, respectively. In conclusion, these data provided information that HA-targeting amiRNA delivered by AdC68 could be pursued as a potential agent for highly pathogenic avian influenza viruses prevention. Abstract Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), account for severe economic losses in the poultry industry. The continuous emergence of a multitude of IBV variants poses many challenges for its diagnosis and control, and live attenuated vaccines, despite their routine use, still plays a significant role in driving IBV evolution, further complicating the epidemiological scenario. Unfortunately, the impact of different vaccination strategies on IB control, epidemiology, and diagnosis has rarely been investigated.This work presents the results of a large-scale diagnostic survey performed in Poland to study IBV molecular epidemiology and how vaccination may affect the viral circulation in the field. To this purpose, 589 samples were collected between May 2017 and January 2019, tested by reverse transcription-PCR for IBV and sequenced. Vaccine and field strains were discriminated based on genetic and anamnestic information.The most commonly detected lineages were 793B (79%) and variant 2 (17.4%), with sporadic detections of QX, Mass, and D274-like strains. Most of the detected strains had a vaccine origin: 46.3% matched one of the applied vaccines, while 36.5% were genetically related to vaccines not implemented in the respective protocol. Besides their practical value for the proper planning of vaccination protocols in Poland, these results suggest that only a fraction (17.2%) of the circulating strains are field ones, imposing a careful assessment of the actual IBV field menaces. Moreover, phenomena like vaccine spreading and persistence seem to occur commonly, stressing the need to further study the epidemiological consequences of the extensive use of live vaccines. Abstract Introduction: Hyponatraemia is the most common electrolyte imbalance seen in clinical practice, and a common laboratory finding in children with communityacquired pneumonia (CAP). This study aimed to identify the incidence of hyponatraemia in cases of CAP, to find predictive tools in order to classify the severity and outcome of CAP and to explore possible differences of clinical importance between the two sexes. Material and methods: The medical files of 54 children (66.4% males), 4.67 ±2.88 years old, were retro-prospectively reviewed. Results: 35/54 (64.8%) children with pneumonia had normal values of sodium at admission, 18/54 (33.3%) had mild hyponatraemia and 1 child (1.9%) moderate hyponatraemia. Increased heart rhythm and tachypnoea at admission were correlated with lower values of sodium (z = -2.664, p = 0.007 and z = -1.705, p = 0.089 respectively). No differences were found between the two sexes concerning the characteristics of pneumonia or the range of sodium in serum at admission. A correlation was found between sodium admission values and: a) C-reactive protein (p = 0.000), and b) leukocyte count (p = 0.006). Sedimentation rate (p = 0.021) was also considered as a possible risk factor affecting the value of sodium at admission to hospital. Finally, a negative association was also observed between the degree of hyponatraemia and the duration of hospitalization (z = -3.398, p = 0.001). Conclusions: Although studies in larger population groups are needed, in our study increased heart rhythm, tachypnoea, leucocyte count, C-reactive protein, and also erythrocyte sedimentation rate could be considered as possible risk factors influencing the degree of hyponatraemia, and thus the outcome of hospitalized children with CAP. Abstract Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe pulmonary infection, with ∼35 % mortality. Spike glycoprotein (S) of MERS-CoV is a key target for vaccines and therapeutics because S mediates viral entry and membrane-fusion to host cells. Here, four different S subunit proteins, receptor-binding domain (RBD; 358-606 aa), S1 (1-751 aa), S2 (752-1296 aa), and SΔTM (1-1296 aa), were generated using the baculoviral system and immunized in mice to develop neutralizing antibodies. We developed 77 hybridomas and selected five neutralizing mAbs by immunization with SΔTM against MERS-CoV EMC/2012 strain S-pseudotyped lentivirus. However, all five monoclonal antibodies (mAb) did not neutralize the pseudotyped V534A mutation. Additionally, one mAb RBD-14F8 did not show neutralizing activity against pseudoviruses with amino acid substitution of L506 F or D509 G (England1 strain, EMC/2012 L506 F, and EMC/2012 D509 G), and RBD-43E4 mAb could not neutralize the pseudotyped I529 T mutation, while three other neutralizing mAbs showed broad neutralizing activity. This implies that the mutation in residue 506-509, 529, and 534 of S is critical to generate neutralization escape variants of MERS-CoV. Interestingly, all five neutralizing mAbs have binding affinity to RBD, although most mAbs generated by RBD did not have neutralizing activity. Additionally, chimeric antibodies of RBD-14F8 and RBD-43E4 with human Fc and light chain showed neutralizing effect against wild type MERS-CoV KOR/KNIH/002, similar to the original mouse mAbs. Thus, our mAbs can be utilized for the identification of specific mutations of MERS-CoV. Abstract The CVTree web server (http://tlife.fudan.edu.cn/ cvtree) presented here is a new implementation of the whole genome-based, alignment-free composition vector (CV) method for phylogenetic analysis. It is more efficient and user-friendly than the previously published version in the 2004 web server issue of Nucleic Acids Research. The development of whole genome-based alignment-free CV method has provided an independent verification to the traditional phylogenetic analysis based on a single gene or a few genes. This new implementation attempts to meet the challenge of ever increasing amount of genome data and includes in its database more than 850 prokaryotic genomes which will be updated monthly from NCBI, and more than 80 fungal genomes collected manually from several sequencing centers. This new CVTree web server provides a faster and stable research platform. Users can upload their own sequences to find their phylogenetic position among genomes selected from the server's inbuilt database. All sequence data used in a session may be downloaded as a compressed file. In addition to standard phylogenetic trees, users can also choose to output trees whose monophyletic branches are collapsed to various taxonomic levels. This feature is particularly useful for comparing phylogeny with taxonomy when dealing with thousands of genomes. Abstract Camels have cultural value in the Arab society and are considered one of the most important animals in the Arabian Peninsula and arid environments, due to the distinct characteristics of their meat and milk. Moreover, there is a great interest in camel racing and beauty shows. Therefore, treatment of elite animals, increasing the number of camels as well as genetic improvement is an essential demand. Because there are unique camels for milk production, meat, or in racing, the need to propagate genetically superior camels is urgent. Recent biotechnological approaches such as stem cells hold great promise for biomedical research, genetic engineering, and as a model for studying early mammalian developmental biology. Establishment of stem cells lines from camels would tremendously facilitate regenerative medicine for genetically superior camels, permit the gene targeting of the camel genome and the generation of genetically modified animal and be a mean for genome conservation for the elite breeds. In this mini-review, we show the current research, future horizons and potential applications for camel stem cells. Abstract China ratified the International Covenant on Economic, Social and Cultural Rights in 2001. It thus bears obligations under Article 12 of the covenant to take appropriate measures at the domestic level to realize the right to health in China. Accountability is an important component of the right to health. This article examines whether the Western concept of accountability, recently imported into China, has the potential to improve the protection of the right to health within China's existing political, legal, and cultural framework. In so doing, it reviews current Chinese institutional mechanisms and considers the use of less formal mechanisms by which duty-bearers might be held accountable in China. More specifically, this article provides an overview of a range of health-related accountability mechanisms, including judicial, political, administrative, professional, and social accountability arrangements. It concludes that although there is the basis of an accountability framework for the right to health in China, the effective operation of accountability mechanisms is hindered by longstanding cultural and political barriers. Abstract Human toxocariasis is generally a benign, self-curing disease, and neurologic involvement is quite exceptional. In this study, we report a case of toxocariasis caused by ingestion of an unusual transport host, namely live slugs. The clinical picture comprised eosinophilic lung involvement with severe neurologic disorders in relation to vasculitis as well as retinal detachment. Abstract Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA's 5 0 -terminal 'cap'. The minimal 'cap0' consists of N7-methylguanosine linked to the first nucleotide via a 5 0 -5 0 triphosphate (ppp) bridge. Cap0 is further modified by 2 0 -O-methylation of the next two riboses, yielding 'cap1' (m7GpppNmN) and 'cap2' (m7GpppNmNm). However, some viral RNAs lack 2 0 -O-methylation, whereas others contain only ppp-at their 5 0 -end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0-and 5 0 ppp-mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K 1/2,app $9 to 23 nM). The 2 0 -O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K 1/2,app $450 nM). The 5 0 -terminal regions of 5 0 ppp-mRNAs were recognized by IFIT5 (K 1/2,app $400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5 0 -terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1-and 5 0 ppp-mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations. Abstract A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.Materials and MethodsSynthetic DNA linkers, restriction endonucleases, and polynucleotide kinase were from New England Biolabs (Beverly, MA). DNA polymerase IKlenow) and T4 DNA Ligase were from Boehringer Mannheim Biochemicals (Indianapolis, IN). [35Slmethionine, [35S]cysteine, and [35S]sulfate were from Amersham Corp. (Arlington Heights, IL). Trans-[35S]label was from ICN Radiochemicals (Irvine, CA). t25I-ACTH, used as a tracer in the ACTH RIA was obtained from the Metabolic Research Lab at The Univer- Abstract OBJECTIVE To study the clinical characteristics of patients in Zhejiang province, China, infected with the 2019 severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) responsible for coronavirus disease 2019 (covid-2019). DESIGN Retrospective case series. SETTING Seven hospitals in Zhejiang province, China. PARTICIPANTS 62 patients admitted to hospital with laboratory confirmed SARS-Cov-2 infection. Abstract Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves.Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs. Abstract Emerging and re-emerging pathogens represent a substantial threat to public health, as demonstrated with numerous outbreaks over the past years, including the 2013-2016 outbreak of Ebola virus in western Africa. Coronaviruses are also a threat for humans, as evidenced in 2002/2003 with infection by the severe acute respiratory syndrome coronavirus (SARS-CoV), which caused more than 8000 human infections with 10% fatality rate in 37 countries. Ten years later, a novel human coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV), associated with severe pneumonia, arose in the Kingdom of Saudi Arabia. Until December 2016, MERS has accounted for more than 1800 cases and 35% fatality rate. Finding an animal model of disease is key to develop vaccines or antivirals against such emerging pathogens and to understand its pathogenesis. Knowledge of the potential role of domestic livestock and other animal species in the transmission of pathogens is of importance to understand the epidemiology of the disease. Little is known about MERS-CoV animal host range. In this paper, experimental data on potential hosts for MERS-CoV is reviewed. Advantages and limitations of different animal models are evaluated in relation to viral pathogenesis and transmission studies. Finally, the relevance of potential new target species is discussed. Abstract To date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, and yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions, and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. This study describes a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity, and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions.Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. We identified here an RNA mutagen-resistant variant of the nsP2 helicase/protease that conferred increased fidelity and yet could not operate in the same manner as high-fidelity polymerases. We show that the alphavirus helicase is a key component of the fidelity-regulating machinery. Our data show that the RNA mutagenic activity of compounds such as ribavirin is coupled to and potentiated by nucleotide depletion and that RNA viruses can fine-tune their replication fidelity when faced with an intracellular environment depleted of nucleotides. Abstract Highlights d 5 early innate markers correlate with antibody response to Ebola vaccine rVSV-ZEBOV d IP-10 on day 3 after vaccination is an independent correlate of antibody induction d RNA-seq analysis identifies early gene expression signature linked to IP-10 Abstract To gain insights into the pathogenesis of influenza A virus (IAV) infections, this study focused on characterizing the inflammatory network and identifying key proteins by combining high-throughput data and computational techniques. We constructed the cell-specific normal and inflammatory networks for H5N1 and H1N1 infections through integrating high-throughput data. We demonstrated that better discrimination between normal and inflammatory networks by network entropy than by other topological metrics. Moreover, we identified different dynamical interactions among TLR2, IL-1b, IL10 and NFkB between normal and inflammatory networks using optimization algorithm. In particular, good robustness and multistability of inflammatory sub-networks were discovered. Furthermore, we identified a complex, TNFSF10/HDAC4/HDAC5, which may play important roles in controlling inflammation, and demonstrated that changes in network entropy of this complex negatively correlated to those of three proteins: TNFa, NFkB and COX-2. These findings provide significant hypotheses for further exploring the molecular mechanisms of infectious diseases and developing control strategies. Abstract Objective: To assess virus-specific hospitalization rates, risk factors for illness severity, and seasonal trends in children hospitalized with acute respiratory infections (ARI).Setting: A government hospital serving low-and middle-income population in Amman, Jordan.Participants: Children under two years old hospitalized with fever and/or respiratory symptoms (n=3168) from 3/16/2010-3/31/2013. Children with chemotherapy-associated neutropenia and newborns who had never been discharged after birth were excluded from the study.Outcome Measures: Hospitalization rates and markers of illness severity: admission to intensive care unit (ICU), mechanical ventilation (MV), oxygen therapy, length of stay (LOS), and death.Of the 3168 subjects, 2581 (82%) had at least one respiratory virus detected, with respiratory syncytial virus (RSV) being the most predominant pathogen isolated. During admission, 1013 (32%) received oxygen therapy, 284(9%) were admitted to ICU, 111(4%) were placed on MV, and 31(1%) children died. Oxygen therapy was higher in RSV only subjects compared to human rhinovirus only (42% vs 29%, p<0.001), adenovirus only (42% vs 21%, p<0.001), and human parainfluenza virus only (42% vs 23% p<0.001) subjects. Underlying medical condition was associated with oxygen therapy (aOR 1.95, 95% CI 1.49-2.56), ICU admission (aOR 2.51, 95%CI 1.71-3.68), MV (1.91, 95% CI 1.11-3.28), and longer LOS (aOR1.71, 95% CI 1.37-2.13). Similarly, younger age was associated with oxygen therapy (0.23, 95% CI 0.17-0.31), ICU admission (aOR 0.47, 95% CI 0.30-0.74), MV (0.28, 95% CI 0.15-0.53), and longer LOS (aOR 0.47, 95% CI 0.38-0.59). Pneumonia was strongly associated with longer LOS (aOR 2.07, 95% CI 1.65-2.60), oxygen therapy (aOR 2.94, 95% CI 2.22-3.89), ICU admission (aOR 3.12, 95% CI 2.16-4.50), and MV (aOR 3.33, 95% CI 1.85-6.00). Virus-specific hospitalization rates ranged from 0.5-10.5 per 1000 children.Respiratory viruses are associated with severe illness in Jordanian children hospitalized with ARI. Prevention strategies such as extended breastfeeding, increased access to palivizumab and RSV vaccine development could help decrease hospitalization rates and illness severity, particularly in young children with underlying medical conditions.For peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Bashir Hospital is one of the three major government hospitals in Amman, which is Jordan's largest city and capital. 12 It was estimated that during the study period, the hospital provided care for ≈55% of all children in Amman (author SF, unpublished data). Additionally, 17,557 children were admitted to the pediatric wards, 11,230 (64%) of whom were under two years of age.After consent, trained local research staff obtained blood either by heel stick or venipuncture, as well as nasal and throat swabs. Standardized questionnaires were used to record demographic, clinical, and socioeconomic data. Parents were queried in Arabic, and bilingual research staff transcribed the information into an Englishlanguage case report form at the time of the interview. After subjects were discharged, charts were abstracted for the following: antibiotic use and type, blood, urine, and/or cerebral spinal fluid (CSF) cultures, organisms isolated, chest radiography and results, oxygen use, intensive care unit (ICU) stay, mechanical ventilation (MV), length of stay (LOS) in the hospital, and discharge status. Abstract Hsueh P-R, Asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARSCoV-2): Facts and myths, Journal of Microbiology, Immunology and Infection, https://doi. Abstract Chronic rhinosinusitis (CRS) is a multifactorial condition in which the microbiota plays a pathogenic role. The nature of the interaction between the microbiota and the local immune system is very complex and has not been fully elucidated. Recent improvements in the microbiological techniques have greatly advanced our understanding of the complex nature of this interaction. This paper summarizes the current state of the rapidly evolving research on this subject. Defining the nature of the role of the microbiota in CRS is important because of the associated therapeutic implications. Abstract Coronaviruses are assembled by budding into smooth membranes of the intermediate ERto-Golgi compartment. We have studied the association of the viral membrane glycoproteins M and S in the formation of the virion envelope. Using coimmunoprecipitation analysis we demonstrated that the M and S proteins of mouse hepatitis virus (MHV) interact specifically forming heteromultimeric complexes in infected cells. These could be detected only when the detergents used for their solubilization from cells or virions were carefully chosen: a combination of nonionic (NP-40) and ionic (deoxycholic acid) detergents proved to be optimal. Pulse-chase experiments revealed that newly made M and S proteins engaged in complex formation with different kinetics. Whereas the M protein appeared in complexes immediately after its synthesis, newly synthesized S protein did so only after a lag phase of >20 min. Newly made M was incorporated into virus particles faster than S, which suggests that it associates with preexisting S molecules. Using the vaccinia virus T7-driven coexpression of M and S we also demonstrate formation of M/S complexes in the absence of other coronaviral proteins. Pulse-chase labelings and coimmunoprecipitation analyses revealed that Abstract Rfam is a collection of RNA sequence families, represented by multiple sequence alignments and covariance models (CMs). The primary aim of Rfam is to annotate new members of known RNA families on nucleotide sequences, particularly complete genomes, using sensitive BLAST filters in combination with CMs. A minority of families with a very broad taxonomic range (e.g. tRNA and rRNA) provide the majority of the sequence annotations, whilst the majority of Rfam families (e.g. snoRNAs and miRNAs) have a limited taxonomic range and provide a limited number of annotations. Recent improvements to the website, methodologies and data used by Rfam are discussed. Rfam is freely available on the Web at Abstract Since the emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrom Coronavirus (MERS-CoV) it has become increasingly clear that bats are important reservoirs of CoVs. Despite this, only 6% of all CoV sequences in GenBank are from bats. The remaining 94% largely consist of known pathogens of public health or agricultural significance, indicating that current research effort is heavily biased towards describing known diseases rather than the 'pre-emergent' diversity in bats. Our study addresses this critical gap, and focuses on resource poor countries where the risk of zoonotic emergence is believed to be highest. We surveyed the diversity of CoVs in multiple host taxa from twenty countries to explore the factors driving viral diversity at a global scale. We identified sequences representing 100 discrete phylogenetic clusters, ninety-one of which were found in bats, and used ecological and epidemiologic analyses to show that patterns of CoV diversity correlate with those of bat diversity. This cements bats as the major evolutionary reservoirs and ecological drivers of CoV diversity. Co-phylogenetic reconciliation analysis was also used to show that host switching has contributed to CoV evolution, and a preliminary analysis suggests that regional variation exists in the dynamics of this process. Overall our study represents a model for exploring global viral diversity and advances our fundamental understanding of CoV biodiversity and the potential risk factors associated with zoonotic emergence. Abstract Purpose: In outbreaks of infectious disease, medical students are easily overlooked in the management of healthcare personnel protection although they serve in clinical clerkships in hospitals. In the early summer of 2015, Middle East respiratory syndrome (MERS) struck South Korea, and students of Sungkyunkwan University School of Medicine (SKKUSOM) were at risk of contracting the disease. The purpose of this report is to share SKKUSOM's experience against the MERS outbreak and provide suggestions for medical schools to consider in the face of similar challenges. Methods: Through a process of reflection-on-action, we examined SKKUSOM's efforts to avoid student infection during the MERS outbreak and derived a few practical guidelines that medical schools can adopt to ensure student safety in outbreaks of infectious disease. Results: The school leadership conducted ongoing risk assessment and developed contingency plans to balance student safety and continuity in medical education. They rearranged the clerkships to another hospital and offered distant lectures and tutorials. Five suggestions are extracted for medical schools to consider in infection outbreaks: instant cessation of clinical clerkships; rational decision making on a school closure; use of information technology; constant communication with hospitals; and open communication with faculty, staff, and students. Conclusion: Medical schools need to take the initiative and actively seek countermeasures against student infection. It is essential that medical schools keep constant communication with their index hospitals and the involved personnel. In order to assure student learning, medical schools may consider offering distant education with online technology. Abstract Identifying recombinant sequences in an era of large genomic databases is challenging as it requires an efficient algorithm to identify candidate recombinants and parents, as well as appropriate statistical methods to correct for the large number of comparisons performed. In 2007, a computation was introduced for an exact nonparametric mosaicism statistic that gave high-precision P values for putative recombinants. This exact computation meant that multiplecomparisons corrected P values also had high precision, which is crucial when performing millions or billions of tests in large databases. Here, we introduce an improvement to the algorithmic complexity of this computation from O(mn 3 ) to O(mn 2 ), where m and n are the numbers of recombination-informative sites in the candidate recombinant. This new computation allows for recombination analysis to be performed in alignments with thousands of polymorphic sites. Benchmark runs are presented on viral genome sequence alignments, new features are introduced, and applications outside recombination analysis are discussed. Abstract Shiga toxin-producing Escherichia coli (STEC) cause gastrointestinal illnesses including non-bloody or bloody diarrhoea, haemorrhagic colitis (HC), and the haemolytic uremic syndrome (HUS). To investigate the occurrence of STEC among grazing dromedaries from Kenya, E. coli isolated from fecal matter collected from 163 dromedaries on a large ranch were screened for the presence of stx1 and stx2. STEC strains were isolated and serotyped. Isolates were subjected to PCR for the subtyping of stx genes and for the detection of eae and ehx. In addition, whole genome sequencing (WGS) was carried out to detect further virulence genes and to determine the multilocus sequence types (MLST). Antimicrobial resistance profiles were determined by disk diffusion.STEC was isolated from 20 (12.3%) of the fecal samples. Thereof, nine (45%) isolates were STEC O156:H25, three (15%) isolates typed STEC O43:H2. The remaining isolates occurred as single serotypes or were O nontypeable. Eleven (55%) of the isolates harboured stx2a, nine (45%) eae, and 14 (70%) ehx, respectively. WGS revealed the presence of iss in 16 (80%), subAB in four (20%) and astA in two (10%) of the isolates, Furthermore, espA, tccP, nleA, nleB, tccP, and tir were found exclusively among STEC O156:H25.Eleven different sequence types (ST) were detected. The most prominent was ST300/ST5343, which comprised STEC O156:H25. All STEC isolates were pan susceptible to a panel of 16 antimicrobial agents. Overall, the results indicate that dromedary camels in Kenya may be reservoirs of STEC, including serotypes possessing virulence markers associated to disease in humans, such as STEC O156:H25. STEC in camels may represent a health hazard for humans with close contact to camels or to consumers of camel derived foodstuffs, such as unpasteurised camel milk. Abstract Objective: A total of 131 cases of avian-originated H7N9 infection have been confirmed in China mainland from February 2013 to May 2013. We calculated the overall burden of H7N9 cases in China as of 31 May 2013 to provide an example of comprehensive burden of disease in the 21st century from an acute animalborne emerging infectious disease.Design: We present an accurate and operable method for estimating the burden of H7N9 cases in China. The main drivers of economic loss were identified. Costs were broken down into direct (outpatient and inpatient examination and treatment) and indirect costs (cost of disability-adjusted life years (DALYs) and losses in the poultry industry), which were estimated based on field surveys and China statistical year book.Setting: Models were applied to estimate the overall burden of H7N9 cases in China.The huge poultry industry losses followed live poultry markets closing down and poultry slaughtering in some areas. Though the proportion of direct medical losses and DALYs losses in the estimate of H7N9 burden was small, the medical costs per case were extremely high (particularly for addressing the use of modern medical devices). A cost-effectiveness assessment for the intervention should be conducted in a future study. Abstract This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.The intraspecies genomic diversity of the single-strand RNA (+) virus species hepatitis A virus (Hepatovirus), hepatitis C virus (Hepacivirus), and hepatitis E virus (Orthohepevirus) was compared. These viral species all can cause liver inflammation (hepatitis), but share no gene similarity. The codon usage of human hepatitis A virus (HAV) is suboptimal for replication in its host, a characteristic it shares with taxonomically related rodent, simian, and bat hepatitis A virus species. We found this codon usage to be strikingly similar to that of Triatoma virus that infects blood-sucking kissing bugs. The codon usage of that virus is well adapted to its insect host. The codon usage of HAV is also similar to other invertebrate viruses of various taxonomic families. An evolutionary ancestor of HAV and related virus species is hypothesized to be an insect virus that underwent a host jump to infect mammals. The similarity between HAV and invertebrate viruses goes beyond codon usage, as they also share amino acid composition characteristics, while not sharing direct sequence homology. In contrast, hepatitis C virus and hepatitis E virus are highly similar in codon usage preference, nucleotide composition, and amino acid composition, and share these characteristics with Human pegivirus A, West Nile virus, and Zika virus. We present evidence that these observations are only partly explained by differences in nucleotide composition of the complete viral codon regions.We consider the combination of nucleotide composition, amino acid composition, and codon usage preference suitable to provide information on possible evolutionary similarities between distant virus species that cannot be investigated by phylogeny. Abstract ultraviolet (uV) radiation induces skin injury, and is associated with the development and formation of melanoma, which is a highly lethal form of skin cancer. juglanin is a natural product, which is predominantly extracted from Polygonum aviculare, and is considered a functional component among its various compounds. juglanin has been reported to exert marked protective effects in various diseases via the inhibition of inflammation and tumor cell growth. the present study aimed to explore the effects of juglanin on human skin cancer induced by uV and to reveal the underlying molecular mechanism. in the present study, immunohistochemical analysis, western blot analysis, RT-qPCR analysis and flow cytometry assays were mainly used in vivo and/or in vitro. the results indicated that in mice, uVB exposure increased susceptibility to carcinogens, and accelerated disease pathogenesis. conversely, juglanin was able to ameliorate this condition via inhibition of inflammation, suppression of cell proliferation and induction of apoptosis via p38/c-jun n-terminal kinase (jnK) blockage, nuclear factor (nf)-κB inactivation and caspase stimulation in vivo. in addition, in vitro, the present study demonstrated that treatment of uVB-stimulated B16f10 melanoma cells with juglanin resulted in a dose-dependent decrease in cell viability, as well as increased apoptosis via the upregulation of caspase expression and poly (adP-ribose) polymerase cleavage. in addition, juglanin markedly attenuated p38/jnK signaling, inactivated the phosphoinositide 3-kinase/protein kinase B pathway and suppressed uVB-induced nf-κB activation. taken together, these results indicated the possibility of applying juglanin in combination with uVB as a potential therapeutic strategy for preventing skin cancer. Abstract Interferon-induced transmembrane protein 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of al arge number of viruses,i ncluding West Nile virus,D engue virus,Ebola virus,and Zika virus.Production of this 14.5 kDa, 133-residue transmembrane protein, especially with essential posttranslational modifications,b yr ecombinant expression is challenging.Inthis report, we document the chemical synthesis of IFTIM3 in multi-milligram quantities (> 15 mg) and the preparation of phosphorylated and fluorescent variants.T he synthesis was accomplished by using KAHA ligations,w hich operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C-terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic,h omogenous IFITM3 and its derivatives for further studying its structure and biological mode of action.Interferon-induced transmembrane protein 3( IFITM3) is a1 33 amino acid membrane-associated protein (MP) that inhibits the replication of pathogenic viruses.This protein was first named fragilis and reported by Saitou and co-workers in 2002, [1] but interest surged when its antiviral properties came to light during the 2009 H1N1 influenza pandemic. Everitt and co-workers [2] reported that patients lacking full IFITM3 were more quickly and severely infected, leading to adramatic increase in mortality.S ubsequent studies have shown that IFITM3 exhibits antiviral activity against al arge number of viruses, [3, 4] including West Nile virus, [5, 6] Dengue virus, [5] [6] [7] Filoviruses (Ebola and Marburg viruses), [8] SARS Coronavirus, [8] Rift Va lley fever virus, [9] HIV-1 [10] and, most recently, Zika virus. [11] IFITM3 and its related proteins (IFITMs) contain two highly hydrophobic intramembrane domains:aconserved loop region, and amore polar N-terminal region that varies in sequence and length (Figure 1a) . Several proposals for its structure,m embrane-spanning topology,a nd mechanism of action have been reported but there is little consensus of how IFITM3 prevents viral replication. [12] Further studies,such as structure determination and investigation of its numerous posttranslational modifications, [13] [14] [15] [16] are hampered in part by lack of access to the pure protein, especially homogeneous material containing the essential posttranslational modifications.In this report, we document the production of milligram quantities of homogeneous IFITM3 and key post-translationally modified variants by total chemical synthesis by using aketoacid-hydroxylamine (KAHA) ligation. [17] Key to the success of this work is the unique nature of the KAHA ligation using 5-oxaproline,w hich operates under acidic conditions ideal for solubilizing hydrophobic peptide segments and delivers more soluble peptide esters compared to amide as the primary ligation product. Theu se of 5-oxaproline results in the introduction of homoserine,anon-canonical amino acid, at the ligation site.W hen carefully chosen, we have found that this is an innocuous mutation of many residues and have shown that it does not disturb folding or biological activity. [18] Thes ynthetic route allows facile incorporation of key posttranslational modifications,i ncluding phosphorylation and the attachment of afluorescent dye.This work establishes access to homogenous IFITM3 and will enable further studies on its structure and mode of action.Our synthetic planning took into account the different structural regions of IFITM3 and took inspiration from amap of key residue constructed by Brass and co-workers. [7] We aimed to synthesize IFITM3 from three segments using two KAHA ligations.F or the first ligation site,w ec hose Ser50Hse,s ince this is located in an exposed loop region that shows considerable variability among IFITM proteins. As the second ligation, we selected Thr95Hse,s ince this site was reported to be one of the few variable sites within an otherwise highly conserved region. This strategy called for the use of av aline a-ketoacid for the ligation, which although somewhat slower than less hindered residues,h as proven to be an excellent choice for KAHA ligations. [18b] Thes ynthesis of segment 3, which contains the IM2 domain, proved to be the most challenging.O wing to its extreme hydrophobicity,wefirst attempted to synthesize it by tert-butyloxycarbonyl solid-phase peptide synthesis (Boc-SPPS), since Boc chemistry is known to be better suited than fluorenylmethyloxycarbonyl solid-phase peptide synthesis (Fmoc-SPPS) for the preparation of hydrophobic Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10. Abstract Pulmonary fibrosis (PF) is a common complication in those interstitial lung diseases patients, which will result in poor prognosis and short survival. Traditional therapeutic methods such as glucocorticoid and cytotoxic drugs are insufficient for treating PF and may cause severe side effects. Recent studies showed that traditional Chinese herbal abstraction such as Tanshinone IIA (TIIA) was displayed significant anti-PF effects in animal models. However, the exact mechanisms underlying the protective effects of TIIA were not fully understood. Here we further investigated the protective effects of TIIA and its mechanisms underlying. PF models of rat were induced by bleomycin (BLM); TIIA was administered subsequently. The PF changes were identified by histopathological analyses. The results showed that BLM resulted in severe PF and alveolar inflammation; together with significant elevation of transforming growth factor-β 1 (TGF-β1). Angiotensin-converting enzyme 2 (ACE-2) together with angiotensin-(1-7) [ANG-(1-7)] were both greatly reduced after BLM administration. TIIA treatment notably attenuated BLM induced PF and inflammation, decreased expression of TGF-β1 and reversed ACE-2 and ANG-(1-7) production in rat lungs. Thus we may draw the conclusion that TIIA may exert protective effects on BLM induced PF in rats, and the ACE-2/ANG-(1-7) axis may ascribe to those protective effects. Abstract To cite: Rabinowitz PMacG, Pappaioanou M, Bardosh KL, et al. A planetary vision for one health. Abstract To determine the feasibility of a randomised controlled trial (RCT) of rhinothermy for the common cold.Design: Open label randomised placebo-controlled feasibility study.Setting: Single centre research institute in New Zealand recruiting participants from the community.Participants: 30 adult participants with symptoms of a common cold, presenting within 48 hours of the onset of symptoms.Interventions: Participants were randomly assigned 2:1 to receive either: 35L/min of 100% humidified air at 41 O C via high flow nasal cannulae (HFNC), two hours per day for up to five days (rhinothermy); or Vitamin C 250mg daily for five days (control).The proportion of screened candidates who were randomised.In all 30/79 (38%, 95% CI 27 to 50) of potential participants screened for eligibility were randomised. Rhinothermy was well tolerated and all randomised participants completed the study (100%, 95% CI 88 to 100%). The reduction from baseline in the modified Jackson score was significantly greater with rhinothermy compared with control at days 2, 3, 4, 5, and 6, with the maximum difference at day 4 (-6.4, 95%CI -9.4 to -3.3). The substantial clinical benefit threshold for modified Jackson score was a 5 unit change.Conclusions: This study shows that an RCT of rhinothermy compared with vitamin C in the treatment of the common cold is feasible. A robust adequately powered RCT is needed, particularly as this feasibility study identified that rhinothermy improved symptoms of the common cold compared with control. Abstract Conclusion International cooperation projects have been an invaluable component of China's response to HIV/AIDS, and China has now been able to take this information and share its experiences with other countries with the help of these same international programmes. Abstract A B S T R A C T Cytomegalovirus (CMV) pneumonia is a rare opportunistic infection in the setting of HIV (Human Immunodeficiency Virus)-infection. Establishing accurate diagnosis of CMV pneumonia in HIV-infection can be challenging. Co-infections by multiple opportunistic pathogens are common and a high degree of clinical vigilance to evaluate for multiple infections, including CMV pneumonia, should be maintained. As there can be a degree of overlap in clinical and radiological features amongst different opportunistic infections affecting the lungs, definitive microbiological and cytohistologic evidences are needed. Reliance on microbiological evidence of CMV in respiratory specimens alone for the diagnosis of CMV pneumonia will lead to an over-diagnosis of the condition and unnecessary treatment.In our case report, we describe a 53-year-old man with recently diagnosed HIV-infection who presented with non-resolving pneumonia. A diagnosis of CMV pneumonia was reached through consistent clinical, radiological, microbiological and cytologic investigations. The patient made a full clinical recovery after being started on anti-CMV treatment. Abstract Citation Yuan S. 2015. Statins may decrease the fatality rate of Middle East respiratory syndrome infection. mBio 6(4):e01120-15. Abstract Here we introduce a culture system for the isolation, passaging and amplification of avian tracheal epithelial (ATE) cells. The ATE medium, which contains chicken embryo extract and fetal bovine serum, supports the growth of ciliated cells, goblet cells and basal cells from chicken tracheas on fibronectin-or matrigel-coated dishes. Non-epithelial cells make up less than 10% of the total population. We further show that ATE cells support the replication and spread of infectious bronchitis virus (IBV). Interestingly, immunocytostaining revealed that basal cells are resistant to IBV infection. We also demonstrate that glycosaminoglycan had no effect on infection of the cells by IBV. Taken together, these findings suggest that primary ATE cells provide a novel cell culture system for the amplification of IBV and the in vitro characterization of viral cytopathogenesis.tracheal epithelial cell / infectious bronchitis virus / basal cell / primary culture / glycosaminoglycan Abstract Plasmacytoid dendritic cells (pDCs) are sensor cells with diverse immune functions, from type I interferon (IFN-I) production to antigen presentation, T cell activation, and tolerance. Regulation of these functions remains poorly understood but could be mediated by functionally specialized pDC subpopulations. We address pDC diversity using a high-dimensional single-cell approach: mass cytometry (CyTOF). Our analysis uncovers a murine pDC-like population that specializes in antigen presentation with limited capacity for IFN-I production. Using a multifaceted crossspecies comparison, we show that this pDC-like population is the definitive murine equivalent of the recently described human AXL + DCs, which we unify under the name transitional DCs (tDCs) given their continuum of pDC and cDC2 characteristics. tDCs share developmental traits with pDCs, as well as recruitment dynamics during viral infection. Altogether, we provide a framework for deciphering the function of pDCs and tDCs during diseases, which has the potential to open new avenues for therapeutic design.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). and O.A.P.; Resources, O.A.P. and B.R.; Writing, R.L., M.A.-H., and J.I.; Visualization, R.L., M.A.-H., Z.L., and J.I.; Funding Acquisition, J.I.; Supervision, J.I.; Project Administration, J.I. DECLARATION OF INTERESTS The authors declare no competing interests. SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.Dendritic cells (DCs) are unique therapeutic targets given their capacity to modulate immune responses. Yet complete alignment of the DC network between species is lacking. Using a multidimensional approach, Leylek et al. identify the mouse homolog of human AXL + DCs, named transitional DCs (tDCs), and reveal their similarities with pDCs.Leylek et al.As a control for homeostatic proliferation, T cells were cultured alone without DCs. Results were expressed as frequency of CTV low CD4 + T cells.Human: PBMCs stained with CFSE (Millipore Sigma) at 37°C in a water bath for 10 minutes before T cell purification using the Pan T Cell Isolation Kit (Miltenyi Biotec) according to manufacturer's instructions. FACS-purified DC subsets were co-cultured with allogeneic T cells in a 1:20 ratio for 6 days. As a control for homeostatic proliferation, T cells were cultured alone without DCs. Results are expressed as frequency of CFSE low CD4 + T cells. Abstract Bioinformatics and genome science (BGS) are gradually gaining roots in Africa, contributing to studies that are leading to improved understanding of health, disease, agriculture and food security. While a few African countries have established foundations for research and training in these areas, BGS appear to be limited to only a few institutions in specific African countries. However, improving the disciplines in Africa will require pragmatic efforts to expand training and research partnerships to scientists in yet-unreached institutions. Here, we discuss the need to expand BGS programmes in Africa, and propose mechanisms to do so. Abstract Background: The Ebola virus has been responsible for numerous outbreaks since the 1970s, with the most recent outbreak taking place between 2014 and 2016 and causing an international public health emergency. Ebola virus disease (EVD) has a high mortality rate and no approved targeted treatment exists to date. A number of established drugs are being considered as potential therapeutic agents for the treatment of EVD. Objective: We aimed to identify potential drug repositioning candidates and to assess the scientific evidence available on their efficacy. Methods: We conducted a systematic literature search in MEDLINE, Embase, and other relevant trial registry platforms for studies published between January 1976 and January 2017. We included drug screening, preclinical studies, and clinical studies on repurposed drugs for the treatment of EVD. The risk of bias for animal studies and nonrandomized clinical studies was assessed. The quality of reporting for case series and case reports was evaluated. Finally, we selected drugs approved by established regulatory authorities, which have positive in vitro study outcomes and at least one additional animal or clinical trial. Results: We identified 3301 publications, of which 37 studies fulfilled our inclusion criteria. Studies were highly heterogeneous in terms of study type, methodology, and intervention. The risk of bias was high for 13 out of 14 animal studies. We selected 11 drugs with potential anti-EVD therapeutic effects and summarized their evidence. Conclusions: Several established drugs may have therapeutic effects on EVD, but the quality and quantity of current scientific evidence is lacking. This review highlights the need for well-designed and conducted preclinical and clinical research to establish the efficacy of potential repurposed drugs against EVD. Abstract Tubuloreticular inclusions (TRIs) are subcellular structures located within the cisternae of endoplasmic reticulum. Formation of TRIs has been linked to the exposure of excess interferon (IFN), either from endogenous or exogenous sources. In renal disease, TRIs have been most commonly associated with systemic lupus erythematosus (SLE), and human immunodeficiency virus-associated nephropathy (HIVAN). Case reports of patients with renal biopsies showing TRIs without underlying SLE or HIV are infrequent in adults, and to our knowledge none have been reported in children. We report 3 pediatric cases in which the renal biopsy showed TRIs on electron microscopy without underlying SLE or HIV infection. The first patient presented at 2 years of age with nephrotic syndrome and renal failure. His renal biopsy revealed focal segmental glomerulosclerosis and TRIs. The second patient presented at 6 months of age with infantile nephrotic syndrome, and his renal biopsy revealed membranous Case Rep Nephrol Dial 2017;7:91-101 Abstract The distinct characteristic of the Middle East Respiratory Syndrome (MERS) outbreak in South Korea is that it not only involves intra-hospital transmission, but it also involves hospital-to-hospital transmission. It has been the largest MERS outbreak outside the Middle East, with 186 confirmed cases and, among them, 36 fatal cases as of July 26, 2015. All confirmed cases are suspected to be hospital-acquired infections except one case of household transmission and two cases still undergoing examination. The Korean health care system has been the major factor shaping the unique characteristics of the outbreak. Taking this as an opportunity, the Korean government should carefully assess the fundamental problems of the vulnerability to hospital infection and make short-as well as long-term plans for countermeasures. In addition, it is hoped that this journal, Epidemiology and Health, becomes a place where various topics regarding MERS can be discussed and shared. Abstract Activation of the type I interferon (IFN) pathway by small interfering RNA (siRNA) is a major contributor to the off-target effects of RNA interference in mammalian cells. While IFN induction complicates gene function studies, immunostimulation by siRNAs may be beneficial in certain therapeutic settings. Various forms of siRNA, meeting different compositional and structural requirements, have been reported to trigger IFN activation. The consensus is that intracellularly expressed shorthairpin RNAs (shRNAs) are less prone to IFN activation because they are not detected by the cell-surface receptors. In particular, lentiviral vector-mediated transduction of shRNAs has been reported to avoid IFN response. Here we identify a shRNA that potently activates the IFN pathway in human cells in a sequence-and 5 0 -triphosphatedependent manner. In addition to suppressing its intended mRNA target, expression of the shRNA results in dimerization of interferon regulatory factor-3, activation of IFN promoters and secretion of biologically active IFNs into the extracellular medium. Delivery by lentiviral vector transduction did not avoid IFN activation by this and another, unrelated shRNA. We also demonstrated that retinoic-acid-inducible gene I, and not melanoma differentiation associated gene 5 or toll-like receptor 3, is the cytoplasmic sensor for intracellularly expressed shRNAs that trigger IFN activation. Abstract Influenza virus (IV) is a continuously evolving virus that widely spreads in humans and contributes to substantial morbidity and mortality. Re-emergence of human infection with avian influenza virus H5N1 poses extra challenge to IV control. Artificial microRNA (amiRNA)-mediated RNA interference has become a powerful antiviral approach due to its high specificity and rapid effect. Here, we designed several amiRNAs targeting the hemagglutinin gene of H5N1, a major determinant of pathogenicity. Expression and delivery efficiency were enhanced by presenting functional amiRNA with chimpanzee adenovirus serotype 68 (AdC68). One amiRNA, HA-1405, significantly limited H5N1 replication in vitro and inhibited 96.7% of clade 2.3.2 replication. AdC68-conjugated HA-1405 treatment remarkably decreased different clades of H5N1 plaque formation in Madin-Darby canine kidney cells. Moreover, prophylactic administration with rAd(HA-1405) markedly alleviated clinical symptoms and reduced ~3to 40-folds of lung viral RNA copies against four clades of H5N1 in Institute of Cancer Research (ICR) mice. Our results further showed that rAd(HA-1405) conferred 70 and 40% immediate protection against lethal clade 2.3.2 and clade 2.3.4 H5N1 challenge, respectively. In conclusion, these data provided information that HA-targeting amiRNA delivered by AdC68 could be pursued as a potential agent for highly pathogenic avian influenza viruses prevention. Abstract Objective: In this study we execute a rational screen to identify Chinese medical herbs that are commonly used in treating viral respiratory infections and also contain compounds that might directly inhibit 2019 novel coronavirus (2019-nCoV), an ongoing novel coronavirus that causes pneumonia. Methods: There were two main steps in the screening process. In the first step we conducted a literature search for natural compounds that had been biologically confirmed as against sever acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus. Resulting compounds were cross-checked for listing in the Traditional Chinese Medicine Systems Pharmacology Database. Compounds meeting both requirements were subjected to absorption, distribution, metabolism and excretion (ADME) evaluation to verify that oral administration would be effective. Next, a docking analysis was used to test whether the compound had the potential for direct 2019-nCoV protein interaction. In the second step we searched Chinese herbal databases to identify plants containing the selected compounds. Plants containing 2 or more of the compounds identified in our screen were then checked against the catalogue for classic herbal usage. Finally, network pharmacology analysis was used to predict the general in vivo effects of each selected herb. Results: Of the natural compounds screened, 13 that exist in traditional Chinese medicines were also found to have potential anti-2019-nCoV activity. Further, 125 Chinese herbs were found to contain 2 or more of these 13 compounds. Of these 125 herbs, 26 are classically catalogued as treating viral respiratory infections. Network pharmacology analysis predicted that the general in vivo roles of these 26 herbal plants were related to regulating viral infection, immune/inflammation reactions and hypoxia response. Conclusion: Chinese herbal treatments classically used for treating viral respiratory infection might contain direct anti-2019-nCoV compounds. Abstract The yeast Kre2p/Mntlp cxl,2-mannosyltransferase is a type II membrane protein with a short cytoplasmic amino terminus, a membrane-spanning region, and a large catalytic luminal domain containing one N-glycosylation site. Anti-Kre2p/Mntlp antibodies identify a 60-kD integral membrane protein that is progressively N-glycosylated in an MNNl-dependent manner. Kre2p/Mntlp is localized in a Golgi compartment that overlaps with that containing the medial-Golgi mannosyltransferase Mnnlp, and distinct from that including the late Golgi protein Kexlp. To determine which regions of Kre2p/Mntlp are required for Golgi localization, Kre2p/Mntlp mutant proteins were assem-bled by substitution of Kre2p domains with equivalent sequences from the vacuolar proteins DPAP B and Pho8p. Chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. The resuits demonstrate that the NH2-terminal cytoplasmic domain is necessary for correct Kre2p Golgi localization whereas, the membrane-spanning and stem domains are dispensable. However, in a test of targeting sufficiency, the presence of the entire Kre2p cytoplasmic tail, plus the transmembrane domain and a 36-amino acid residue luminal stem region was required to localize a Pho8p reporter protein to the yeast Golgi. T HE Golgi apparatus plays a fundamental role in glycan processing and sorting of newly synthesized proteins in the secretory pathway of eukaryotic cells. The Golgi apparatus of a typical mammalian cell is composed of a polarized stack of membranous saccules that are differentiated into functionally distinct subcompartments. After the addition of core N-linked sugar moieties in the endoplasmic reticulum, glycoproteins proceed through the cis-Golgi network, the cis-, medial-, and transcisternae, and the trans-Golgi network, where further modifications take place. These include the addition of O-linked sugars and the elaboration, in a protein-specific manner, of complex N-linked carbohydrate structures. The Golgi apparatus also constitutes a major organelle responsible for protein trafficking where particular proteins are directed to precise cellular locations. Two specific Golgi subcompartments have been found to be involved in protein sorting. The cis-Golgi network constitutes the site from which many resident ER proteins are retrieved, and the main function of the TGN is to direct glycoproteins exiting the Golgi complex to either the lysosome or the cell surface ( Abstract Acute respiratory distress syndrome (ARDS) is a common acute and severe disease in clinic. Recent studies indicated that Cxc chemokine ligand 5 (CXCL5), an inflammatory chemokine, was associated with tumorigenesis. The present study investigated the role of the CXCL5/Cxc chemokine receptor 2 (CXCR2) bio-axis in ARDS, and explored the underlying molecular mechanism.The pathological morphology of lung tissue and degree of pulmonary edema were assessed by hematoxylin-eosin staining and pulmonary edema score, respectively. Real-time PCR and Western blot analysis were performed to detect the expression levels of CXCL5, CXCR2, Matrix metalloproteinases 2 (MMP2), and Matrix metalloproteinases 9 (MMP9) in lung tissues. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the expression levels of CXCL5 and inflammatory factors (IL-1b, IL-6, TNF-a, and IL-10) in serum.The results demonstrated that diffuse alveolar damage and pulmonary edema appeared in lipopolysaccharide (LPS)-induced ARDS and were positively correlated with the severity of ARDS. In addition, CXCL5 and its receptor CXCR2 were overexpressed by upregulation of MMP2 and MMP9 in lung tissues of ARDS. In addition, CXCL5 neutralizing antibody effectively alleviated inflammatory response, diffuse alveolar damage, and pulmonary edema, and decreased the expression levels of MMP2 and MMP9 compared to LPS-induced ARDS.We found that CXCL5/CXCR2 accelerated the progression of ARDS, partly by upregulation of MMP2 and MMP9 in lung tissues with the release of inflammatory factors.Abbreviations: ARDS -acute respiratory distress syndrome; CXCL5 -Cxc chemokine ligand 5; CXCR2 -Cxc chemokine receptor 2; MMP2 -matrix metalloproteinases 2; MMP9 -matrix metalloproteinases 9; LPS -lipopolysaccharide; UCC -uterine cervix cancer; H&E staining -hematoxylin-eosin staining; W/D -wet-to-dry; ELISA assay -enzyme-linked immunosorbent assay; SDS-PAGE -sulfate polyacrylamide gel electrophoresis; PVDF -polyvinylidene fluoride; TBST -tris-buffered saline plus 0.1% Tween 20Full-text PDF: https://www.medscimonit.com/abstract/index/idArt/915835 Abstract The -1 programmed ribosomal frameshifts (PRF), which are used by many viruses, occur at a heptanucleotide slippery sequence and are currently thought to involve the tRNAs interacting with the ribosomal P-and A-site codons. We investigated here whether the tRNA occupying the ribosomal E site that precedes a slippery site influences -1 PRF. Using the human immunodeficiency virus type 1 (HIV-1) frameshift region, we found that mutating the E-site codon altered the -1 PRF efficiency. When the HIV-1 slippery sequence was replaced with other viral slippery sequences, mutating the E-site codon also altered the -1 PRF efficiency. Because HIV-1 -1 PRF can be recapitulated in bacteria, we used a bacterial ribosome system to select, by random mutagenesis, 16S ribosomal RNA (rRNA) mutations that modify the expression of a reporter requiring HIV-1 -1 PRF. Three mutants were isolated, which are located in helices 21 and 22 of 16S rRNA, a region involved in translocation and E-site tRNA binding. We propose a novel model where -1 PRF is triggered by an incomplete translocation and depends not only on the tRNAs interacting with the P-and A-site codons, but also on the tRNA occupying the E site.Present address: Dominic Dulude, Centre de recherche, Hoˆpital Ste-Justine, 3175, chemin de la Coˆte-Ste-Catherine, Abstract The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) project is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature and diagnostic criteria for nonproliferative and proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and diagnostic criteria for classifying lesions in the digestive system including the salivary glands and the exocrine pancreas of laboratory rats and mice. Most lesions are illustrated by color photomicrographs. The standardized nomenclature, the diagnostic criteria, and the photomicrographs are also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and age related lesions as well as lesions induced by exposure to test items. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature and diagnostic criteria for the digestive system will decrease misunderstandings among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. Abstract Graphical Abstract Highlights d We identify the murine homolog of human AXL + DCs, now called transitional DCs (tDCs) d tDC development depends on Tcf4, similar to pDCs d tDCs are inefficient at IFN-I production but can efficiently activate T cells d During influenza infection, tDCs and pDCs accumulate in the lung Abstract Rapamycin and its derivatives are specific inhibitors of mammalian target of rapamycin (mTOR) kinase and, as a result, are wellestablished immunosuppressants and antitumorigenic agents. Additionally, this class of drug promotes gene delivery by facilitating lentiviral vector entry into cells, revealing its potential to improve gene therapy efforts. However, the precise mechanism was unknown. Here, we report that mTOR inhibitor treatment results in down-regulation of the IFN-induced transmembrane (IFITM) proteins. IFITM proteins, especially IFITM3, are potent inhibitors of virus-cell fusion and are broadly active against a range of pathogenic viruses. We found that the effect of rapamycin treatment on lentiviral transduction is diminished upon IFITM silencing or knockout in primary and transformed cells, and the extent of transduction enhancement depends on basal expression of IFITM proteins, with a major contribution from IFITM3. The effect of rapamycin treatment on IFITM3 manifests at the level of protein, but not mRNA, and is selective, as many other endosome-associated transmembrane proteins are unaffected. Rapamycin-mediated degradation of IFITM3 requires endosomal trafficking, ubiquitination, endosomal sorting complex required for transport (ESCRT) machinery, and lysosomal acidification. Since IFITM proteins exhibit broad antiviral activity, we show that mTOR inhibition also promotes infection by another IFITM-sensitive virus, Influenza A virus, but not infection by Sendai virus, which is IFITM-resistant. Our results identify the molecular basis by which mTOR inhibitors enhance virus entry into cells and reveal a previously unrecognized immunosuppressive feature of these clinically important drugs. In addition, this study uncovers a functional convergence between the mTOR pathway and IFITM proteins at endolysosomal membranes. fusion | endosome | IFITM | interferon | virus Abstract Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis (48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure sms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB. Abstract Statins lower the hyperlipidemia and reduce the incidence of cardiovascular events and related mortality. A 60-year-old man who was diagnosed with a transient ischemic attack was started on acetyl-L-carnitine, cilostazol, and rosuvastatin. After rosuvastatin treatment for 4 weeks, the patient presented with sudden onset fever, cough, and dyspnea. His symptoms were aggravated despite empirical antibiotic treatment. All infectious pathogens were excluded based on results of culture and polymerase chain reaction of the bronchoscopic wash specimens. Chest radiography showed diffuse ground-glass opacities in both lungs, along with several subpleural ground-glass opacity nodules; and a foamy alveolar macrophage appearance was confirmed on bronchoalveolar lavage. We suspected rosuvastatin-induced lung injury, discontinued rosuvastatin and initiated prednisolone 1 mg/kg tapered over 2weeks. After initiating steroid therapy, his symptoms and radiologic findings significantly improved. We suggest that clinicians should be aware of the potential for rosuvastatin-induced lung injury. Abstract The success of lentiviral vectors in curing fatal genetic and acquired diseases has opened a new era in human gene therapy. However, variability in the efficacy and safety of this therapeutic approach has been reported in human patients. Consequently, lentiviral-vector-based gene therapy is limited to incurable human diseases, with little understanding of the underlying causes of adverse effects and poor efficacy. To assess the role that host genetic variation has on efficacy of gene therapy, we characterized lentiviral-vector gene therapy within a set of 12 collaborative cross mouse strains. Lentiviral vectors carrying the firefly luciferase cDNA under the control of a liver-specific promoter were administered to female mice, with total-body and hepatic luciferase expression periodically monitored through 41 weeks post-vector administration. Vector copy number per diploid genome in mouse liver and spleen was determined at the end of this study. We identified major strain-specific contributions to overall success of transduction, vector biodistribution, maximum luciferase expression, and the kinetics of luciferase expression throughout the study. Our results highlight the importance of genetic variation on gene-therapeutic efficacy; provide new models with which to more rigorously assess gene therapy approaches; and suggest that redesigning preclinical studies of gene-therapy methodologies might be appropriate. Abstract W e are gratified that Ron Fouchier has joined (1) the important effort to quantify the risks (2-5) of the creation of potential pandemic pathogens, including ferret-transmissible variants of influenza A/H5N1. However, we disagree with many aspects of his assessment.As in our article and Fouchier's letter, here we proceed through the calculation, starting with probability of laboratory-acquired infections and the conditional probability of sparking a pandemic given such an infection and concluding with the consequences thereof. We then discuss some more general considerations.Fouchier bases his calculations on one of the sources we also used, the tabulation by Henkel et al. of reports of accidents involving select agents in the United States between 2004 and 2010 (6). However, he argues that the risk in his laboratory is considerably lower than the lower bound obtained from these reports of 0.2% per laboratory-year in a biosafety level 3 (BSL3) laboratory. He states, "These estimates, however, do not take into account specific pathogen types or research settings. This is crucial, because working practices in, e.g., virology and microbiology laboratories are different and because each biosafety laboratory is unique" (1). He proposes an alternative calculation based on 0 viral laboratoryacquired infections (LAI) in BSL3 labs over 2,044 lab-years in BSL2, -3, and -4 labs with select agents (6) and suggests that the proper value is Ͻ1/2,044 lab-years, or Ͻ5 ϫ 10 Ϫ4 /lab-year.These numbers are both conceptually and statistically invalid. While bacteriology and virology labs certainly perform some different activities, neither the references cited by Fouchier nor any other evidence of which we are aware justifies the relevant claim: that BSL3 bacteriology labs are more accident-prone than BSL3 virology labs over a given time span. Absent any such evidence, the proper comparison would be BSL3 LAI/BSL3 lab-years. Unfortunately, BSL3 lab-years are not publicly available. Therefore, in our original calculation, we used 2,044 lab-years in BSL2, -3, and -4 labs as a denominator to calculate a lower bound on the risk, with LAI in BSL3 as the numerator. Fouchier's suggestion to use the same (too-large) denominator to form an upper bound is inappropriate and is made more so by excluding bacterial LAI from the numerator but keeping bacterial lab-years in the denominator.If one does choose to use 0 viral LAIs as the numerator, one would need to specify the number of viral BSL3 lab-years for a proper denominator. In this case, given the uncertainty surrounding a rare event, the proper way to account for 0 observed events is not to say that the true rate is less than one divided by the number of lab-years, but that the true rate has a 95% confidence interval Citation Lipsitch M, Inglesby TV. 2015. Reply to "Studies on influenza virus transmission between ferrets: the public health risks revisited." mBio 6(1):e00041-15. Abstract Treatment of emerging RNA viruses is hampered by the high mutation and replication rates that enable these viruses to operate as a quasispecies. Declining honey bee populations have been attributed to the ectoparasitic mite Varroa destructor and its affiliation with Deformed Wing Virus (DWV). In the current study we use next-generation sequencing to investigate the DWV quasispecies in an apiary known to suffer from overwintering colony losses. We show that the DWV species complex is made up of three master variants. Our results indicate that a new DWV Type C variant is distinct from the previously described types A and B, but together they form a distinct clade compared with other members of the Iflaviridae. The molecular clock estimation predicts that Type C diverged from the other variants ∼ 319 years ago. The discovery of a new master variant of DWV has important implications for the positive identification of the true pathogen within global honey bee populations. Abstract Methods: Based on the effects of 4 shRNAs targeting different regions of HTNV genomic RNA on viral replication, the most effective RNA interference fragments of the S and M genes were constructed in pSilencer-3.0-H1 vectors, and designated pSilencer-S and pSilencer-M, respectively. The antiviral effect of pSilencer-S/M against HTNV was evaluated in both HTNV-infected Vero-E6 cells and mice. Results: In HTNV-infected Vero-E6 cells, pSilencer-S and pSilencer-M targeted the viral nucleocapsid proteins and envelope glycoproteins, respectively, as revealed in the immunofluorescence assay. Transfection with pSilencer-S or pSilencer-M (1, 2, and 4 μg) markedly inhibited the viral antigen expression in dose-and time-dependent manners. Transfection with either plasmid (2 μg) significantly decreased HTNV-RNA level at 3 day postinfectin (dpi) and the progeny virus titer at 5 dpi. In mice infected with lethal doses of HTNV, intraperitoneal injection of pSilencer-S or pSilencer-M (30 μg) considerably increased the survival rates and mean time to death, and significantly reduced the mean virus yields and viral RNA level, and alleviated virus-induced pathological lesions in lungs, brains and kidneys. Conclusion: Plasmid-based shRNAs potently inhibit HTNV replication in vitro and in vivo. Our results provide a basis for development of shRNA as therapeutics for HTNV infections in humans. Abstract This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.A new strain of the H1N1 subtype of influenza A virus resulted in a pandemic outbreak. In South Korea, cases of pandemic influenza have increased. Therefore, we explored perception or preventive behaviors for this virus in hospital employees and outpatients. Materials and Methods: Data was collected from hospital employees and outpatients at three university hospitals located in Daegu, Gyeongju in South Korea between the 21st and 30th of September, 2009 using a self-administrated questionnaire. We estimated perception by components of The Health Belief Model (HBM), preventive behaviors consisted of avoidance behaviors, and the recommended behaviors by the Korea Center of Disease Control and Prevention (KCDC). Desire for vaccination was identified. Results: The 1,837 participants comprised hospital employees (n = 880, 47.9%) and outpatients (n = 957, 52.1%). Of all hospital employees, 491 (55.8%) and 708 (80.5%) perceived susceptibility of the pandemic influenza and benefits of the preventive behaviors, respectively. Among all outpatients, 490 (51.2%) and 651 (68.0%) perceived susceptibility of the pandemic influenza and benefits of the preventive behaviors, respectively. Recommended preventative behaviors were adopted by 674 (76.6%) of hospital employees and 631 (65.9%) of outpatients. Vaccination was desired by 479 (54.4%) of hospital employees and 484 (50.6%) of outpatients. Factors influencing preventative behaviors included gender, economic status (for hospital employees) and educational level (for outpatients). All HBM components except perception of barriers were associated with the preventive behaviors in both groups. Conclusion: The majority of the surveyed hospital employees and outpatients perceived the benefits of preventive behaviors for pandemic influenza and performed them. Abstract Laws are fundamental tools that regulate and manage various issues to protect the rights of the people in a society. Legislation on disease surveillance enables agencies to regulate and manage public health, including preventing the spread of infectious diseases. We assessed the Infectious Disease Prevention and Control Act of Korea (IDPCA) through the lens of biosurveillance to understand its effectiveness in protecting public health. In addition, the relevant legislation and regulations of the United States and the World Health Organization were examined. The evaluation concludes that the current IDPCA is limited in terms of providing guidance for early detection of and response to hazards using integrated data and an information-sharing system. Further revision of the laws is needed to enable early detection and warning of potential threats to public health. Abstract Conidiobolomycosis is a rare fungal disease of both humans and animals, occurring mainly in tropical and subtropical climates. We describe a disseminated fungal infection in a young, apparently immunocompetent dog who initially presented for antibiotic resistant pneumonia. Histopathology and mycology identified a Conidiobolus sp., further confirmed as Conidiobolus incongruus through DNA sequencing of D1/D2 regions. This is the first report of this species causing disease in dogs and the fifth reported infection in animals. Abstract Background: Ticks are primary vectors for many well-known disease-causing agents that affect human and animal populations globally such as tick-borne encephalitis, Crimean-Congo hemorrhagic fever and African swine fever. In this study, viral metagenomics was used to identify what viruses are present in Rhipicephalus spp. ticks collected in the Zambezi Valley of Mozambique. Methods: The RNA was amplified with sequence-independent single primer amplification (SISPA) and high-throughput sequencing was performed on the Ion Torrent platform. The generated sequences were subjected to quality check and classfied by BLAST. CodonCode aligner and SeqMan were used to assemble the sequences. Results: The majority of viral sequences showed closest sequence identity to the Orthomyxoviridae family, although viruses similar to the Parvoviridae and Coronaviridae were also identified. Nearly complete sequences of five orthomyxoviral segments (HA, NP, PB1, PB2, and PA) were obtained and these showed an amino acid identity of 32-52% to known quaranjaviruses. The sequences were most closely related to the Wellfleet Bay virus, detected and isolated from common eider during a mortality event in the USA. Conclusions: In summary, this study has identified a highly divergent virus with in the Orthomyxoviridae family associated with Rhipicephalus ticks from Mozambique. Further genetic and biological studies are needed in order to investigate potential pathogenesis of the identified orthomyxovirus.ARTICLE HISTORY Abstract Modern livestock production became highly intensive and large scaled to increase production efficiency. This production environment could add stressors affecting the health and growth of animals. Major stressors can include environment (air quality and temperature), nutrition, and infection. These stressors can reduce growth performance and alter immune systems at systemic and local levels including the gastrointestinal tract. Heat stress increases the permeability, oxidative stress, and inflammatory responses in the gut. Nutritional stress from fasting, antinutritional compounds, and toxins induces the leakage and destruction of the tight junction proteins in the gut. Fasting is shown to suppress pro-inflammatory cytokines, whereas deoxynivalenol increases the recruitment of intestinal pro-inflammatory cytokines and the level of lymphocytes in the gut. Pathogenic and viral infections such as Enterotoxigenic E. coli (ETEC) and porcine epidemic diarrhea virus can lead to loosening the intestinal epithelial barrier. On the other hand, supplementation of Lactobacillus or Saccharaomyces reduced infectious stress by ETEC. It was noted that major stressors altered the permeability of intestinal barriers and profiles of genes and proteins of pro-inflammatory cytokines and chemokines in mucosal system in pigs. However, it is not sufficient to fully explain the mechanism of the gut immune system in pigs under stress conditions. Correlation and interaction of gut and systemic immune system under major stressors should be better defined to overcome aforementioned obstacles. (This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract A 7-year-old neutered female domestic shorthaired cat born in Poland and then moved to Japan presented to the local clinic with recent onset of convulsive cluster seizures and status epilepticus. Magnetic resonance imaging revealed bilateral swelling of the hippocampus with T2 hyperintensity and contrast enhancing image, suggesting hippocampal necrosis. The cat completely recovered after treatment with antiepileptic drugs (AED) and administration of prednisolone (1 mg/kg PO q24h for 4 days and tapered). However, cluster seizures reoccurred and developed into status epilepticus despite increasing doses of AED. Although the convulsions were resolved by other AEDs, stupor and renal failure developed, and the cat was euthanized. Pathological findings were consistent with hippocampal necrosis. Immunological analysis for leucine-rich glioma inactivated 1 (LGI1) autoantibodies was negative, but antibodies against DCC (deleted in colorectal carcinoma) known as netrin-1 receptor were found. This report describes a case of feline autoimmune limbic encephalitis and hippocampal necrosis that were presumably associated with DCC autoantibodies. Abstract Background: Development an effective vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) is urgent and limited information is available on vaccination in nonhuman primate (NHP) model. We herein report of evaluating a recombinant receptor-binding domain (rRBD) protein vaccine in a rhesus macaque model. Methods: Nine monkeys were randomly assigned to high-dose, low-dose and mock groups,which were immunized with different doses of rRBD plus alum adjuvant or adjuvant alone at different time points (0, 8, 25 weeks). Immunological analysis was conducted after each immunisation. Monkeys were challenged with MERS-CoV at 14 days after the final immunisation followed by observation for clinical signs and chest X-rays. Nasal, oropharyngeal and rectal swabs were also collected for analyses. Monkeys were euthanized 3 days after challenge and multiple specimens from tissues were collected for pathological, virological and immunological tests. Conclusion: Robust and sustained immunological responses (including neutralisation antibody) were elicited by the rRBD vaccination. Besides, rRBD vaccination alleviated pneumonia with evidence of reduced tissue impairment and clinical manifestation in monkeys. Furthermore, the rRBD vaccine decreased viral load of lung, trachea and oropharyngeal swabs of monkeys. These data in NHP paves a way for further development of an effective human vaccine against MERS-CoV infection. Abstract Canine kobuviruses (CaKVs) are newly recognized picornaviruses that have been recently detected in dogs in the U.S.A., Italy, U.K., the Republic of Korea and Tanzania. To trace the evolution of CaKV strains, a total of 201 fecal samples from rectal swabs of diarrheic dogs, which were obtained from May 2014 to April 2015 in northeast China, were detected by reverse transcription-PCR targeting a partial (504 bp) fragment of the 3D gene. Furthermore, a phylogenetic analysis of the CaKV strains identified in northeast China was conducted based on the partial 3D gene sequence. The results indicated that 36 fecal samples (17.91%, 36/201) were positive for CaKV, in which the co-infection rates of canine coronavirus, canine parvovirus-2 and canine bocavirus were 58.33%, 41.67%, and 11.11%, respectively. Sequence comparison of the partial 3D gene revealed nucleotide homologies of 94.4-100%, 95.6-98.6%, 94.3-97.6%, 94.4-96.3% and 93.3-95.1% within the 36 Chinese CaKV strains, and between the 36 Chinese CaKV strains and four CaKV reference strains from South Korea, Italy, U.S.A. and Tanzania, respectively. A phylogenetic tree revealed that the 36 Chinese CaKV strains formed one specific CaKV lineage with CaKVs that have recently been identified in other countries. The 36 Chinese CaKV strains were closely related to CaKV reference strains from Asia and Europe, but differed genetically from CaKV reference strains from North America and Africa. This study provides evidence that CaKVs circulate in diarrhoetic dogs in China and that they exhibit substantial genetic diversity and high co-infection rates with other enteric viruses. Abstract Background: Micro-organisms transmitted from vertebrate animalsincluding livestockto humans account for an estimated 60% of human pathogens. Micro-organisms can be transmitted through inhalation, ingestion, via conjunctiva or physical contact. Close contact with animals is crucial for transmission. The role of intensity and type of contact patterns between livestock and humans for disease transmission is poorly understood. In this systematic review we aimed to summarise current knowledge regarding patterns of human-livestock contacts and their role in micro-organism transmission. Methods: We included peer-reviewed publications published between 1996 and 2014 in our systematic review if they reported on human-livestock contacts, human cases of livestock-related zoonotic diseases or serological epidemiology of zoonotic diseases in human samples. We extracted any information pertaining the type and intensity of human-livestock contacts and associated zoonoses. Results: 1522 papers were identified, 75 were included: 7 reported on incidental zoonoses after brief animalhuman contacts (e.g. farm visits), 10 on environmental exposures and 15 on zoonoses in developing countries where backyard livestock keeping is still customary. 43 studies reported zoonotic risks in different occupations. Occupations at risk included veterinarians, culling personnel, slaughterhouse workers and farmers. For culling personnel, more hours exposed to livestock resulted in more frequent occurrence of transmission. Slaughterhouse workers in contact with live animals were more often positive for zoonotic micro-organisms compared to co-workers only exposed to carcasses. Overall, little information was available about the actual mode of micro-organism transmission. Conclusions: Little is known about the intensity and type of contact patterns between livestock and humans that result in micro-organism transmission. Studies performed in occupational settings provide some, but limited evidence of exposure response-like relationships for livestock-human contact and micro-organism transmission. Better understanding of contact patterns driving micro-organism transmission from animals to humans is needed to provide options for prevention and thus deserves more attention. Abstract This review highlights recent developments in HIV-1 antibody engineering and discusses the effects of increased polyreactivity on serum half-lives of engineered antibodies.Recent studies have uncovered a wealth of information about the relationship between the sequences and efficacies of anti-HIV-1 antibodies through a combination of bioinformatics, structural characterization and in vivo studies. This knowledge has stimulated efforts to enhance antibody breadth and potency for therapeutic use. Although some engineered antibodies have shown increased polyreactivity and short halflives, promising efforts are circumventing these problems.Antibodies are desirable as therapeutics due to their ability to recognize targets with both specificity and high affinity. Furthermore, the ability of antibodies to stimulate Fc-mediated effector functions can increase their utility. Thus, mAbs have become central to strategies for the treatment of various diseases. Using both targeted and library-based approaches, antibodies can be engineered to improve their therapeutic properties. This article will discuss recent antibody engineering efforts to improve the breadth and potency of anti-HIV-1 antibodies. The polyreactivity of engineered HIV-1 bNAbs and the effect on serum half-life will be explored along with strategies to overcome problems introduced by engineering antibodies. Finally, advances in creating bispecific anti-HIV-1 reagents are discussed. Abstract Korean Red Ginseng (KRG) is a heat-processed ginseng developed by the repeated steaming and airdrying of fresh ginseng. Compared with fresh ginseng, KRG has been shown to possess greater pharmacological activities and stability because of changes that occur in its chemical constituents during the steaming process. In addition to anticancer, anti-inflammatory, and immune-modulatory activities, KRG and its purified components have also been shown to possess protective effects against microbial infections. Here, we summarize the current knowledge on the properties of KRG and its components on infections with human pathogenic viruses such as respiratory syncytial virus, rhinovirus, influenza virus, human immunodeficiency virus, human herpes virus, hepatitis virus, norovirus, rotavirus, enterovirus, and coxsackievirus. Additionally, the therapeutic potential of KRG as an antiviral and vaccine adjuvant is discussed. Abstract Background and Objectives: Noise levels and room acoustic parameters at a tertiary referral hospital, Seoul National University Hospital (SNUH) in Korea, are investigated. Materials and Methods: Through a questionnaire, acoustically problematic rooms are identified. Noise levels in emergency rooms (ERs) and intensive care units (ICUs) are measured over about three days. Acoustically critical and problematic rooms in the otolaryngology department are measured including examination rooms, operating rooms, nurse stations, receptions, and patient rooms. Results: The A-weighted equivalent noise level, LAeq, ranges from 54 to 56 dBA, which is at least 10 dB lower than the noise levels of 65 to 73 dBA measured in American ERs. In an ICU, the noise level for the first night was 66 dBA, which came down to 56 dBA for the next day. The noise levels during three different ear surgeries vary from 57 to 62 dBA, depending on the use of surgical drills and suctions. The noise levels in a patient room is found to be 47 dBA, while the nurse stations and the receptions have high noise levels up to 64 dBA. The reverberation times in an operation room, examination room, and single patient room are found to be below 0.6 s. Conclusions: At SNUH, the nurse stations and receptions were found to be quite noisy. The ERs were quieter than in the previous studies. The measured reverberation times seemed low enough but some other nurse stations and examination rooms were not satisfactory according to the questionnaire. J Audiol Otol 2019;23(2):76-82 KEY WORDS:0 Hospital noise · ER noise · ICU noise · Ear surgery noise · Reverberation time.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.www.ejao.org 77Cho WH, et al. Abstract increasing research has demonstrated that expression of brain and muscle arnT-like 1 (BMal1) and other circadian clock genes can be regulated by drugs and toxicants. We previously demonstrated that icariin, extracted from Herba epimedii, sromotes osteogenic differentiation. However, the mechanism underlying the association between icariin and BMal1 in osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMScs) remains unclear. The present study was designed with an aim to clarify the association between icariin and BMal1 in osteogenic differentiation of BMScs. The cell counting Kit-8 assay was used to evaluate cell proliferation. The expression of bone morphogenetic protein 2 (BMP2), runX family transcription factor 2 (runX2), alkaline phosphatase (alP), osteocalcin (oc) and BMal1 in BMScs was evaluated by reverse transcription-quantitative Pcr and western blotting. alP and alizarin red S (arS) staining were also performed. icariin promoted BMSc proliferation, and upregulated expression of osteogenic genes and BMal1. in addition, expression of the osteogenic genes BMP2, runX2, alP and oc were upregulated by BMal1 overexpression. Furthermore, we confirmed that BMal1 deficiency suppressed osteogenic differentiation in BMScs. Finally, arS staining of BMal1 -/-BMScs revealed that BMal1 was an essential intermediary in matrix mineralization during osteogenic differentiation. in conclusion, these results demonstrated that icariin promoted osteogenic differentiation through BMal1-BMP2 signaling in BMScs. The present study thus described a novel target of icariin that has potential applications in the treatment of osteogenic disorders. Abstract Prion diseases, also called transmissible spongiform encephalopathies (TSEs), lead to neurological dysfunction in animals and are fatal. Infectious prion proteins are causative agents of many mammalian TSEs, including scrapie (in sheep), chronic wasting disease (in deer and elk), bovine spongiform encephalopathy (BSE; in cattle), and CreutzfeldteJakob disease (CJD; in humans). BSE, better known as mad cow disease, is among the many recently discovered zoonotic diseases. BSE cases were first reported in the United Kingdom in 1986. Variant CJD (vCJD) is a disease that was first detected in 1996, which affects humans and is linked to the BSE epidemic in cattle. vCJD is presumed to be caused by consumption of contaminated meat and other food products derived from affected cattle. The BSE epidemic peaked in 1992 and decreased thereafter; this decline is continuing sharply owing to intensive surveillance and screening programs in the Western world. However, there are still new outbreaks and/or progression of prion diseases, including atypical BSE, and iatrogenic CJD and vCJD via organ transplantation and blood transfusion. This paper summarizes studies on prions, particularly on prion molecular mechanisms, BSE, vCJD, and diagnostic procedures. Risk perception and communication policies of the European Union for the prevention of prion diseases are also addressed to provide recommendations for appropriate government policies in Korea. Abstract Gram-negative pathogen-induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF), a traditional Chinese medicine (TCM) formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosainduced acute pneumonia in mice were evaluated. The mechanisms by which four typical antiinflammatory ingredients from QF, arctigenin (ATG), cholic acid (CLA), chlorogenic acid (CGA) and sinapic acid (SPA), regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6) and chemokines (IL-8 and RANTES), reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In Abstract Rationale: Pneumocystis jirovecii causes severe pneumonia in immunocompromised hosts. Human immunodeficiency virus infection, malignancy, solid organ or hematopoietic cell transplantation, and primary immune deficiency compose the risk factors for Pneumocystis pneumonia (PCP) in children, and PCP can be an initial clinical manifestation of primary immune deficiency.Patient concerns: A 5-month-old infant presented with cyanosis and tachypnea. He had no previous medical or birth history suggesting primary immune deficiency. He was diagnosed with interstitial pneumonia on admission.Diagnoses: He was diagnosed with PCP, and further evaluations revealed underlying X-linked hyper-IgM syndrome.Interventions: He was treated with trimethoprim/sulfamethoxazole for PCP, and eventually received allogeneic hematopoietic cell transplantation for hyper-IgM syndrome.Outcomes: Twenty months have passed after transplantation without severe complications.Lessons: PCP should be considered in infants presenting with severe interstitial pneumonia even in the absence of evidence of immune deficiency. Primary immune deficiency should also be suspected in infants diagnosed with PCP.Abbreviations: HD = hospital day, HIGM = hyper-IgM, HIV = human immunodeficiency virus, PCP = Pneumocystis pneumonia, PCR = polymerase chain reaction, PID = primary immune deficiency, TMP/SMX = trimethoprim/sulfamethoxazole. Abstract Using a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4 + CD25 + FOXP3 + regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-b1 and TGF-b2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments. Abstract BMJ Open publishes all reviews undertaken for accepted manuscripts. Reviewers are asked to complete a checklist review form (http://bmjopen.bmj.com/site/about/resources/checklist.pdf) and are provided with free text boxes to elaborate on their assessment. These free text comments are reproduced below.ARTICLE DETAILSProtocol for a randomised, single-blind, two-arm, parallel-group controlled trial of the efficacy of rhinothermy delivered by nasal high flow therapy in the treatment of the common cold Abstract Itraconazole (ITZ) is a well-known antifungal agent that also has anti-cancer activity. In this study, we identified ITZ as a broad-spectrum inhibitor of enteroviruses (e.g. poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e. shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as novel molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs. Abstract SIRT7 is an NAD + -dependent protein deacetylase that regulates cell growth and proliferation. Previous studies have shown that SIRT7 is required for RNA polymerase I (Pol I) transcription and pre-rRNA processing. Here, we took a proteomic approach to identify novel molecular targets and characterize the role of SIRT7 in non-nucleolar processes. We show that SIRT7 interacts with numerous proteins involved in transcriptional regulation and RNA metabolism, the majority of interactions requiring ongoing transcription. In addition to its role in Pol I transcription, we found that SIRT7 also regulates transcription of snoRNAs and mRNAs. Mechanistically, SIRT7 promotes the release of P-TEFb from the inactive 7SK snRNP complex and deacetylates CDK9, a subunit of the elongation factor P-TEFb, which activates transcription by phosphorylating serine 2 within the Cterminal domain (CTD) of Pol II. SIRT7 counteracts GCN5-directed acetylation of lysine 48 within the catalytic domain of CDK9, deacetylation promoting CTD phosphorylation and transcription elongation. Abstract A long-term animal experiment involving inoculation with bovine coronavirus (BCoV) was conducted to verify its persistent infection in cattle. Three colostrum-deprived Holstein calves were housed separately in individual rooms of a high-containment facility and inoculated with the BCoV strain Kumamoto/1/07. Until the end of the experiment (1,085, 700 and 280 days, respectively), viral RNAs were detected sporadically by RT-PCR and nested PCR from plasma, nasal discharge, and feces. Seroconversion and titer changes were validated by hemagglutination inhibition tests and neutralization tests. Among the samples, nasal discharge showed a higher viral positivity than feces, which seemed to be associated with positive detection in the plasma. These data demonstrate the existence of persistent infection of BCoV in the respiratory tissues of cattle. Abstract Infrared thermal imaging (IRT) is a non-invasive, non-contact technique which allows one to measure and visualize infrared radiation. In medicine, thermal imaging has been used for more than 50 years in various clinical settings, including Raynaud's phenomenon and systemic sclerosis. Imaging and quantification of surface body temperature provides an indirect measure of the microcirculation's overall performance. As such, IRT is capable of confirming the diagnosis of Raynaud's phenomenon, and, with additional cold or heat challenge, of differentiating between the primary and secondary condition. In systemic sclerosis IRT has a potential role in assessing disease activity and monitoring treatment response. Despite certain limitations, thermal imaging can find a place in clinical practice, and with the introduction of small, low-cost infrared cameras, possibly become a part of routine rheumatological evaluation. Abstract The RNA Virus Database is a database and web application describing the genome organization and providing analytical tools for the 938 known species of RNA virus. It can identify submitted nucleotide sequences, can place them into multiple whole-genome alignments (in species where more than one isolate has been fully sequenced) and contains translated genome sequences for all species. It has been created for two main purposes: to facilitate the comparative analysis of RNA viruses and to become a hub for other, more specialised virus Web sites. It is available at the following four mirrored sites. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging infectious disease of growing global importance, has caused severe acute respiratory disease in more than 1600 people, resulting in almost 600 deaths. The high case fatality rate, growing geographic distribution and vaguely defined epidemiology of this novel pathogen have created an urgent need for effective public health countermeasures, including safe and effective treatment strategies. Despite the relatively few numbers of cases to date, research and development of MERS-CoV therapeutic candidates is advancing quickly. This review surveys the landscape of these efforts and assesses their potential for use in affected populations. Abstract A demyelinating disease induced in Dr. Friedrich's current address is Brookdale Abstract Rationale: Denatonium benzoate is a useful indicator to ensure that the respirator being used by an individual forms a tight enough seal to adequately protect against unwanted airborne exposure. Although the relative risk for adverse effects of fit testing using denatonium benzoate is low, the absolute number of workers with adverse reactions may nevertheless be sizeable.Patient concerns: A 34-year-old female nurse rapidly developed shortness of breath, cough, and agitation after denatonium benzoate fit testing. She had a history of allergy to shrimp, crab, mite, and disinfecting products (containing quaternary ammonium).Diagnoses: Due to typical symptoms of asthma after exposure to denatonium benzoate aerosol without any other apparent cause, serial pulmonary function tests indicating obstructive lung function and a higher concentration of immunoglobulin antibody E, she was diagnosed with allergic asthma.Interventions: This patient was treated with omalizumab (Xolair), corticosteroid, b 2 agonist, montelukast, and Symbicort turbuhaler.Outcomes: The patient showed quick responses after treatment with diphenhydramine (intramuscularly), fenoterol HBr (inhalation), and prednisolone (oral). Approximately 2 weeks later, she suffered from difficulty breathing and asthmatic symptoms again when she was exposed to polished wax and disinfectant. She was treated with omalizumab (Xolair), corticosteroid, b 2 agonist, montelukast, and Symbicort turbuhaler. The patient was in stable condition with improvement in symptoms during follow-up.Lessons: There may be potentially important health risks when healthcare workers are exposed to denatonium benzoate. Individuals who have a history of allergy to disinfecting products (containing quaternary ammonium) should avoid exposure of denatonium benzoate. More advanced research is needed in the future.Abbreviations: ASM = airway smooth muscle, HCWs = healthcare workers, IgE = immunoglobulin antibody E, SARS = severe acute respiratory syndrome. Abstract Human rhinoviruses (HRV) are one of the major causes of common cold in humans and are also associated with acute asthma and bronchial illness. Heat-shock protein 90 (Hsp90), a molecular chaperone, is an important host factor for the replication of single-strand RNA viruses. In the current study, we examined the effect of the Hsp90 inhibitor pochonin D, in vitro and in vivo, using a murine model of human rhinovirus type 1B (HRV1B) infection. Our data suggested that Hsp90 inhibition significantly reduced the inflammatory cytokine production and lung damage caused by HRV1B infection. The viral titer was significantly lowered in HRV1B-infected lungs and in Hela cells upon treatment with pochonin D. Infiltration of innate immune cells including granulocytes and monocytes was also reduced in the bronchoalveolar lavage (BAL) by pochonin D treatment after HRV1B infection. Histological analysis of the lung and respiratory tract showed that pochonin D protected the mice from HRV1B infection. Collectively, our results suggest that the Hsp90 inhibitor, pochonin D, could be an attractive antiviral therapeutic for treating HRV infection.Original Article Abstract Recoding' is a term used to describe non-standard read-out of the genetic code, and encompasses such phenomena as programmed ribosomal frameshifting, stop codon readthrough, selenocysteine insertion and translational bypassing. Although only a small proportion of genes utilize recoding in protein synthesis, accurate annotation of 'recoded' genes lags far behind annotation of 'standard' genes. In order to address this issue, provide a service to researchers in the field, and offer training data for developers of gene-annotation software, we have gathered together known cases of recoding within the Recode database. Recode-2 is an improved and updated version of the database. It provides access to detailed information on genes known to utilize translational recoding and allows complex search queries, browsing of recoding data and enhanced visualization of annotated sequence elements. At present, the Recode-2 database stores information on approximately 1500 genes that are known to utilize recoding in their expression-a factor of approximately three increase over the previous version of the database. Recode-2 is available at Abstract Spatio-temporal aspects in the propagation of infectious pathogens of humans are reviewed. Mathematical modelling of these issues using metapopulation models is presented. Abstract Eosinophilic enteritis (EOE) is a type of inflammatory bowel disease and is characterized clinically by chronic obstinate diarrhea. Three Japanese Black (JB) fattening cattle (2 males and 1 female) on different cattle farms presented with chronic episodic diarrhea without fever or dehydration. Soft reddish spherical carneous tissues (1−3 cm) were occasionally excreted within the diarrheic feces. Administration of antibiotics, antidiarrheal drugs and vermicides had no therapeutic effect, but dexamethasone improved the fecal characteristics. The symptoms persisted until the animals were slaughtered at 27-30 months of age. Histopathological examination of the intestines revealed marked eosinophilic infiltration in the lamina propria and submucosa. From these findings, we diagnosed these cattle as the first cases of EOE in Jb cattle. KEY WORDS: eosinophilic enteritis, inflammatory bowel disease, Japanese Black cattle Abstract Objective: This study investigated the sources of stress, corresponding symptoms, and stress relief among nurses of the first Chinese anti-Ebola medical team during the Sierra Leone aid mission. Method: A purposive sampling method was used and 10 nurses were selected from the first Chinese anti-Ebola medical team that was dispatched to Sierra Leone. Data were collected via phone and semistructured interviews, then analyzed using Colaizzi's seven-step method. Results: The data showed three major themes: (1) The causes of stress during the Sierra Leone aid mission mainly related to unsafety, responsibility, and unfamiliarity; (2) Physical, cognitive, emotional, and behavioral symptoms were documented; (3) Nurses experienced relief from stress after the mission. Conclusion: Targeted measures, proper responses and good community support can effectively lower stress among nurses on anti-Ebola missions. Abstract Cancer is a genetic disease where genetic variations cause abnormally functioning genes that appear to alter expression. Proteins, the final products of gene expression, determine the phenotypes and biological processes. Therefore, detecting gene expression levels can be used for cancer diagnosis, prognosis, and treatment prediction in a clinical setting. In this review, we investigated six gene expression assay systems (qRT-PCR, DNA microarray, nCounter, RNA-Seq, FISH, and tissue microarray) that are currently being used in clinical cancer studies. Some of these methods are also commonly used in a modified way; for example, detection of DNA content or protein expression. Herein, we discuss their principles, sample preparation, design, quantification and sensitivity, data analysis, time for sample preparation and processing, and cost. We also compared these methods according to their sample selection, particularly for the feasibility of using formalin-fixed paraffin-embedded (FFPE) samples, which are routinely archived for clinical cancer studies. We intend to provide a guideline for choosing an assay method with respect to its oncological applications in a clinical setting. Abstract Filoviruses cause severe and fatal viral hemorrhagic fever in humans. Filovirus research has been extensive since the 2014 Ebola outbreak. Due to their high pathogenicity and mortality, live filoviruses require Biosafety Level-4 (BSL-4) facilities, which have restricted the development of anti-filovirus vaccines and drugs. An HIV-based pseudovirus cell infection assay is widely used for viral entry studies in BSL-2 conditions. Here, we successfully constructed nine in vitro pseudo-filovirus models covering all filovirus genera and three in vivo pseudo-filovirus-infection mouse models using Ebola virus, Marburg virus, and Lloviu virus as representative viruses. The pseudo-filovirus-infected mice showed visualizing bioluminescence in a dose-dependent manner. A bioluminescence peak in mice was reached on day 5 post-infection for Ebola virus and Marburg virus and on day 4 post-infection for Lloviu virus. Two known filovirus entry inhibitors, clomiphene and toremiphene, were used to validate the model. Collectively, our study shows that all genera of filoviruses can be well-pseudotyped and are infectious in vitro. The pseudo-filovirus-infection mouse models can be used for in vivo activity evaluation of anti-filovirus drugs. This sequential in vitro and in vivo evaluation system of filovirus entry inhibitors provides a secure and efficient platform for screening and assessing anti-filovirus agents in BSL-2 facilities. Abstract Rat microsomal aldehyde dehydrogenase (msALDH) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (Miyauehi, K., R. Masaki, S. Taketani, A. Yamamoto, A. Akayama, and Y. Tashiro. 1991. J. Biol. Chem. 266:19536-19542). This membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination. When expressed from eDNA in COS-1 cells, wild-type msALDH is localized exclusively in the well-developed ER. The removal of the hydrophobic Abstract Novel reassortant avian influenza H7N9 virus and pandemic 2009 H1N1 (H1N1pdm) virus cause human infections, while avian H7N2 and swine H1N1 virus mainly infect birds and pigs, respectively. There is no robust in vitro model for assessing the infectivity of emerging viruses in humans. Based on a recently established method, we generated long-term expanding 3D human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. We report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. In addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. We also established improved 2D monolayer culture conditions for the differentiated airway organoids. To demonstrate the ability of differentiated airway organoids to identify human-infective virus, 3D and 2D differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, H7N9/Ah versus H7N2 and H1N1pdm versus an H1N1 strain isolated from swine (H1N1sw). The humaninfective H7N9/Ah virus replicated more robustly than the poorly human-infective H7N2 virus; the highly human-infective H1N1pdm virus replicated to a higher titer than the counterpart H1N1sw. Collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. These differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human. airway organoid | proximal differentiation | influenza virus | infectivity Abstract Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 XZ_ap cDNA library. Three related clones, designated SY2, SY10, and SYll, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the XZ_ap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SYll protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SYll represented 58.7-and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SYll cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SYll to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins. Abstract The expression of any gene must be precisely controlled for appropriate function. This expression can be controlled at various levels. This includes epigenetic regulation through DNA methylation or histone modifications. At the posttranscriptional level, regulation can be via alternative splicing or controlling messenger RNA (mRNA) stability. RNA cleavage is one way to control mRNA stability. For example, microRNA (miRNA)-induced mRNA cleavage has long been recognised in plants. RNA cleavage also appears to be widespread in other kingdoms of life, and it is now clear that mRNA cleavage plays critical functions in animals. Although miRNA-induced mRNA cleavage can occur in animals, it is not a widespread mechanism. Instead, mRNA cleavage can be induced by a range of other mechanisms, including by endogenous short inhibitory RNAs (endo-siRNAs), as well as the Ribonuclease III (RNase III) enzymes Drosha and Dicer. In addition, RNA cleavage induced by endo-siRNAs and PIWI-interacting RNAs (piRNAs) is important for genome defence against transposons. Moreover, several RNase has been identified as important antiviral mediators. In this review, we will discuss these various RNA endonucleolytic cleavage mechanisms utilised by animals to regulate the expression of genes and as a defence against retrotransposons and viral infection. Abstract Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle degeneration, caused by the absence of dystrophin. Exon skipping by antisense oligonucleotides (ASOs) has recently gained recognition as therapeutic approach in DMD. Conjugation of a peptide to the phosphorodiamidate morpholino backbone (PMO) of ASOs generated the peptide-conjugated PMOs (PPMOs) that exhibit a dramatically improved pharmacokinetic profile. When tested in animal models, PPMOs demonstrate effective exon skipping in target muscles and prolonged duration of dystrophin restoration after a treatment regime. Herein we summarize the main pathophysiological features of DMD and the emergence of PPMOs as promising exon skipping agents aiming to rescue defective gene expression in DMD and other neuromuscular diseases. The listed PPMO laboratory findings correspond to latest trends in the field and highlight the obstacles that must be overcome prior to translating the animal-based research into clinical trials tailored to the needs of patients suffering from neuromuscular diseases. Abstract Genome Detective is a web-based, user-friendly software application to quickly and accurately assemble all known virus genomes from next generation sequencing datasets. This application allows the identification of phylogenetic clusters and genotypes from assembled genomes in FASTA format. Since its release in 2019, we have produced a number of typing tools for emergent viruses that have caused large outbreaks, such as Zika and Yellow Fever Virus in Brazil. Here, we present The Genome Detective Coronavirus Typing Tool that can accurately identify the novel severe acute respiratory syndrome (SARS) related coronavirus (SARS-CoV-2) sequences isolated in China and around the world. The tool can accept up to 2,000 sequences per submission and the analysis of a new whole genome sequence will take approximately one minute. The tool has been tested and validated with hundreds of whole genomes from ten coronavirus species, and correctly classified all of the SARS-related coronavirus (SARSr-CoV) and all of the available public data for SARS-CoV-2. The tool also allows tracking of new viral mutations as the outbreak expands globally, which may help to accelerate the development of novel diagnostics, drugs and vaccines to stop the COVID-19 disease. Abstract A hallmark of translation in human immunodeficiency virus type 1 (HIV-1) is a -1 programmed ribosome frameshifting event that produces the Gag-Pol fusion polyprotein. The constant Gag to Gag-Pol ratio is essential for the virion structure and infectivity. Here we show that the frameshifting efficiency is modulated by Leu-tRNA Leu that reads the UUA codon at the mRNA slippery site. This tRNA Leu isoacceptor is particularly rare in human cell lines derived from T-lymphocytes, the cells that are targeted by HIV-1. When UUA decoding is delayed, the frameshifting follows an alternative route, which maintains the Gag to Gag-Pol ratio constant. A second potential slippery site downstream of the first one is normally inefficient but can also support -1-frameshifting when altered by a compensatory resistance mutation in response to current antiviral drug therapy. Together these different regimes allow the virus to maintain a constant -1-frameshifting efficiency to ensure successful virus propagation. Figure 1. -1FS on HIV-1 gag-pol mRNA. (A) Scheme of the gag-pol frameshifting site. Slippery site (SS1) and the putative second slippery site (pSS2) are highlighted in green; the stimulatory mRNA structure element downstream of the SS1 is indicated as a stem-loop (SL1). Amino acids incorporated into 0-frame and -1-frame peptides as well as the potential -1FS routes and in vivo efficiencies are shown below the frameshifting sites. (B) Top panel: Amino acids incorporated into 0-and -1-frames are shown above the mRNA sequence. Bottom panel: -1FS efficiency with the wild-type (wt) mRNA and U 4 C derivative with disrupted SS1 measured at limiting amounts of Leu-tRNA NAA Leu (molar ratio 0.3 tRNA to 70S ribosome) at the end of translation (2 min). The 0-frame is the sum of MQANF and MQANFLG peptides, -1-frame corresponds to MQANFFR/FLR peptides. MQANF was identified based on its position on the chromatogram while MQANFFR/FLR and MQANFLG products were quantified using [ 14 C]Arg and [ 3 H]Gly, respectively. (C) Concentration dependence of -1FS efficiency on the Leu-tRNA NAA Leu (tRNA NAA Leu , closed circles) or a mixture of tRNA Leu isoacceptors reading CUN codons (tRNA NAG Leu , open circles). -1FS product was detected using [ 14 C]Arg. (D) Change in the FS regime with the Leu-tRNA NAA Leu concentration. The ratio of FFR route (open circles) versus FLR (closed circles) route was calculated from peptides with different radioactive labels as follows. The sum of FFR and FLR frameshifting products was calculated using [ 14 C]Arg. To determine the amount of FLR, the mRNA was translated to the 0-frame peptide fMet-Gln-Asn-Phe-Leu-Gly-Lys-Ile (MQANFLGKI). The presence of Ile allows for separation between 0-frame MQANFLGKI and -1-frame MQANFLR peptides. The FFR peptide was then determined by subtracting the FLR from the total Arg-containing product. (E) -1FS efficiency in the presence of varying concentrations of Gly-tRNA Gly in the presence of excess Arg-tRNA Arg (2 M) (green squares) or with varying concentrations of Arg-tRNA Arg in the presence of 3 or 6 M Gly-tRNA Gly (red and light red squares, respectively). Abstract This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.This article is a part of a series of reviews covering Fc Receptors appearing in Volume 268 of Immunological Reviews.Summary: Antibodies are key molecules in the fight against infections. Although previously thought to mediate protection solely in the extracellular environment, recent research has revealed that antibody-mediated protection extends to the cytosolic compartment of cells. This postentry viral defense mechanism requires binding of the antibody to a cytosolic Fc receptor named tripartite motif containing 21 (TRIM21). In contrast to other Fc receptors, TRIM21 shows remarkably broad isotype specificity as it does not only bind IgG but also IgM and IgA. When viral pathogens coated with these antibody isotypes enter the cytosol, TRIM21 is rapidly recruited and efficient neutralization occurs before the virus has had the time to replicate. In addition, inflammatory signaling is induced. As such, TRIM21 acts as a cytosolic sensor that engages antibodies that have failed to protect against infection in the extracellular environment. Here, we summarize our current understanding of how TRIM21 orchestrates humoral immunity in the cytosolic environment. Abstract The present study was carried out to characterize pathogenic E. coli in apparently healthy and diarrheic neonatal calves with special reference to the hybrid E. coli strains and evaluate their clinical and hematobiochemical consequences. One hundred and seventy calves (age 1-30 days) were divided into two groups: apparently healthy (n = 70) and diarrheic (n=100). Animals were subjected to thorough clinical, hematobiochemical and bacteriological examinations. Clinically, diarrheic calves showed various degree of diarrhea with the presence of cardinal signs of dehydration in moderate and severe cases. There was a significant increase (p<0.05) in the hemogram parameters with uremia and hyperkalemia in calves with severe diarrhea. The O-H serotyping of cultural and biochemically positive isolates identified 31 isolates belonging to 12 serotypes including and O128:H2. Molecular characterization of E. coli isolates on three toxin genes: heat-stable enterotoxin (sta), shiga toxin type 1 and 2 (stx1 and stx2) revealed two well-known with high frequency of enterohemorrhagic E. coli (EHEC). Molecular analysis also showed a number of E. coli isolates that carry sta and stx1 or sta and stx2 gene and belonged to O8, and O127:H6, O86 and O128:H2. These isolates were identified as hybrid E. coli strains (ETEC-STEC) and found in both apparently healthy and diarrheic calves. In conclusion, the present study identified high frequency of pathogenic E. coli in both apparently healthy and diarrheic calves. Serological and molecular analysis of E. coli isolates showed that high frequency of EHEC and presence of a new phenotype, STEC-ETEC hybrid, revealing their importance in the etiopathogenesis of diarrhea in calves and reinforcing the role of these animals as a reservoir of potentially pathogenic E. coli for humans. Abstract Vaccination continues to have a major impact on the health of humans and animals. Furthermore, vaccination of animals is proving to be effective in reducing transmission to humans. Understanding linkages between innate and adaptive immunity are improving formulations of new, as well as existing, vaccines, making them more effective. Abstract Respiratory virus infections, such as those mediated by influenza virus, parainfluenza virus, respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus (SARS-CoV), rhinovirus, and adenovirus, are responsible for substantial morbidity and mortality, especially in children and older adults. Furthermore, the potential emergence of highly pathogenic strains of influenza virus poses a significant public health threat. Thus, the development of vaccines capable of eliciting long-lasting protective immunity to those pathogens is a major public health priority. CD8 + Tissue-resident memory T (T RM ) cells are a newly defined population that resides permanently in the nonlymphoid tissues including the lung. These cells are capable of providing local protection immediately after infection, thereby promoting rapid host recovery. Recent studies have offered new insights into the anatomical niches that harbor lung CD8 + T RM cells, and also identified the requirement and limitations of T RM maintenance. However, it remains controversial whether lung CD8 + T RM cells are continuously replenished by new cells from the circulation or permanently lodged in this site. A better understanding of how lung CD8 + T RM cells are generated and maintained and the tissue-specific factors that drive local T RM formation is required for optimal vaccine development. This review focuses on recent advance in our understanding of CD8 + T RM cell establishment and maintenance in the lung, and describes how those processes are uniquely regulated in this tissue. Abstract This study reports on two Hanwoo (a native Korean breed of cattle) calves, a 3-and 6-month-old presenting with diarrhea, anorexia and blindness. Ophthalmoscopic examination revealed bilateral papilledema in both calves. Reverse-transcription polymerase chain reaction tests for bovine viral diarrhea virus, rotavirus and coronavirus were all negative. The levels of serum vitamin A in the two affected calves were 0.317 µg/dl and 0.481 µg/dl, respectively. These values are much lower than the normal vitamin A levels; therefore, the calves were diagnosed with hypovitaminosis A. Abstract We report the complete genome sequences of a buffalo coronavirus (BufCoV HKU26) detected from the faecal samples of two domestic water buffaloes (Bubalus bubalis) in Bangladesh. They possessed 98-99% nucleotide identities to bovine coronavirus (BCoV) genomes, supporting BufCoV HKU26 as a member of Betacoronavirus 1. Nevertheless, BufCoV HKU26 possessed distinct accessory proteins between spike and envelope compared to BCoV. Sugar-binding residues in the N-terminal domain of S protein in BCoV are conserved in BufCoV HKU26.New Microbes and New Infections Abstract Physical entanglement, and particularly knots arise spontaneously in equilibrated polymers that are sufficiently long and densely packed. Biopolymers are no exceptions: knots have long been known to occur in proteins as well as in encapsidated viral DNA. The rapidly growing number of RNA structures has recently made it possible to investigate the incidence of physical knots in this type of biomolecule, too. Strikingly, no knots have been found to date in the known RNA structures. In this Point of View Article we discuss the absence of knots in currently available RNAs and consider the reasons why knots in RNA have not yet been found, despite the expectation that they should exist in Nature. We conclude by singling out a number of RNA sequences that, based on the properties of their predicted secondary structures, are good candidates for knotted RNAs. Abstract Community-acquired pneumonia (CAP) is the leading cause of death worldwide.Despite the vast diversity of respiratory microbiota, Streptococcus pneumoniae remains the most prevalent pathogen among etiologic agents. Despite the significant decrease in the mortality rates for lower respiratory tract infections in recent decades, CAP ranks third as a cause of death in Brazil. Since the latest Guidelines on CAP from the Sociedade Brasileira de Pneumologia e Tisiologia (SBPT, Brazilian Thoracic Association) were published (2009), there have been major advances in the application of imaging tests, in etiologic investigation, in risk stratification at admission and prognostic score stratification, in the use of biomarkers, and in the recommendations for antibiotic therapy (and its duration) and prevention through vaccination. To review these topics, the SBPT Committee on Respiratory Infections summoned 13 members with recognized experience in CAP in Brazil who identified issues relevant to clinical practice that require updates given the publication of new epidemiological and scientific evidence. Twelve topics concerning diagnostic, prognostic, therapeutic, and preventive issues were developed. The topics were divided among the authors, who conducted a nonsystematic review of the literature, but giving priority to major publications in the specific areas, including original articles, review articles, and systematic reviews. All authors had the opportunity to review and comment on all questions, producing a single final document that was approved by consensus. Abstract Vectors for expression of h9/14, h9/14A5, h9-3A/14 were described in (1,2).Plasmids encoding the eukaryotic translation initiation factors eIF1, 1A, 4A, 4B, 4G, 5, 5B and Escherichia coli Met-tRNA synthetase were described in (3). pGL3R-βglo, pGL3R-βact, pGL3R-L1, pGL3R-HCV, pGL3R-EMCV, pGl3R-CrPV were the kind gift of Dr. Sergey Dmitriev (4). PCR fragments amplified from these constructs and containing the Firefly luciferase ORF fused to 5ʼ-UTRs of β-globin, β-actin, LINE-1, HCV IRES or CrPV IRES under the control of SP6 promoter was inserted to pUC18 plasmid digested by HindIII and EcoRI to generate pUCβglo, pUCβact and pUCL1, and by PstI and EcoRI to generate pUCHCV and pUCCrPV. Vectors for in vivo expression of monocistronic mRNAs containing Firefly luciferase ORF fused to 5ʼ-UTRs of β-globin, β-actin and LINE-1 were prepared by insertion of PCR fragment containing respective 5ʼ-UTRs to pGL3R-EMCV digested by AflII and NcoI to remove upstream Renilla luciferase ORF and EMCV IRES. Plasmid pCrPV-VHLM was constructed by insertion of T7 promoter, CrPV IRES nucleotides 6028 to 6216 followed by a short ORF encoding Val-His-Leu-Met tripeptide, UAA stop codon and 100 nt random sequence taken from β-glucuronidase (GUS) gene, into the pUC18 vector. Plasmids pscAluYb8, pscAluYf2, pscAluYj4 were obtained from the plasmids pscAlu/α-feto and pSscAlu/LDL (1) using quick change protocol. pSAlu110 and pSAlu151 contained the Alu portion of 7SL RNA gene (nucleotides 1-74; 271-299 and 1-99; 251-299, respectively) with a closing loop GATT following position 74 or 99, preceded by the T7 promoter.[ 32 P]-MVHL-stop mRNA synthesis [ 32 P]-UTP was added to the transcription reaction. Preprolactin and MVHL-stop mRNAs were synthesized with SP6 RNA polymerase from plasmids pSP-BP4 (8) and MVHL-stop (9) linearized with EcoRI and XhoI. Capped and uncapped RNAs encoding Firefly luciferase fused to various 5ʼ-UTRs were synthesized with SP6 RNA polymerase from plasmids pUCβglo, pUCβact, pUCL1, pUCCHV and pUCCrPV linearized with EcoRI. HCV-NSʼ and CrPV-VHLM mRNAs were synthesized with T7 RNA polymerase from plasmids pXL.HCV(40-373).NSʼ (10) and pCrPV-VHLM linearized with EcoRI and SspI, respectively. The mRNAs were purified by LiCl precipitation, G50 sepharose chromatography, ethanol precipitation, and resuspended in water. Non-coding AluY A , scAluY A , scAluY L , and 4.5S RNAs were synthesized with T7 RNA polymerase from plasmids pPAlu, pPscAlu/a feto, pSscAlu/LDL and pS4.5S (1) linearized with SspI, SpeI, SpeI and Andreev, D.E., Dmitriev, S.E., Terenin, I.M., Prassolov, V.S., Merrick, W.C. and Shatsky, I.N. (2009) Differential contribution of the m7G-cap to the 5' end-dependent translation initiation of mammalian mRNAs. Nucleic Acids Res., 37, 6135-6147. 5. Dewannieux, M., Esnault, C. and Heidmann, T. (2003) LINE-mediated retrotransposition of marked Alu sequences. Nat. Genet., 35, 41-48. 6. Gurevich, V.V., Pokrovskaya, I.D., Obukhova, T.A. and Zozulya, S.A. (1991) Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. Anal. Biochem., 195, 207-213. 7. Milligan, J.F. and Uhlenbeck, O.C. (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol., 180, 51-62. 8. Siegel, V. and Walter, P. (1988) Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP. Cell, 52, 39-49. 9. Alkalaeva, E.Z., Pisarev, A.V., Frolova, L.Y., Kisselev, L.L. and Pestova, T.V. (2006) In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3. Cell, 125, 1125-1136. Abstract Dovepress submit your manuscript | www.dovepress.com Dovepress 2573 O r I g I N a l r e s e a r c h open access to scientific and medical research Open Access Full Text Article People's republic of china; 2 shandong Provincial Key laboratory of carbohydrate chemistry and glycobiology, shandong University, Jinan 250012, shandong, People's republic of china; 3 school of Pharmaceutical sciences, shandong University, Jinan 250012, shandong, People's republic of china *These authors contributed equally to this workIn the past few years, significant progress has been made in inhibiting neovascularization at the tumor site, cutting off the nutrient supply of the tumor, and inhibiting tumor growth and metastasis. However, many proteins/peptides have the disadvantage of poor stability, short half-life, and uncertain targeting ability. Chemical modification can be used to overcome these disadvantages; many polyethylene glycol-modified proteins/peptides have been approved by US FDA. The purpose of this study was to obtain a novel anti-angiogenic chondroitin sulfate (CS)-peptide nanoparticle conjugate with efficient anti-neovascularization and tumor targeting ability and an acceptable half-life.The CS-ES2-AF nanoparticle conjugate was synthesized and characterized using 1 H-nuclear magnetic resonance spectroscopy, transmission electron microscopy, and particle size and zeta potential analyzer. The anti-angiogenic ability was studied using MTT, migration, tube formation, and chick chorioallantoic membrane assays. The targeting ability of CS-ES2-AF was studied by ELISA, surface plasmon resonance, and bioimaging. The pharmacokinetics was also studied. Results: The CS-ES2-AF could self-assemble into stable nanoparticles in aqueous solution, which significantly enhances its anti-neovascularization activity, tumor targeting more explicit, and prolongs its half-life. Conclusion: CS is an effective protein/peptide modifier, and CS-ES2-AF displayed good potential in tumor targeting therapy. Abstract METHODSSubjects with CF were recruited from the two CF centres by personal invitation from the Investigators (SCB, CEW) or the Study Coordinators (MEW, JC). All CF patients were >12-years of age, had chronic Pseudomonas aeruginosa infection and at least one positive sputum culture in the prior 12-months. The majority of patients were studied during clinical stability, however, four patients were studied close to the completion of intravenous antibiotics for logistic reasons (e.g. travelling long distance, avoid missing school attendance). Healthy control participants were recruited from contact with staff at both healthcare facilities including relatives of the staff for the younger controls. Nineteen CF patients with CF and 10 healthy controls participated. Two patients and two controls performed only one of the experiment days.The Distance Rig consisted of an expandable wind tunnel that allowed high-efficiency particulate air (HEPA)-filtered air to be introduced upstream of the subject;[1] ensuring unidirectional air flow of particle-free air and permitting measurement of cough aerosols at distances of 0 to 4-metres from the subject without interference from other particle sources ( Figure E1 ). The air velocity was maintained at 0.1-metres/sec, typical of a mechanicallyventilated indoor environment, such as a hospital.[2-4] The Rig was positively pressurized to prevent room air contamination.A six-stage Andersen Impactor (Thermo Scientific, Franklin, MA) captured and sized viable cough aerosols in the six stages between 0.6 and >7 m.[5] A vacuum pump was used to draw 28.3 L/min of air through the Impactor. The pump flow was checked daily using a rotameter, and all Andersen Impactor O-rings were inspected for wear. Exhaust air from the pump was HEPA-filtered.A Lasair II-110 (Particle Measuring Systems, Boulder, CO) optical particle counter (OPC) measured real-time particle concentration in six channels between 0.1 and >5 µm using a sample flow of 28.3 L/min, permitting detection of very low concentrations. The OPC verified that there were no room air particles present before each test, subjects' lungs were free of residual room air, and no contamination of aerosols occurred during testing. It also confirmed that the total particle concentration in the Rig was <0.01 particles per cm 3 (p/cc) prior to the subject coughing, which was approximately 10 4 times lower than the room air concentration and similar to an ISO 4 cleanroom.The Andersen Impactor and OPC samples were collected through a common 12.5 cm circular, sharp-edged isokinetic inlet. It was set parallel to the airflow and provided 100% particle aspiration efficiency at the tunnel air velocity of 0.1 metres/sec. [6] Smoke visualization tests confirmed sample extraction by the inlet was uniform. One metre of conductive tubing transported samples from the inlet to the Andersen Impactor and OPC. The tubing residence time was 1-sec for Andersen samples and 0.06-sec for OPC samples.Sample losses due to gravitational settling and inertial impaction were from 0 to 8% and 0 to 1%, respectively, and diffusion losses were negligible.[6] Air velocity was monitored continuously using a 9535 hot-wire anemometer (TSI Inc., St. Paul, MN). Temperature and water vapour concentration were measured simultaneously work environments are related to indoor air symptoms. J Occup Environ Med 2007;49(6):641-50. 3. Baldwin PE, Maynard AD. A survey of windspeeds in indoor workplaces. Ann Occup Hyg 1998;42(5):303-13.4. de Dear RJ, Fountain ME. Field experiments on occupant comfort and office thermal environments in a hot-humid climate. ASHRAE Transactions 1994;100(2):457-75. 5. Wainwright CE, France MW, O'Rourke P, et al. Cough-generated aerosols of Pseudomonas aeruginosa and other Gram-negative bacteria from patients with cystic fibrosis. Thorax 2009;64(11):926-31. 6. Baron PA, Willeke K. Aerosol Measurement: Principles, Techniques, and Applications: Wiley, 2005. 7. Goldberg LJ. Naval biomedical research laboratory, programmed environment, aerosol facility. Appl Microbiol 1971;21(2):244-52. 8. Goldberg LJ, Watkins HMS, Boerke EE, et al. The use of a rotating drum for the study of aerosols over extended periods of time. Am J Epidemiol 1958;68:85-93. 9. Gruel RL, Reid CR, Allemann RT. The optimum rate of drum rotation for aerosol aging. J Aerosol Sci 1987;18(1):17-22. 10. Clifton IJ, Fletcher LA, Beggs CB, et al. An aerobiological model of aerosol survival of different strains of Pseudomonas aeruginosa isolated from people with cystic fibrosis. J Cyst Fibros 2010;9(1):64-68. Abstract Despite the significant progress in the recent efforts toward developing an effective vaccine against toxoplasmosis, the search for new protective vaccination strategy still remains a challenge and elusive goal because it becomes the appropriate way to prevent the disease. Various experimental approaches in the past few years showed that developing a potential vaccine against the disease can be achievable. The combination of multi-epitopes expressing different stages of the parasite life cycle has become an optimal strategy for acquiring a potent, safe, and effective vaccine. Epitope-based vaccines have gained attention as alternative vaccine candidates due to their ability of inducing protective immune responses. This mini-review highlights the current status and the prospects of Toxoplasma gondii vaccine development along with the application of epitope-based vaccine in the future parasite immunization as a novel under development and evaluation strategy.ARTICLE HISTORY Abstract South Korea was free of the Middle East Respiratory Syndrome (MERS) until 2015. The MERS outbreak in South Korea during 2015 was the largest outbreak of the Coronavirus outside the Middle East. The major characteristic of this outbreak is inter-or intra-hospital transmission. This recent MERS outbreak in South Korea is examined and assessed in this paper. The main objectives of the study is to characterize the pattern of the MERS outbreak in South Korea based on a basic reproductive ratio, the probability of ultimate extinction of the disease, and the spatio-temporal proximity of occurrence between patients. The survival function method and stochastic branching process model are adapted to calculate the basic reproductive ratio and the probability of ultimate extinction of the disease. We further investigate the occurrence pattern of the outbreak using a spatio-temporal autocorrelation function. Abstract Background: The natural history and consequences of severe H1N1 influenza infection among cancer patients are not yet fully characterized. We describe eight cases of H1N1 infection in cancer patients admitted to the intensive care unit of a referral cancer center.Patients and methods: Clinical data from all patients admitted with acute respiratory failure due to novel viral H1N1 infection were reviewed. Lung tissue was submitted for viral and bacteriological analyses by real-time RT-PCR, and autopsy was conducted on all patients who died.Results: Eight patients were admitted, with ages ranging from 55 to 65 years old. There were five patients with solid organ tumors (62.5%) and three with hematological malignancies (37.5%). Five patients required mechanical ventilation and all died. Four patients had bacterial bronchopneumonia. All deaths occurred due to multiple organ failure. A milder form of lung disease was present in the three cases who survived. Lung tissue analysis was performed in all patients and showed diffuse alveolar damage in most patients. Other lung findings were necrotizing bronchiolitis or extensive hemorrhage.Conclusions: H1N1 viral infection in patients with cancer can cause severe illness, resulting in acute respiratory distress syndrome and death. More data are needed to identify predictors of unfavorable evolution in these patients. Abstract The lack of vaccines against several important viral diseases necessitates the development of therapeutics to save lives and control epidemics. In recent years, therapeutic antibodies have received considerable attention due to their good safety profiles and clinical success when used against viruses such as respiratory syncytial virus, Ebola virus and Hendra virus. The binding affinity of these antibodies can directly impact their therapeutic efficacy. However, we and others have also demonstrated that the subtype of Fc-gamma receptors (FcgRs) engaged influences the stoichiometric requirement for virus neutralization. Hence, the development of therapeutic antibodies against infectious diseases should consider the FcgRs engaged and Fc-effector functions involved. This review highlights the current state of knowledge about FcgRs and FcgR effector functions involved in virus neutralization, with emphasis on factors that can affect FcgR engagement. A better understanding of Fc-FcgR interactions during virus neutralization will allow development of therapeutic antibodies that are efficacious and can be administered with minimal side effects. Abstract Objective In this study, we aimed to describe the processes of both the donning and the doffing of personal protective equipment for Ebola and evaluate contamination during the doffing process.We recruited study participants among physicians and nurses of the emergency department of Samsung Medical Center in Seoul, Korea. Participants were asked to carry out doffing and donning procedures with a helper after a 50-minute brief training and demonstration based on the 2014 Centers for Disease Control and Prevention protocol. Two separate cameras with high-density capability were set up, and the donning and doffing processes were videotaped. A trained examiner inspected all video recordings and coded for intervals, errors, and contaminations defined as the outside of the equipment touching the clinician's body surface.Overall, 29 participants were enrolled. Twenty (68.9%) were female, and the mean age was 29.2 years. For the donning process, the average interval until the end was 234.2 seconds (standard deviation [SD], 65.7), and the most frequent errors occurred when putting on the outer gloves (27.5%), respirator (20.6%), and hood (20.6%). For the doffing process, the average interval until the end was 183.7 seconds (SD, 38.4), and the most frequent errors occurred during disinfecting the feet (37.9%), discarding the scrubs (17.2%), and putting on gloves (13.7%), respectively. During the doffing process, 65 incidences of contamination occurred (2.2 incidents/ person). The most vulnerable processes were removing respirators (79.2%), removing the shoe covers (65.5%), and removal of the hood (41.3%).Conclusion A significant number of contaminations occur during the doffing process of personal protective equipment. Clin Exp Emerg Med 2015;2(3):162-167 http://dx. Abstract Ebola hemorrhagic fever (EHF) was first reported in 1976 with two concurrent outbreaks of acute viral hemorrhagic fever centered in Yambuku (near the Ebola river), Democratic Republic of Congo, and in Nzara, Sudan. The current outbreak of the Ebola virus was started by reporting the first case in March 2014 in the forest regions of southeastern Guinea. Due to infection rates raising over 13,000% within a 6-month period, Ebola is now considered as a global public health emergency and on August 8 th , 2014 the World Health Organization (WHO) declared the epidemic to be a Public Health Emergency of International Concern. With more than 5000 involved cases and nearly 3000 deaths, this event has turned into the largest and most dangerous Ebola virus outbreak in the world. Based on the above-mentioned, the present article aimed to review the virologic characteristics, transmission, clinical manifestation, diagnosis, treatment, and prevention of Ebola virus disease. Abstract N-myc & STAT Interactor, NMI, is a protein that has mostly been studied for its physical interactions with transcription factors that play critical roles in tumor growth, progression and metastasis. NMI is an inducible protein, thus its intracellular levels and location can vary dramatically, influencing a diverse array of cellular functions in a context-dependent manner. The physical interactions of NMI with its binding partners have been linked to many aspects of tumor biology including DNA damage response, cell death, epithelial-to-mesenchymal transition and stemness. Thus, discovering more details about the function(s) of NMI could reveal key insights into how transcription factors like c-Myc, STATs and BRCA1 are contextually regulated. Although a normal, physiological function of NMI has not yet been discovered, it has potential roles in pathologies ranging from viral infection to cancer. This review provides a timely perspective of the unfolding roles of NMI with specific focus on cancer progression and metastasis. Abstract is a comprehensive virus genome/segment database. We extracted 18 418 complete virus genomes/ segments from the International Nucleotide Sequence Database Collaboration (INSDC, http:// www.insdc.org/) by DNA Data Bank of Japan (DDBJ), EMBL and GenBank and stored them in our system. The list of registered viruses is arranged hierarchically according to taxonomy. Keyword searches can be performed for genome/segment data or biological features of any virus stored in GIB-V. GIB-V is equipped with a BLAST search function, and search results are displayed graphically or in list form. Moreover, the BLAST results can be used online with the ClustalW feature of the DDBJ. All available virus genome/segment data can be collected by the GIB-V download function. GIB-V can be accessed at no charge at Abstract 19 Abstract Alcoholic liver disease (ALD) is characterized by macrophage and neutrophil leukocyte recruitment and activation in the liver. Damage-and pathogen-associated molecular patterns contribute to a self-perpetuating proinflammatory state in ALD. Triggering receptor expressed on myeloid cells 1 (TREM-1) is a surface receptor that amplifies inflammation induced by toll-like receptors (TLRs) and is expressed on neutrophils and monocytes/macrophages. We hypothesized that TREM-1 signaling contributes to proinflammatory pathway activation in ALD. Using an in vivo ALD model in mice, we tested the effects of ligand-independent TREM-1 inhibitory peptides that were formulated into human high-density lipoprotein (HDL)-mimicking complexes GF9-HDL and GA/E31-HDL. As revealed in vitro, macrophages endocytosed these rationally designed complexes through scavenger receptors. A 5-week alcohol feeding with the Lieber-DeCarli diet in mice resulted in increased serum alanine aminotransferase (ALT), liver steatosis, and increased proinflammatory cytokines in the liver. TREM-1 messenger RNA (mRNA) expression was significantly increased in alcohol-fed mice, and TREM-1 inhibitors significantly reduced this increase. TREM-1 inhibition significantly attenuated alcohol-induced spleen tyrosine kinase (SYK) activation, an early event in both TLR4 and TREM-1 signaling. The TREM-1A LD has been a long-standing public health problem globally, yet the pathomechanisms leading to alcoholic hepatitis, a severe and often deadly form of the disease, are only partially understood. The direct effects of alcohol and its metabolites on hepatocytes result in steatosis and Abstract Background: Neonatal diarrhea accounts for more than 50% of total deaths in dairy calves. Few population-based studies of cattle have investigated how the microbiota is impacted during diarrhea.Objectives: To characterize the fecal microbiota and predict the functional potential of the microbial communities in healthy and diarrheic calves.Methods: Fifteen diarrheic calves between the ages of 1 and 30 days and 15 age-matched healthy control calves were enrolled from 2 dairy farms. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene (Illumina, San Diego, CA).Results: Significant differences in community membership and structure were identified among healthy calves from different farms. Differences in community membership and structure also were identified between healthy and diarrheic calves within each farm. Based on linear discriminant analysis effect size (LEfSe), the genera Bifidobacterium, Megamonas, and a genus of the family Bifidobacteriaceae were associated with health at farm 1, whereas Lachnospiraceae incertae sedis, Dietzia and an unclassified genus of the family Veillonellaceae were significantly associated with health at farm 2. The Phylogenetic Investigation of Communities Reconstruction of Unobserved States (PICRUSt) analysis indicated that diarrheic calves had decreased abundances of genes responsible for metabolism of various vitamins, amino acids, and carbohydrate.Clinical Relevance: The fecal microbiota of healthy dairy calves appeared to be farm specific as were the changes observed during diarrhea. The differences in microbiota structure and membership between healthy and diarrheic calves suggest that dysbiosis can occur in diarrheic calves and it is associated with changes in predictive metagenomic function. Abstract Objective When faced with an emergent epidemic with high mortality and morbidity potential, policy makers must decide what public health interventions to deploy at different stages of the outbreak. However, almost nothing is known about how the public view these interventions or how they trade off risks (of disease) with inconvenience (of interventions). In this paper, we aim to understand public perceptions on pandemic interventions, as well as to identify if there are any distinct respondent preference classes. Design A discrete choice experiment. setting This study was fielded in Singapore between November 2012 and February 2013. Participants A random sample of 500 Singapore residents aged 21 and over, including 271 women and 229 men, was analysed. Outcome measures Demographic information was collected from each participant. Participants were also shown a series of pairs of alternatives, each combining interventions and morbidity, mortality and cost outcomes and declared a preference for one combination. A random utility model was developed to determine the individual's preference for interventions and a hierarchical cluster analysis was performed to identify distinct respondent preference classes. Abstract Context: Hand, foot and mouth disease (HFMD) is a widespread pediatric disease caused primarily by human enterovirus 71 (EV-A71) and Coxsackievirus A16 (CV-A16). Objective: This study reports a systematic review of the epidemiology of HFMD in Asia. Data Sources: PubMed, Web of Science and Google Scholar were searched up to December 2014. Study Selection: Two reviewers independently assessed studies for epidemiologic and serologic information about prevalence and incidence of HFMD against predetermined inclusion/exclusion criteria. Data Extraction: Two reviewers extracted answers for 8 specific research questions on HFMD epidemiology. The results are checked by 3 others. Results: HFMD is found to be seasonal in temperate Asia with a summer peak and in subtropical Asia with spring and fall peaks, but not in tropical Asia; evidence of a climatic role was identified for temperate Japan. Risk factors for HFMD include hygiene, age, gender and social contacts, but most studies were underpowered to adjust rigorously for confounding variables. Both community-level and school-level transmission have been implicated, but their relative importance for HFMD is inconclusive. Epidemiologic indices are poorly understood: No supporting quantitative evidence was found for the incubation period of EV-A71; the symptomatic rate of EV-A71/Coxsackievirus A16 infection was from 10% to 71% in 4 studies; while the basic reproduction number was between 1.1 and 5.5 in 3 studies. The uncertainty in these estimates inhibits their use for further analysis. Limitations: Diversity of study designs complicates attempts to identify features of HFMD epidemiology. Conclusions: Knowledge on HFMD remains insufficient to guide interventions such as the incorporation of an EV-A71 vaccine in pediatric vaccination schedules. Research is urgently needed to fill these gaps.Key Words: hand, foot and mouth disease, EV-A71, CV-A16, epidemiology (Pediatr Infect Dis J 2016;35:e285-e300) H and, foot and mouth disease (HFMD) has become an endemic childhood disease in East and Southeast Asia. Its main etiologic agents are human enterovirus 71 (EV-A71) and Coxsackievirus 16 (CV-A16). Although usually mild-with symptoms limited to >38°C fever, malaise, rashes on the volar regions of the hands and feet, herpangina and difficulty eating and drinking-more rarely, infection can lead to complications of the nervous or cardiopulmonary systems. Such cases can result in long-term sequelae such as cognitive and motor disorders 1,2 or death, usually from pulmonary edema or brainstem encephalitis. 3 Although complications are rare, the number of children being infected in high-incidence countries such as China (≈2.7 M cases in 2014 3 ) means the death toll can be substantial (384 deaths in China in 2014 3 ). The EV-A71 virus seems to be responsible for more severe outcomes, while CV-A16 and other Coxsackieviruses, such as CV-A2, CV-A6 and CV-A10, usually present milder symptoms that resolve within a few weeks. [4] [5] [6] There are nearly 25 years of literature from Asia that describes the epidemiology of HFMD, drawing on pediatric cohorts, national surveillance systems, outbreak investigations and clinical data, and from disparate countries that span stages of economic development and with climates that range from tropical to temperate. This diversity complicates attempts to identify general features of HFMD epidemiology and conceals gaps in the body of knowledge of this important pediatric disease.The objective of this paper is to provide a robust systematic review of the epidemiology of HFMD that informs public health policy making about HFMD epidemics. The review covers 3 major areas: (1) history and seasonality of HFMD, and the efforts in predictive modeling; (2) risk factors for infection, to guide control and (3) global epidemiologic parameters, such as the incubation period and basic reproduction number, which may determine the effectiveness of control policies. Abstract To assess relationships between xanthine oxidase (XOD) and nephropathogenic infectious bronchitis virus (NIBV) infection, 240 growing layers (35 days old) were randomly divided into two groups (infected and control) of 120 chickens each. Each chicken in the control and infected group was intranasally inoculated with 0.2 mL sterile physiological saline and virus, respectively, after which serum antioxidant parameters and renal XOD mRNA expression in growing layers were evaluated at 8, 15 and 22 days post-inoculation (dpi). The results showed that serum glutathione peroxidase and superoxide dismutase activities in the infected group were significantly lower than in the control group at 8 and 15 dpi (p ＜ 0.01), while serum malondialdehyde concentrations were significantly higher (p ＜ 0.01). The serum uric acid was significantly higher than that of the control group at 15 dpi (p ＜ 0.01). In addition, the kidney mRNA transcript level and serum activity of XOD in the infected group was significantly higher than that of the control group at 8, 15 and 22 dpi (p ＜ 0.05). The results indicated that NIBV infection could cause the increases of renal XOD gene transcription and serum XOD activity, leading to hyperuricemia and reduction of antioxidants in the body. Abstract Fig. S1. Related to Fig. 1. The morphologies of WT CVB3 and CVB3 3A-H57Y ROs in BGM cells are indistinguishable. BF738735 treatment does not affect Golgi apparatus architecture. A) BGM cells infected with WT CVB3 and prepared for EM by HPF-FS reveal typical enterovirus ROs, including single-membrane tubules, DMVs and multilamellar vesicles (left to right). B, C). BGM cells either untreated (B) or treated with BF738735 for 7 hours (C) were fixed for EM analysis. Representative images are shown illustrating that the morphology of the Golgi apparatus was unaffected by treatment. Scale bars, 200 nm (A) or 1 µm (B, C).Fig. S5. Related to Fig. 6. Innate antiviral responses during CVB3 3A-H57Y infection. U2OS or HEK293T cells (KO for PKR, MAVS and AGO where indicated) were infected with CVB3-Rluc 3A-H57Y at MOI 0.01. Following infection, the inoculum was removed and fresh medium with or without BF738735 inhibitor was added to the cells. Cells were lysed to determine the intracellular amounts of luciferase as a measure of genome replication. Values represent mean values of triplicates ± standard error of the means. Abstract Emerging infectious diseases (EIDs) are a significant burden on global economies and public health. Most present personal protective equipment used to prevent EID transmission and infections is typically devoid of antimicrobial activity. We report on green bioprotective nanofibrous membranes (RNMs) with rechargeable antibacterial and antiviral activities that can effectively produce biocidal reactive oxygen species (ROS) solely driven by the daylight. The premise of the design is that the photoactive RNMs can store the biocidal activity under light irradiation and readily release ROS under dim light or dark conditions, making the biocidal function "always online." The resulting RNMs exhibit integrated properties of fast ROS production, ease of activity storing, long-term durability, robust breathability, interception of fine particles (>99%), and high bactericidal (>99.9999%) and virucidal (>99.999%) efficacy, which enabled to serve as a scalable biocidal layer for protective equipment by providing contact killing against pathogens either in aerosol or in liquid forms. The successful synthesis of these fascinating materials may provide new insights into the development of protection materials in a sustainable, self-recharging, and structurally adaptive form. Abstract With the spread of novel coronavirus (2019-nCoV) pneumonia, chest high-resolution computed tomography (HRCT) has been one of the key diagnostic tools. To achieve early and accurate diagnostics, determining the radiological characteristics of the disease is of great importance. In this small scale research we retrospectively reviewed and selected six cases confirmed with 2019-nCoV infection in West China Hospital and investigated their initial and follow-up HRCT features, along with the clinical characteristics. The 2019-nCoV pneumonia basically showed a multifocal or unifocal involvement of ground-glass opacity (GGO), sometimes with consolidation and fibrosis. No pleural effusion or lymphadenopathy was identified in our presented cases. The follow-up CT generally demonstrated mild to moderate progression of the lesion, with only one case showing remission by the reducing extent and density of the airspace opacification. Abstract Objective: Established preclinical disease models are essential for not only studying aetiology and/or pathophysiology of the relevant diseases but more importantly also for testing prevention and/or treatment concept(s). The present study proposed and established a detailed induction and assessment protocol for a unique and cost-effective preclinical steroid-associated osteonecrosis (SAON) in rats with pulsed injections of lipopolysaccharide (LPS) and methylprednisolone (MPS). Methods: Sixteen 24-week-old male SpragueeDawley rats were used to induce SAON by one intravenous injection of LPS (0.2 mg/kg) and three intraperitoneal injections of MPS (100 mg/kg) with a time interval of 24 hour, and then, MPS (40 mg/kg) was intraperitoneally injected three times a week from week 2 until sacrifice. Additional 12 rats were used as normal controls. Two and six weeks after induction, animals were scanned by metabolic dual energy Xray absorptiometry for evaluation of tissue composition; serum was collected for bone turnover markers, Microfil perfusion was performed for angiography, the liver was collected for histopathology and bilateral femora and bilateral tibiae were collected for histological examination. Results: Three rats died after LPS injection, i.e., with 15.8% (3/19) mortality. Histological evaluation showed 100% incidence of SAON at week 2. Dual energy X-ray absorptiometry showed significantly higher fat percent and lower lean mass in SAON group at week 6. Micro-computed tomography (Micro-CT) showed significant bone degradation at proximal tibia 6 weeks after SAON induction. Angiography illustrated significantly less blood vessels in the Journal of Orthopaedic Translation (2018) 13, 13e24 proximal tibia and significantly more leakage particles in the distal tibia 2 weeks after SAON induction. Serum amino-terminal propeptide of type I collagen and osteocalcin were significantly lower at both 2 and 6 weeks after SAON induction, and serum carboxy-terminal telopeptide was significantly lower at 6 weeks after SAON induction. Histomorphometry revealed significantly lower osteoblast surface and higher marrow fat fraction and oedema area in SAON group. Hepatic oedema appeared 2 weeks after SAON induction, and lipid accumulation appeared in the liver of SAON rats 6 weeks after SAON induction. Conclusion: The present study successfully induced SAON in rats with pulsed injection of LPS and MPS, which was well simulating the clinical feature and pathology. Apart from available large animal models, such as bipedal emus or quadrupedal rabbits, our current SAON small model in rats could be a cost-effective preclinical experimental model to study body metabolism, molecular mechanism of SAON and potential drugs developed for prevention or treatment of SAON. The translational potential of this article: The present study successfully induced SAON in a small animal model in rats with pulsed injection of LPS and MPS. The evaluation protocols with typical histopathologic ON features and advanced evaluation approaches to identify the metabolic disorders of SAON could be used in future rat SAON studies. The SAON rat model is a suitable and cost-effective animal model to study molecular mechanism of SAON and potential drugs developed for prevention and treatment of SAON. Abstract and South Korea (2015). No specific vaccine has yet been reported against MERS. Purpose: To address the urgent need for an MERS vaccine, in the present study, we have designed two multi-epitope vaccines (MEVs) against MERS utilizing several in silico methods and tools. Methods: The design of both the multi-epitope vaccines (MEVs) are composed of cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes, screened form thirteen different proteins of MERS-CoV. Both the MEVs also carry potential B-cell linear epitope regions, B-cell discontinuous epitopes as well as interferon-γ-inducing epitopes. Human β-defensin-2 and β-defensin-3 were used as adjuvants to enhance the immune response of MEVs. To design the MEVs, short peptide molecular linkers were utilized to link screened most potential CTL epitopes, HTL epitopes and the adjuvants. Tertiary models for both the MEVs were generated, refined, and further studied for their molecular interaction with toll-like receptor 3. The cDNAs of both MEVs were generated and analyzed in silico for their expression in a mammalian host cell line (human). Results: Screened CTL and HTL epitopes were found to have high propensity for stable molecular interaction with HLA alleles molecules. CTL epitopes were also found to have favorable molecular interaction within the cavity of transporter associated with antigen processing. The selected CTL and HTL epitopes jointly cover upto 94.0% of worldwide human population. Both the CTL and HTL MEVs molecular models have shown to have stable binding and complex formation propensity with toll-like receptor 3. The cDNA analysis of both the MEVs have shown high expression tendency in mammalian host cell line (human). Conclusion: After multistage in silico analysis, both the MEVs are predicted to elicit humoral as well as cell mediated immune response. Epitopes of the designed MEVs are predicted to cover large human population worldwide. Hence both the designed MEVs could be tried in vivo as potential vaccine candidates against MERS. submit your manuscript | www.dovepress.com Dovepress Dovepress 2378 srivastava et al Abstract China's rapid and sustained economic growth offers an opportunity to ask whether the advantages of growth diffuse throughout an economy, or remain localized in areas where the growth has been the greatest. A critical policy area in China has been the health system, and health inequality has become an issue that has led the government to broaden national health insurance programs. This study investigates whether health system resources and performance have converged over the past 30 years across China's 31 provinces. To examine geographic variation of health system resources and performance at the provincial level, we measure the degree of sigma convergence and beta convergence in indicators of health system resources (structure), health services utilization (process), and outcome. All data are from officially published sources: the China Health Statistics Year Book and the China Statistics Year Book. Sigma convergence is found for resource indicators, whereas it is not observed for either process or outcome indicators, indicating that disparities only narrowed in health system resources. Beta convergence is found in most indicators, except for 2 procedure indicators, reflecting that provinces with poorer resources were catching up. Convergence found in this study probably reflects the mixed outcome of government input, and market forces. Thus, left alone, the equitable distribution of health care resources may not occur naturally during a period of economic growth. Governmental and societal efforts are needed to reduce geographic health variation and promote health equity. Abstract Major hurdles for survival after lung transplantation are rejections and infectious complications. Adequate methods for monitoring immune suppression status are lacking. Here, we evaluated quantification of torque teno virus (TTV) and Epstein-Barr virus (EBV) as biomarkers for defining the net state of immunosuppression in lung-transplanted patients.Methods. This prospective single-center study included 98 patients followed for 2 years after transplantation. Bacterial infections, fungal infections, viral respiratory infections (VRTI), cytomegalovirus (CMV) viremia, and acute rejections, as well as TTV and EBV levels, were monitored.Results. The levels of torque teno virus DNA increased rapidly after transplantation, likely due to immunosuppressive treatment. A modest increase in levels of Epstein-Barr virus DNA was also observed after transplantation. There were no associations between either TTV or EBV and infectious events or acute rejection, respectively, during follow-up. When Tacrolimus was the main immunosuppressive treatment, TTV DNA levels were significantly elevated 6-24 months after transplantation as compared with Cyclosporine treatment.Conclusions. Although replication of TTV, but not EBV, appears to reflect the functionality of the immune system, depending on the type of immunosuppressive treatment, quantification of TTV or EBV as biomarkers has limited potential for defining the net state of immune suppression. Abstract Highlights d ITZ, an antifungal and anticancer agent, is a broad-spectrum enterovirus inhibitor d OSBP and ORP4 are identified as novel targets of ITZ d ITZ binds OSBP and inhibits OSBP-mediated lipid exchange at membrane contact sites d ITZ also inhibits hepatitis C virus replication SUMMARY Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs. Abstract Data from all reported cases of 2009 pandemic influenza A (H1N1) were obtained from the China Information System for Disease Control and Prevention. The spatiotemporal distribution patterns of cases were characterized through spatial analysis. The impact of travel-related risk factors on invasion of the disease was analyzed using survival analysis, and climatic factors related to local transmission were identified using multilevel Poisson regression, both at the county level. The results showed that the epidemic spanned a large geographic area, with the most affected areas being in western China. Significant differences in incidence were found among age groups, with incidences peaking in school-age children. Overall, the epidemic spread from southeast to northwest. Proximity to airports and being intersected by national highways or freeways but not railways were variables associated with the presence of the disease in a county. Lower temperature and lower relative humidity were the climatic factors facilitating local transmission after correction for the effects of school summer vacation and public holidays, as well as population density and the density of medical facilities. These findings indicate that interventions focused on domestic travel, population density, and climatic factors could play a role in mitigating the public health impact of future influenza pandemics. Abstract Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in piglets. However, the biological characteristics of PDCoV are unclear. In this study, the hemagglutination (HA) abilities of two PDCoV strains (CH-01 and HNZK-04) were investigated. Our results showed that PDCoV has the ability to agglutinate rabbit erythrocytes after virion pretreatment with trypsin or neuraminidase. Additionally, the HA assay results showed a significant positive correlation with the infectious viral titer. Our results suggest that assessing the HA activity of PDCoV may be a useful diagnostic method for investigating and surveilling PDCoV infections. Abstract Despite consistent efforts to protect public health there is still a heavy burden of viral disease, both in the United States and abroad. In addition to conventional medical treatment, there is a need for a holistic approach for early detection and prevention of viral outbreaks at a population level. One-Health is a relatively new integrative approach to the solving of global health challenges. A key component to the One-Health approach is the notion that human health, animal health, and environmental health are all innately interrelated. One-Health interventions, initiated by veterinary doctors, have proven to be effective in controlling outbreaks, but thus far the applications focus on zoonotic viruses transmitted from animals to humans. Environmental engineers and environmental scientists hold a critical role in the further development of One-Health approaches that include water-related transport and transmission of human, animal, and zoonotic viruses. In addition to waterborne viruses, the proposed approach is applicable to a wide range of viruses that are found in human excrement since contaminated water-based surveillance systems may be used for early detection of viral disease. This paper proposes a greater One-Health based framework that involves water-related pathways. The first step in the proposed framework is the identification of critical exposure pathways of viruses in the water environment. Identification of critical pathways informs the second and third steps, which include water-based surveillance systems for early detection at a population level and implementation of intervention approaches to block the critical pathways of exposure. Abstract Respiratory pathogens are thought to be involved in the pathogenesis and exacerbations of asthma at all ages; however, little is known about the airway microbiome in the elderly.Aim of the study: To identify respiratory pathogens in the induced sputum (IS) of elderly asthmatics, and to determine the association between pathogens and the markers of asthma activity.Material and methods: Twenty-nine subjects with stable asthma, 15 above 65 years of age and 14 aged 30-49 years, underwent clinical evaluation, fractional exhaled nitric oxide measurement, and sputum induction. Pathogens were detected by multiplex reverse transcription polymerase chain reaction. The periostin concentration of IS supernatants was measured by enzyme-linked immunosorbent assay. Serum eosinophil cationic protein and total IgE levels were measured by ImmunoCAP.Conclusions: Despite the existence of differences in some clinical and inflammatory characteristics of asthma between elderly and non-elderly asthmatics, the pathogen detection rates in the IS from the two groups are similar. Abstract were analyzed. Results: Of 23,534 travelers, 61% were non-occupational and 39% occupational. Business travelers were more likely to be men, had short times to departure and shorter trip durations, and commonly refused influenza, meningococcal, and hepatitis B vaccines. Most business travelers indicated that employers suggested the pre-travel health consultation, whereas non-occupational travelers sought consultations because of travel health concerns. Conclusions: Sub-groups of occupational travelers have characteristic profiles, with business travelers being particularly distinct. Employers play a role in encouraging business travelers to seek pretravel consultations. Such consultations, even if scheduled immediately before travel, can identify vaccination gaps and increase coverage. Abstract The Pneumonia Etiology Research for Child Health (PERCH) project is the largest multicountry etiology study of childhood pneumonia since the Board on Science and Technology in International Development studies of the 1980s. However, it is not the only recent or ongoing pneumonia etiology study, and even with seven sites, it cannot capture all epidemiologic settings in the developing world. Funding providers, researchers and policymakers rely on the best available evidence to strategically plan programs, new research directions and interventions. We aimed to describe the current landscape of recent pneumonia etiology studies in children under 5 years of age in the developed and developing world, as ascertained by a literature review of relevant studies with data since the year 2000 and a survey of researchers in the field of childhood pneumonia. We collected information on the study population, study design, case definitions, laboratory samples and methods and identified pathogens. A literature review identified 88 studies with child pneumonia etiology results. As of June 2010, our survey of researchers identified an additional 65 ongoing and recently completed child pneumonia etiology studies. This demonstrates the broad existing context into which the PERCH study must be placed. However, the landscape analysis also reveals a multiplicity of case definitions, levels of clinician involvement, facility types, specimen collection, and laboratory techniques. It reinforces the need for the standardization of methods and analyses for present and future pneumonia etiology studies in order to optimize their cumulative potential to accurately describe the microbial causes of childhood pneumonia.Each year, approximately 1.6 million children die from pneumonia [1]. The Pneumonia Etiology Research for Child Health (PERCH) study is the largest multisite study of childhood pneumonia since the Board of Science and Technology for International Development (BOSTID) studies were done in the 1980s [2] . The goal of PERCH is to identify the expected etiologies of pneumonia in 2015, a time when the burden of the major causes of bacterial pneumonia in the developing world, Streptococcus pneumoniae and Haemophilus influenzae type b (Hib), will likely be significantly reduced by widespread introduction and use of conjugate vaccines. Moreover, PERCH capitalizes upon new molecular diagnostic techniques that were not available 2 decades ago when the BOSTID studies were carried out. Another salient difference between PERCH and the BOSTID studies is that the 7 sites participating in PERCH will follow a highly standardized protocol, which includes standardization of enrollment criteria, specimen collection, and laboratory testing.Although PERCH is the largest multicountry, childhood pneumonia etiology study in developing countries that has been conducted in the past 2 decades, it is not the only contemporary pneumonia etiology study, and cannot capture the entire complexity of all epidemiologic settings. In recent years, many developed and developing country sites have initiated pneumonia studies that provide etiology data. These studies will Abstract Accurate and rapid diagnosis of highly pathogenic avian influenza A H5N1 is of critical importance for the effective clinical management of patients. Here, we developed a rapid and simultaneous detection toolkit for influenza A H5 subtype viruses in human samples based on a bioconjugate of quantum dots (QDs) assembly and a smartphone-based rapid dual fluorescent diagnostic system (SRDFDS). Methods: Two types of QDs were assembled on a latex bead to enhance the detection sensitivity and specificity of influenza A infection (QD580) and H5 subtype (QD650). The dual signals of influenza A and H5 subtype of H5N1-infected patients were detected simultaneously and quantified separately by SRDFDS equipped with two emission filters. Results: Our results showed a high sensitivity of 92.86% (13/14) and 78.57% (11/14), and a specificity of 100% (38/38, P < 0.0001) and 97.37% (37/38) for influenza A and H5 subtype detection, respectively. Conclusion: Therefore, our multiplex QD bioconjugates and SRDFDS-based influenza virus detection toolkit potentially provide accurate and meaningful diagnosis information with improved detection accuracies and sensitivities for H5N1 patients. Abstract Supplementary material and methodsHepAD38 cells were kindly provided by Dr. Christoph Seeger, Fox Chase Center (USA) and cultured as described previously. HepG2-NTCP cells were provided by Dr. Wang-Shick Ryu, Yonsei University (South Korea) and were maintained as described previously [1]. Briefly, HepG2-NTCP were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% of fetal bovine serum (FBS), 5 mM L-Glutamine, 50 U/mL penicillin and 50 µg/mL streptomycin.For immunofluorescence analysis (IFA) at day 6 post-infection, cells were fixed overnight with 4% paraformaldehyde and permeabilized with 0.075% Triton X-100 in PBS and then blocked with 5% bovine serum albumin (BSA) in PBS at 4°C for 16 h. Cells were incubated with rabbit anti-HBc antibody (DAKO, USA) for 2 h, repeatedly washed and subsequently incubated with secondary antibody, anti-rabbit-IgG conjugated with Alexa Fluor 488. Cell nuclei were stained with Hoechst33342. 25 sub-layout images per well, covering an entire area of a single well, were captured by automated confocal microscopy OPERA (Perkin Elmer). Images were Abstract Background-The ongoing yellow fever (YF) epidemic in Angola strains the global vaccine supply, prompting WHO to adopt dose sparing for its vaccination campaign in Kinshasa in July-August 2016. Although a 5-fold fractional-dose vaccine is similar to standard-dose vaccine in safety and immunogenicity, efficacy is untested. There is an urgent need to ensure the robustness of fractional-dose vaccination by elucidating the conditions under which dose fractionation would reduce transmission.Methods-We estimate the effective reproductive number for YF in Angola using disease natural history and case report data. With simple mathematical models of YF transmission, we calculate the infection attack rate (IAR, the proportion of population infected over the course of an epidemic) under varying levels of transmissibility and five-fold fractional-dose vaccine efficacy for two vaccination scenarios: (i) random vaccination in a hypothetical population that is completely susceptible; (ii) the Kinshasa vaccination campaign in July-August 2016 with different age cutoff for fractional-dose vaccines.Findings-We estimate the effective reproductive number early in the Angola outbreak was between 5·2 and 7·1. If vaccine action is all-or-nothing (i.e. a proportion VE of vaccinees receives This manuscript version is made available under the CC BY-NC-ND 4.0 license.Correspondence to: Joseph T. Wu; Marc Lipsitch. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.JTW, CMP, and ML reviewed the literature and designed the study. JTW and ML developed the mathematical model. JTW ran the mathematical model. JTW, CMP, GML, and ML interpreted the model results and approved the final version.complete and the remainder receive no protection), n-fold fractionation can dramatically reduce IAR as long as efficacy VE exceeds 1/n. This benefit threshold becomes more stringent if vaccine action is leaky (i.e. the susceptibility of each vaccinee is reduced by a factor that is equal to the vaccine efficacy VE). The age cutoff for fractional-dose vaccines chosen by the WHO for the Kinshasa vaccination campaign (namely, 2 years) provides the largest reduction in IAR if the efficacy of five-fold fractional-dose vaccines exceeds 20%.Interpretation-Dose fractionation is a very effective strategy for reducing infection attack rate that would be robust with a large margin for error in case fractional-dose VE is lower than expected.Wu et al. Abstract Even though coronavirus infection of humans is not normally associated with severe diseases, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome showed that highly pathogenic coronaviruses can enter the human population. Shortly thereafter, in Holland in 2004, another novel human coronavirus (HCoV-NL63) was isolated from a seven-month old infant suffering from respiratory symptoms. This virus has subsequently been identified in various countries, indicating a worldwide distribution. HCoV-NL63 has been shown to infect mainly children and the immunocommpromised, who presented with either mild upper respiratory symptoms (cough, fever and rhinorrhoea) or more serious lower respiratory tract involvement such as bronchiolitis and croup, which was observed mainly in younger children. In fact, HCoV-NL63 is the aetiological agent for up to 10% of all respiratory diseases. This review summarizes recent findings of human coronavirus HCoV-NL63 infections, including isolation and identification, phylogeny and taxonomy, genome structure and transcriptional regulation, transmission and pathogenesis, and detection and diagnosis. Abstract The immunoglobulin-like family of CD66 antigens, present on human neutrophils and epithelial cells, are used as receptors for adhesins expressed by the pathogenic Neisseriae . N . gonorrhoeae strain MS11 can express 11 isoforms of these adhesins, called opacity-related (Opa) proteins. Each MS11 Opa protein recognizes a distinct spectrum of CD66 receptors. CD66-Opa binding is mediated by the NH 2 -terminal domain of the receptor and occurs through protein-protein interactions. In this report, we have investigated the molecular basis for the binding between the CD66 and Opa protein families by mapping amino acids in CD66 receptors that determine Opa protein binding. We performed homologue scanning mutagenesis between CD66e, which binds multiple Opa variants, and CD66b, which binds none, and tested both loss-offunction by CD66e and gain-of-function by CD66b in solution assays and in assays involving full-length receptors expressed by epithelial cells. We found that three residues in the CD66e N-domain are required for maximal Opa protein receptor activity. Opa proteins that recognize the same spectrum of native CD66 molecules showed differential binding of receptors with submaximal activity, indicating that the binding characteristics of these Opa proteins are actually slightly different. These data provide a first step toward resolving the structural requirements for Opa-CD66 interaction. Abstract Drug Design, Development and Therapy 2013:7 945-952 Drug Design, Development and Therapy Dovepress submit your manuscript | www.dovepress.com Dovepress 945 R e v i e w open access to scientific and medical research Open Access Full Text ArticleAbstract: Microneedles were first conceptualized for drug delivery many decades ago, overcoming the shortages and preserving the advantages of hypodermic needle and conventional transdermal drug-delivery systems to some extent. Dissolving and biodegradable microneedle technologies have been used for transdermal sustained deliveries of different drugs and vaccines. This review describes microneedle geometry and the representative dissolving and biodegradable microneedle delivery methods via the skin, followed by the fabricating methods. Finally, this review puts forward some perspectives that require further investigation. Abstract We report a case of virus-induced acute respiratory distress syndrome (ARDS) treated with parenteral vitamin C in a patient testing positive for enterovirus/rhinovirus on viral screening. This report outlines the first use of high dose intravenous vitamin C as an interventional therapy for ARDS, resulting from enterovirus/rhinovirus respiratory infection. From very significant preclinical research performed at Virginia Commonwealth University Abstract Context: Coptidis rhizome (CR), also known as Huanglian in Chinese, is the rhizome of Coptis chinensis Franch., C. deltoidea C.Y. Cheng et Hsiao, or C. teeta Wall (Ranunculaceae). It has been widely used to treat bacillary dysentery, diabetes, pertussis, sore throat, aphtha, and eczema in China. Objectives: The present paper reviews the latest advances of CR, focusing on the botany, phytochemistry, traditional usages, pharmacokinetics, pharmacology and toxicology of CR and its future perspectives. Methods: Studies from 1985 to 2018 were reviewed from books; PhD. and MSc. dissertations; the state and local drug standards; PubMed; CNKI; Scopus; the Web of Science; and Google Scholar using the keywords Coptis, Coptidis Rhizoma, Huanglian, and goldthread. Results: Currently, 128 chemical constituents have been isolated and identified from CR. Alkaloids are the characteristic components, together with organic acids, coumarins, phenylpropanoids and quinones. The extracts/compounds isolated from CR cover a wide pharmacological spectrum, including antibacterial, antivirus, antifungal, antidiabetic, anticancer and cardioprotective effects. Berberine is the most important active constituent and the primary toxic component of CR.Conclusions: As an important herbal medicine in Chinese medicine, CR has the potential to treat various diseases. However, further research should be undertaken to investigate the clinical effects, toxic constituents, target organs and pharmacokinetics, and to establish criteria for quality control, for CR and its related medications. In addition, the active constituents, other than alkaloids, in both raw and processed products of CR should be investigated.ARTICLE HISTORY Abstract Background: Respiratory syncytial virus (RSV) is recognized as a serious pathogen in people with chronic cardiopulmonary conditions. Immunoprophylaxis might be considered for adults at high-risk for frequent and severe RSV infection. Thus, we studied the incidence of RSV-related medically attended acute respiratory illness (MARI) in adults with severe chronic obstructive pulmonary disease (COPD) and/or congestive heart failure (CHF).Methods: Subjects ≥50 years of age with Gold Class III/IV COPD and/or American Heart Association class III/IV CHF and exposure to children ≥once per month were recruited. Subjects were evaluated over 1.5 to 2.5 years for RSV-associated MARI, defined as polymerase chain reaction (PCR) and/or seroresponse.Results: Four hundred forty-five subjects were enrolled between October 2011 and May 2012. Overall, 99 RSV infections were documented by PCR or serology for a cumulative incidence of 22.2%. Of these, 42 (9.4%) subjects had protocol-specified RSV-MARI for an incidence of 4.68/100 patient-seasons. All-cause MARI was common (63.85/100 patient-seasons) with rhinovirus most commonly identified.Conclusion: RSV infection was common in adults with severe COPD and/or advanced CHF. Given the severity of underlying cardiopulmonary diseases in the study population, most illnesses were surprisingly mild. Thus, active immunization rather than passive immunoprophylaxis with monoclonal antibodies may be a more cost-effective strategy.disease control, epidemiology, immunoprophylaxis, respiratory syncytial virus (RSV), seasonal incidence, virus classification Abstract The influence of CD25 + CD4 + regulatory T cells (Treg) on acute and chronic viral infection of the central nervous system (CNS) was examined using a glial tropic murine coronavirus. Treg in the CNS were highest during initial T cell mediated virus control, decreased and then remained relatively stable during persistence. Anti-CD25 treatment did not affect CNS recruitment of inflammatory cells. Viral control was initially delayed; however, neither the kinetics of viral control nor viral persistence were affected. By contrast, the absence of Treg during the acute phase resulted in increased demyelination during viral persistence. These data suggest that CNS inflammation, progression of viral control and viral persistence are relatively independent of CD25 + CD4 + Treg. However, their absence during acute infection alters the ability of the host to limit tissue damage. Abstract We implemented a carbapenem-saving strategy in hemato-oncology patients from 2013, using an empirical combination of piperacillin-tazobactam and amikacin for high-risk hemato-oncology patients with febrile neutropenia, who remain hemodynamically unstable > 72 hours despite initial cefepime treatment. All-cause mortality was not different between the two periods (6.54 and 6.57 deaths per 1,000 person-day, P = 0.926). Group 2 carbapenem use significantly decreased after strategy implementation (78.43 vs. 67.43 monthly days of therapy, P = 0.018), while carbapenem-resistant gram-negative bacilli did not show meaningful changes during the study period. Our carbapenem-saving strategy could effectively suppress carbapenem use without an increase of overall mortality.To reduce unnecessary antibiotic use and improve outcomes, development of facility-specific guidelines for fever and neutropenia in hemato-oncology patients is required. 1 Our center, a 1,950-bed tertiary care hospital, used cefepime as an initial empirical antibiotic for hematooncology patients with febrile neutropenia according to the Infectious Diseases Society of America. 2 Empirical antibiotics were escalated to group 2 carbapenems (Gr2Cs) for highrisk patients who remain hemodynamically unstable despite initial treatment. 2 However, this approach raised concerns about carbapenem overuse. From 2013, we implemented a carbapenem-saving strategy using an empirical combination of piperacillin-tazobactam and amikacin (PTZ/AMK) as a bridging regimen before using Gr2C. To evaluate the effects of this strategy, we reviewed antibiotic use, antimicrobial resistance, and overall mortality before and after strategy implementation.Our strategy was based on in vitro data showing that PTZ/AMK combination covered 92.8% of extended spectrum β-lactamase-producing Escherichia coli isolates. 3 Since aminoglycosides were rarely used in our hospital, amikacin susceptibility was also relatively well preserved Abstract Graphical Abstract Highlights d Influenza A virus PA-X targets the majority of host mRNAs for destruction d Downregulation by PA-X correlates with the number of splice sites in a transcript d Splicing renders RNAs susceptible to PA-X d The cellular CFIm complex interacts with PA-X and contributes to PA-X activity Correspondence craig.mccormick@dal.ca (C.M.), d.khaperskyy@dal.ca (D.A.K.), marta.gaglia@tufts.edu (M.M.G.) In Brief Gaucherand et al. uncover a unique relationship between RNA degradation by the influenza A virus ribonuclease PA-X and host RNA splicing, which allows PA-X to selectively target host RNAs for destruction. Abstract We investigated the sensitivity of human rotavirus rapid antigen detection (RAD) kits, RT-PCR and next-generation DNA sequencing (NGS) for detection of bovine group A rotavirus (RVA). The Dipstick 'Eiken' Rota (Dipstick) showed the highest sensitivity out of the seven RAD kits against all selected strains in limited dilution analyses, which was consistent with the results for equine rotavirus previously reported. RT-PCR had 10 0 -10 3 -fold higher sensitivity than the Dipstick. NGS using thirteen RT-PCR-negative fecal samples revealed that all samples yielded RVA reads and especially that two of them covered all 11 genome segments. Moreover, mapping reads to reference sequences allowed genotyping. The NGS would be sensitive and useful for analysis of less dependent on specific primers and screening of genotypes. Abstract Highlights d ISG screening identifies direct and indirect antiretroviral proteins d Interferon-g inhibits HIV-1 through IDO1-mediated tryptophan depletion d TRIM56 enhances the antiretroviral potential of interferon-a Abstract Emerging infectious diseases continue to impose unpredictable burdens on global health and economy. Infectious disease surveillance and pandemic preparedness are essential to mitigate the impact of future threats. Global surveillance networks provide unprecedented monitoring data on plant, animal and human infectious diseases. Using such sources, we report on current major One Health threats and update on their epidemiological status. Abstract We present a unique and informative instance of respiratory syncytial virus (RSV) infection associated with antiphospholipid syndrome (APS), and discuss this case in the context of the literature addressing the immunopathogenesis of APS associated with diverse infections. We describe the case of a 43-yearold man with no significant past medical history who presented with the acute onset of fever, hemoptysis, and extensive bullous, ecchymotic lesions in both lower extremities. Punch biopsy of the lesion demonstrated thrombotic vasculopathy. Further evaluation revealed serum antiphospholipid antibodies as well as a positive RSV PCR in a nasal swab specimen. Clinical manifestations, positive laboratory and pathological findings were strongly suggestive of APS associated with a recent RSV infection. When an infectious etiology is considered for APS, RSV should also be included in the differential diagnosis.2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).We present what, to our knowledge, is the first reported case of antiphospholipid syndrome (APS) associated with Respiratory Syncytial Virus (RSV) infection. APS is a disorder characterized by vascular thrombosis, or pregnancy-related morbidity, in the presence of antiphospholipid (aPL) antibodies [1]. APS is classified as either primary or secondary, and secondary APS is associated with autoimmune disease, infection, medication or malignancy [2] . RSV is a common causative agent of acute viral upper and lower respiratory tract infection throughout all age groups. Despite the common occurrence of RSV respiratory infections, an association or possible causal relationship between RSV and APS has not to our knowledge been discussed previously. We report a case where progressive, extensive ecchymoses appeared to be the initial manifestation of APS occurring in temporal association with, and seemingly triggered by, a recent episode of RSV infection.A 43-year-old male with no significant past medical history presented during the month of December with a 4-day history of fever (maximum temperature 38.8 C), shaking chills, hemoptysis and worsening dyspnea. He had experienced intermittent night sweats, pain and swelling of both wrists and ankle joints over the last several months. He also reported a 10 kg weight loss during this time, but never sought medical attention. He had noted a painful ecchymotic lesion on his left calf one day prior to the admission. Over the subsequent 24 h, the patient developed similar lesions on his abdomen, back, right upper arm and right calf. The patient's 3 year old son had recently had a respiratory illness.On physical examination, the patient had a temperature of 35.3 C, blood pressure of 117/75 mmHg, pulse rate of 112 beats/ min, respiratory rate of 29 breaths/min, and an oxygen saturation of 96% on room air. There were bilateral lung wheezes, and multiple painful ecchymosis with several hemorrhagic bullae over his trunk and extremities (Fig. 1) . CBC on admission showed WBC 9000/mm3 (87% neutrophils) and Hemoglobin 13.4 g/dL, and comprehensive metabolic panel including electrolytes, liver and renal function was all within normal range. The significant laboratory data included high lactate dehydrogenase of 799 IU/L, erythrocyte sedimentation rate of 81 mm/h, and C-reactive protein level of 38.5 mg/dL. The Xray and computed tomography (CT) of the * Corresponding author. Present address: Azumabashi 1-23-30, River Pier Azumabashi Room 2707, Sumida-ku, Abstract Rapid vaccine development in response to an outbreak of a new emerging infectious disease (EID) is a goal targeted by public health agencies worldwide. This goal becomes more complicated when there are no standardized sets of viral and immunological assays, no accepted and well-characterized samples, standards or reagents, and no approved diagnostic tests for the EID pathogen. The diagnosis of infections is of critical importance to public health, but also in vaccine development in order to track incident infections during clinical trials, to differentiate natural infection responses from those that are vaccine-related and, if called for by study design, to exclude subjects with prior exposure from vaccine efficacy trials. Here we review emerging infectious disease biological standards development, vaccine clinical assay development and trial execution with the recent experiences of MERS-CoV and Zika virus as examples. There is great need to establish, in advance, the standardized reagents, sample panels, controls, and assays to support the rapid advancement of vaccine development efforts in response to EID outbreaks.ARTICLE HISTORY Abstract Non-structural protein 1 (NS1) is an essential virulence factor of the highly pathogenic H5N1 avian influenza virus and of the apoptosis associated with the pathogenesis of H5N1. Previous studies have revealed that the NS1 protein is able to induce apoptosis via an extrinsic pathway. However, it remains unclear whether the intrinsic pathway is also associated with this apoptosis. The present study used a clone of the NS1 gene from avian influenza A/Jiangsu/1/2007 and observed the localization of the NS1 protein and cytochrome c release from mitochondria and the change of mitochondrial membrane potential (MMP) in lung cancer cells. Cytotoxicity was detected using an MTT assay and the number of apoptotic cells was counted using a flow cytometer. Following the isolation of mitochondria, western blotting was performed to compare cytochrome c release from the mitochondria in cells before and after apoptosis. The change of MMP was detected using JC-1 staining. Furthermore, the results reveal that the majority of the NS1 protein was localized in the cell nucleus, and that it may induce apoptosis of human lung epithelial cells. The apoptosis occurred with marked cytochrome c release from mitochondria and a change of the MMP. This indicated that the NS1 protein may be associated with apoptosis induced by an intrinsic mitochondrial pathway. Abstract Studies of gene fusions in solid tumors are not as extensive as in hematological malignancies due to several technical and analytical problems associated with tumor heterogeneity. Nevertheless, there is a growing interest in the role of fusion genes in common epithelial tumors after the discovery of recurrent TMPRSS2:ETS fusions in prostate cancer. Among all of the reported fusion partners in the ETS gene family, TMPRSS2:ERG is the most prevalent one. Here, we present a simple and sensitive microarray-based assay that is able to simultaneously determine multiple fusion variants with a single RT-PCR in impure RNA specimens. The assay detected TMPRSS2:ERG fusion transcripts with a detection sensitivity of _10 cells in the presence of more than 3000 times excess normal RNA, and in primary prostate tumors having no`1% of cancer cells. The ability to detect multiple transcript variants in a single assay is critically dependent on both the primer and probe designs. The assay should facilitate clinical and basic studies for fusion gene screening in clinical specimens, as it can be readily adapted to include multiple gene loci. Abstract To introduce and summarize current research in the field of Public Health and Epidemiology Informatics. Methods: The 2017 literature concerning public health and epidemiology informatics was searched in PubMed and Web of Science, and the returned references were reviewed by the two section editors to select 14 candidate best papers. These papers were then peer-reviewed by external reviewers to provide the editorial team with an enlightened vision to select the best papers. Results: Among the 843 references retrieved from PubMed and Web of Science, two were finally selected as best papers. The first one analyzes the relationship between the disease, social/ mass media, and public emotions to understand public overreaction (leading to a noticeable reduction of social and economic activities) in the context of a nation-wide outbreak of Middle East Respiratory Syndrome (MERS) in Korea in 2015. The second paper concerns a new methodology to de-identify patient notes in electronic health records based on artificial neural networks that outperformed existing methods. Conclusions: Surveillance is still a productive topic in public health informatics but other very important topics in Public Health are appearing. For example, the use of artificial intelligence approaches is increasing. Abstract To develop a more effective vaccination strategy for reducing the impact of respiratory syncytial virus (RSV) infection, especially in young infants (<6 months old), it is necessary to understand the transmission dynamics of RSV.Methods. We conducted a community-based prospective cohort study from 2014 to 2016 in Biliran Province, the Philippines, on children <5 years old. We collected nasopharyngeal swabs from symptomatic children with acute respiratory infection (ARI) during household visits and at health facilities. In households (n = 181) with RSV-positive ARI cases (RSV-ARI), we also identified ARI episodes among other children <5 years old in the same household. In addition, we determined the serial interval to estimate the basic reproduction number (R 0 ), the average number of secondary cases generated by a single primary case.Results. In the 181 households analyzed, we found 212 RSV-ARI in 152 households with a single case and 29 households with multiple cases, which included 29 1st RSV-ARI and 31 2nd RSV-ARI. We also found possible index cases among children <5 years old in the same household for 29.0% (18 of 62) of young infants with RSV-ARI. The estimated mean serial interval was 3.2 days, and R 0 was estimated to be 0.92-1.33 for RSV-A and 1.04-1.76 for RSV-B, which varied between different times (2014 and 2015) and places.Conclusions. Young infants are likely to acquire RSV infection from older children in the same household. Therefore, vaccination targeting older children might protect infants from RSV infection. Abstract We have combined in vivo and in vitro approaches to investigate the function of CENP-B, a major protein of human centromeric heterochromatin . Expression of epitope-tagged deletion derivatives of CENP-B in HeLa cells revealed that a single domain <158 residues from the amino terminus of the protein is sufficient to localize CENP-B to centromeres. Centromere localization was abolished if as few as 28 amino acids were removed from the amino terminus of CENP-B. The centromere localization signal of CENP-B T HE centromere (primary constriction) of human mitotic chromosomes consists ofa compacted network of heterochromatin containing highly repeated satellite DNA sequences and their associated proteins. The kinetochore, the site of microtubule attachment to the chromosomes (Luykx, 1965 ; Brinkley and Stubblefield, 1966; Jokelainen, 1967) , is situated at the surface of the centromeric heterochromatin . The kinetochore is now believed to be the location of the mechanochemical motors (such as dynein) (Pfarr et al ., 1990 ; Steuer et al., 1990; Wordernan et al., 1991) that move chromosomes in mitosis . Both gene expression and recombination are strongly suppressed in regions of heterochromatin (Brown, 1966; Henikoff, 1981; Daniels et al., 1986) . The interrelationship between the kinetochore and the underlying heterochromatin is unknown .Centromeric heterochromatin is largely composed of a-satellite DNA, a highly polymorphic family of repetitive DNA sequences that comprises N5 % of the human genome (Willard and Waye, 1987) . The role ofa-satellite DNA in human chromosome structure and function is not known . The consensus monomer for a-satellite DNA (171 by in length) is typically found in higher-order repeats that may be as large as 35-mers (Choo et al., 1991). Within a repeat, adjacent monomers commonly show up to 40% sequence divergence (Willard and Waye, 1987) . Arrays ofa-satellite DNA apparently span contiguous regions of the centromeric DNA of two to three megabases (Wevrick and Willard, 1989 ; Jabs et al., 1989) .In humans, only a single heterochromatin protein has been identified to date. This protein, CENP-B, was originally discovered when centromeric autoantigens (termed CENP antigens) recognized by sera of patients with scleroderma spectrum disease were identified by immunoblotting (Earnshaw Abstract Beginning in 2003, highly pathogenic avian influenza (HPAI) H5N1 virus spread across Southeast Asia, causing unprecedented epidemics. Thailand was massively infected in 2004 and 2005 and continues today to experience sporadic outbreaks. While research findings suggest that the spread of HPAI H5N1 is influenced primarily by trade patterns, identifying the anthropogenic risk factors involved remains a challenge. In this study, we investigated which anthropogenic factors played a role in the risk of HPAI in Thailand using outbreak data from the ''second wave'' of the epidemic (3 July 2004 to 5 May 2005) in the country. We first performed a spatial analysis of the relative risk of HPAI H5N1 at the subdistrict level based on a hierarchical Bayesian model. We observed a strong spatial heterogeneity of the relative risk. We then tested a set of potential risk factors in a multivariable linear model. The results confirmed the role of freegrazing ducks and rice-cropping intensity but showed a weak association with fighting cock density. The results also revealed a set of anthropogenic factors significantly linked with the risk of HPAI. High risk was associated strongly with densely populated areas, short distances to a highway junction, and short distances to large cities. These findings highlight a new explanatory pattern for the risk of HPAI and indicate that, in addition to agro-environmental factors, anthropogenic factors play an important role in the spread of H5N1. To limit the spread of future outbreaks, efforts to control the movement of poultry products must be sustained.avian influenza / epidemiology / poultry farming / spatial analysis / Thailand Abstract Contact networks are convenient models to investigate epidemics, with nodes and links representing potential hosts and infection pathways, respectively. The outcomes of outbreak simulations on networks are driven both by the underlying epidemic model, and by the networks' structural properties, so that the same pathogen can generate different epidemic dynamics on different networks. Here we ask whether there are general properties that make a contact network intrinsically vulnerable to epidemics (that is, regardless of specific epidemiological parameters). By conducting simulations on a large set of modelled networks, we show that, when a broad range of network topologies is taken into account, the effect of specific network properties on outbreak magnitude is stronger than that of fundamental pathogen features such as transmission rate, infection duration, and immunization ability. Then, by focusing on a large set of real world networks of the same type (potential contacts between field voles, Microtus agrestis), we showed how network structure can be used to accurately assess the relative, intrinsic vulnerability of networks towards a specific pathogen, even when those have limited topological variability. These results have profound implications for how we prevent disease outbreaks; in many real world situations, the topology of host contact networks can be described and used to infer intrinsic vulnerability. Such an approach can increase preparedness and inform preventive measures against emerging diseases for which limited epidemiological information is available, enabling the identification of priority targets before an epidemic event. Abstract The Middle East respiratory syndrome (MERS) is proposed to be a zoonotic disease; however, the reservoir and mechanism for transmission of the causative agent, the MERS coronavirus, are unknown. Dromedary camels have been implicated through reports that some victims have been exposed to camels, camels in areas where the disease has emerged have antibodies to the virus, and viral sequences have been recovered from camels in association with outbreaks of the disease among humans. Nonetheless, whether camels mediate transmission to humans is unresolved. Here we provide evidence from a geographic and temporal survey of camels in the Kingdom of Saudi Arabia that MERS coronaviruses have been circulating in camels since at least 1992, are distributed countrywide, and can be phylogenetically classified into clades that correlate with outbreaks of the disease among humans. We found no evidence of infection in domestic sheep or domestic goats.Arabia, where the index case and the majority of cases of MERS have been reported. Citation Alagaili AN, Briese T, Mishra N, Kapoor V, Sameroff SC, de Wit E, Munster VJ, Hensley LE, Zalmout IS, Kapoor A, Epstein JH, Karesh WB, Daszak P, Mohammed OB, Lipkin WI. 2014. Middle East respiratory syndrome coronavirus infection in dromedary camels in Saudi Arabia. mBio 5(2):e00884-14. Abstract Developing and delivering appropriate, affordable, well-adapted medicines for HIV/AIDS remains an urgent challenge: as first-line therapies fail, increasing numbers of people require costly second-line therapy; one-third of ARVs are not available in pediatric formulations; and certain key first-and second-line triple fixed-dose combinations do not exist or sufficient suppliers are lacking. UNITAID aims to help solve these problems through an innovative initiative for the collective management of intellectual property (IP) rights -a patent pool for HIV medicines. The idea behind a patent pool is that patent holders -companies, governments, researchers or universities -voluntarily offer, under certain conditions, the IP related to their inventions to the patent pool. Any company that wants to use the IP to produce or develop medicines can seek a license from the pool against the payment of royalties, and may then produce the medicines for use in developing countries (conditional upon meeting agreed quality standards). The patent pool will be a voluntary mechanism, meaning its success will largely depend on the willingness of pharmaceutical companies to participate and commit their IP to the pool. Generic producers must also be willing to cooperate. The pool has the potential to provide benefits to all. Abstract Protein localization to the TGN was inves-1. Abbreviations used in this paper: CGN, cis-Golgi network; DPAP A, dipeptidyl aminopeptidase A; TGN, trans-Golgi network. Abstract Over the past few years, nucleosides have maintained a prominent role as one of the cornerstones of antiviral and anticancer therapeutics, and many approaches to nucleoside drug design have been pursued. One such approach involves flexibility in the sugar moiety of nucleosides, for example, in the highly successful anti-HIV and HBV drug tenofovir. In contrast, introduction of flexibility to the nucleobase scaffold has only more recently gained significance with the invention of our fleximers. The history, development, and some biological relevance for this innovative class of nucleosides are detailed herein. Abstract Purpose: Sexually transmitted infections (STIs), representing a major global health problem, are caused by different microbes, including bacteria, viruses, and protozoa. Unfortunately, infections of different sexually transmitted pathogens often present similar clinical symptoms, so it is almost impossible to distinguish them clinically. Therefore, the aim of the current study was to develop a sensitive, multitarget, and high-throughput method that can detect various agents responsible for STIs.Methods: We developed and tested a 23-plex PCR coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) assay (sexually transmitted infection-mass spectrometry, STI-MS) that simultaneously targets 11 different agents, including 8 most common clinical pathogens related to STIs (HSV-1, HSV-2, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, and Haemophilus ducreyi) and 3 controversial microorganisms as pathogens (Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum).The results showed that the STI-MS approach can accurately detect the expected agents, without cross-reaction with other organisms. The limit of detection of each STI-MS assay was ranged from 1.739 to 10.009 copies/reaction, using probit analyses. The verification rate for each target organism of the STI-MS ranged from a minimum of 89.3% to a maximum of 100%, using conventional assays and ultrasensitive digital PCR to confirm the STI-MS-positive results. To further evaluate the clinical performance of this assay, 241 clinical specimens (124 urethral/cervical swabs and 117 urine) were tested in parallel using the STI-MS assay and monoplex real-time PCR for each agent. The overall validation parameters of STI-MS were extremely high including sensitivity (from 85.7% to 100%), specificity (from 92.3% to 100%), PPV (from 50% to 100%), and NPV (from 99.1% to 100%) for each target. Conclusion: STI-MS is a useful high-throughput screening tool for detecting mixed infections of STIs and has great potential for application in large-scale epidemiological programs for specific microorganisms of STI. Abstract Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic viral disease with a disease fatality rate between 15% and 70%. Despite the wide range of distribution, the virus (CCHFV) is basically endemic in Africa, Asia, eastern Europe, and the Middle East. Acute febrile illness associated with petechiae, disseminated intravascular coagulation, and multiple-organ failure are the main symptoms of the disease. With all these fatal effects, CCHFV is considered a huge threat as no successful therapeutic approach is currently available for the treatment of this disease. In the present study, we have used the immunoinformatics approach to design a potential epitope-based vaccine against the RNA-dependent RNA polymerase-L of CCHFV. Both the T-cell and B-cell epitopes were assessed, and the epitope "DCSSTPPDR" was found to be the most potential one, with 100% conservancy among all the strains of CCHFV. The epitope was also found to interact with both type I and II major histocompatibility complex molecules and is considered nonallergenic as well. In vivo study of our proposed peptide is advised for novel universal vaccine production, which might be an effective path to prevent CCHF disease. Abstract Bovine Respiratory Disease is the most costly disease that affects beef and dairy cattle industry. Its etiology is multifactorial, arising from predisposing environmental stress conditions as well as the action of several different respiratory pathogens. This situation has hindered the development of effective control strategies. Although different type of vaccines are available, many currently marketed vaccines are based on inactivated cultures of the main viral and bacterial agents involved in this pathology. The molecular composition of commercial veterinary vaccines is a critical issue. The present work aims to define at the proteomic level the most relevant valence of a line of commercial respiratory vaccines widely used in Central and South America. Since Mannheimia haemolytica is responsible for most of the disease associated morbid-mortality, we focused on the main proteins secreted by this pathogen, in particular Leukotoxin A, its main virulence factor. By Western blot analysis and mass spectrometry, Leukotoxin A was identified as a major component of (http://creativecommons.org/licenses/by-nc-nd/4.0/). M. haemolytica culture supernatants. We also identified other ten M. haemolytica proteins, including outer membrane proteins, periplasmic transmembrane solute transporters and iron binding proteins, which are relevant to achieve protective immunity against the pathogen. This work allowed a detailed molecular characterization of this vaccine component, providing evidence of its quality and efficacy. Furthermore, our results contributed to the identification of several proteins of interest as subunit vaccine candidates. Abstract Taiwan, has undergone several phases of modification in structure and activation. The main organizing principles of the CDCMN are centralized isolation of patients with severe highly infectious diseases and centralization of medical resources, as well as a network of designated regional hospitals like those in other countries. The CDCMN is made up of a command system, responding hospitals, and supporting hospitals. It was tested and activated in response to the H1N1 influenza pandemic in 2009-10 and the Ebola outbreak in West Africa in 2014-2016, and it demonstrated high-level functioning and robust capacity. In this article, the history, structure, and operation of the CDCMN is introduced globally for the first time, and the advantages and challenges of this system are discussed. The Taiwanese experience shows an example of a collaboration between the public health system and the medical system that may help other public health authorities plan management and hospital preparedness for highly infectious diseases. Abstract Purpose: Chest radiography is often performed on patients hospitalized with typical clinical manife stations of bronchiolitis. We aimed to determine the proportion of subjects with pathologic chest radio graphic findings and the clinical predictors associated with pathologic chest radiographic findings in young children admitted with the typical presentation of bronchiolitis. Methods: We obtained the following data at admission: sex, age, neonatal history, past history of hospitalization for respiratory illnesses, heart rate, respiratory rate, the presence of fever, total duration of fever, oxygen saturation, laboratory parameters (i.e., complete blood cell count, highsensitivity Creactive protein [hsCRP], etc.), and chest radiography. Results: The study comprised 279 young children. Of these, 26 had a chest radiograph revealing opacity (n=24) or atelectasis (n=2). Multivariate logistic regression analysis showed that after adjustment for confounding factors, the clinical predictors associated with pathologic chest radiographic findings in young children admitted with bronchiolitis were elevated hsCRP level (>0.3 mg/dL) and past history of hospitalization for respiratory illnesses (all P<0.05). Conclusion: The current study suggests that chest radiographs in young children with typical clinical manifestations of bronchiolitis have limited value. Nonetheless, young children with clinical factors such as high hsCRP levels at admission or past history of hospitalization for respiratory illnesses may be more likely to have pathologic chest radiographic findings. Abstract Citation Klotz LC. 2015. Comments on Fouchier's calculation of risk and elapsed time for escape of a laboratory-acquired infection from his laboratory. mBio 6(2):e00268-15. Abstract The targeting signals of two yeast integral membrane dipeptidyl aminopeptidases (DPAPs), DPAP B and DPAP A, which reside in the vacuole and the Golgi apparatus, respectively, were analyzed. No single domain of DPAP B is required for delivery to the vacuolar membrane, because removal or replacement of either the cytoplasmic, transmembrane, or lumenal domain did not affect the protein's transport to the vacuole. DPAP A was localized by indirect immunofluorescence to non-vacuolar, punctate structures characteristic of the yeast Golgi apparatus. The 118-amino acid cytoplasmic domain of DPAP A is sufficient for retention of the protein in these structures, since replacement of the cytoplasmic domain of DPAP B with that of DPAP A resulted in an immunolocalization pattern indistinguishable from that of wild type DPAP A. Overproduction of DPAP A resulted in its mislocaliza-tion to the vacuole, because cells expressing high levels of DPAP A exhibited vacuolar as well as Golgi staining. Deletion of 22 residues of the DPAP A cytoplasmic domain resulted in mislocaiization of the mutant protein to the vacuole. Thus, the cytoplasmic domain of DPAP A is both necessary and sufficient for Golgi retention, and removal of the retention signal, or saturation of the retention apparatus by overproducing DPAP A, resulted in transport to the vacuole. Like wild type DPAP B, the delivery of mutant membrane proteins to the vacuole was unaffected in the secretory vesicle-blocked secl mutant; thus, transport to the vacuole was not via the plasma membrane followed by endocytosis. These data are consistent with a model in which membrane proteins are delivered to the vacuole along a default pathway. Abstract Citation Alagaili AN, Briese T, Karesh WB, Daszak P, Lipkin WI. 2014. Reply to "Concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of Middle East respiratory syndrome (MERS)." mBio 5(4):e01482-14. Abstract chain variable fragment; VH, variable region of the heavy chain; VL, variable region of the light chain the development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected "LYmph Node Derived Antibody Libraries" (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. to demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv) 2 . We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential. www.landesbioscience.com mAbs 131 RepoRt RepoRt Abstract Emerging and reemerging diseases that result from pathogen host shifts are a threat to the health of humans and their domesticates. RNA viruses have extremely high mutation rates and thus represent a significant source of these infectious diseases. In the present study, we showed that a plant-pathogenic RNA virus, tobacco ringspot virus (TRSV), could replicate and produce virions in honeybees, Apis mellifera, resulting in infections that were found throughout the entire body. Additionally, we showed that TRSV-infected individuals were continually present in some monitored colonies. While intracellular life cycle, species-level genetic variation, and pathogenesis of the virus in honeybee hosts remain to be determined, the increasing prevalence of TRSV in conjunction with other bee viruses from spring toward winter in infected colonies was associated with gradual decline of host populations and winter colony collapse, suggesting the negative impact of the virus on colony survival. Furthermore, we showed that TRSV was also found in ectoparasitic Varroa mites that feed on bee hemolymph, but in those instances the virus was restricted to the gastric cecum of Varroa mites, suggesting that Varroa mites may facilitate the spread of TRSV in bees but do not experience systemic invasion. Finally, our phylogenetic analysis revealed that TRSV isolates from bees, bee pollen, and Varroa mites clustered together, forming a monophyletic clade.IMPORTANCE Pathogen host shifts represent a major source of new infectious diseases. Here we provide evidence that a pollenborne plant virus, tobacco ringspot virus (TRSV), also replicates in honeybees and that the virus systemically invades and replicates in different body parts. In addition, the virus was detected inside the body of parasitic Varroa mites, which consume bee hemolymph, suggesting that Varroa mites may play a role in facilitating the spread of the virus in bee colonies. This study represents the first evidence that honeybees exposed to virus-contaminated pollen could also be infected and raises awareness of potential risks of new viral disease emergence due to host shift events. About 5% of known plant viruses are pollen transmitted, and these are potential sources of future host-jumping viruses. The findings from this study showcase the need for increased surveillance for potential host-jumping events as an integrated part of insect pollinator management programs. YP. 2014. Systemic spread and propagation of a plant-pathogenic virus in European honeybees, Apis mellifera. mBio 5(1):e00898-13. Abstract The effect of environmental acidification on Ibaraki virus (IBAV) infection was tested using endosomal inhibitory chemicals and low pH treatment. Treatment of target cells with endosomal inhibitors significantly decreased the progeny virus production. IBAV outer capsid proteins, VP5 and VP2, were removed from virion when purified IBAV was exposed to low pH environment. Further experiment showed that the exposure to low pH buffer facilitated IBAV infection when the cellular endosomal pathway was impaired by bafilomycin A1. Results obtained in this study suggest that acidic environment is essential to initiate IBAV infection. Abstract For peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open For peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open +33 549 444 600 WORD COUNT 4120 words ABSTRACT Introduction: Severe trauma represents the leading cause of mortality worldwide. While 80% of deaths occur within the first 24 hours after trauma, 20% occur later and are mainly due to healthcare-associated infections, including ventilator-associated pneumonia (VAP).Preventing underinflation of the tracheal cuff is recommended to reduce microaspiration, which play a major role in the pathogenesis of VAP. Automatic devices facilitate the regulation of tracheal cuff pressure, and their implementation has the potential to reduce VAP. The objective of this work is to determine whether continuous regulation of tracheal cuff pressure using a pneumatic device reduces the incidence of VAP compared to intermittent control in severe trauma patients.This multicentre, randomised, controlled and open-label trial will include patients suffering from severe trauma that are admitted within the first 24 hours, who require invasive mechanical ventilation to longer than 48 hours. Their tracheal cuff pressure will be monitored either once every 8 hours (control group) or continuously using a pneumatic device (intervention group). The primary endpoint is the proportion of patients that develop VAP in the intensive care unit (ICU) at day-28. The secondary endpoints include the proportion of patients that develop VAP in the ICU, early (≤7 days) or late (>7 days) VAP, time until the first VAP diagnosis, the number of ventilator-free days and antibiotic-free days, the length of stay in the ICU, the proportion of patients with ventilator associated events and that die during their ICU stay. Ethics and dissemination: This protocol has been approved by the ethics committee of Poitiers University Hospital, and will be carried out according to the principles of the Declaration of Helsinki and the Good Clinical Practice guidelines. The results of this study will For peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open pneumonia in mechanically ventilated severe trauma patients: the AGATE study protocol Abstract This Perspectives in Veterinary Medicine article seeks to define, describe putative causes, and discuss key diagnostic tests for primary and secondary bronchiolar disorders to propose a classification scheme in cats with support from a literature review and case examples. The small airways (bronchioles with inner diameters <2 mm), located at the transitional zone between larger conducting airways and the pulmonary acinus, have been overlooked as major contributors to clinical syndromes of respiratory disease in cats. Because the trigger for many bronchiolar disorders is environmental and humans live in a shared environment with similar susceptibility, understanding these diseases in pet cats has relevance to One Health. Thoracic radiography, the major imaging modality used in the diagnostic evaluation of respiratory disease in cats, has low utility in detection of bronchiolar disease. Computed tomography (CT) with paired inspiratory and expiratory scans can detect pathology centered on small airways. In humans, treatment of bronchiolar disorders is not well established because of heterogeneous presentations and often late definitive diagnosis. A review of the human and veterinary medical literature will serve as the basis for a proposed classification scheme in cats. A case series of cats with CT or histopathologic evidence of bronchiolar lesions or both, either as a primary disorder or secondary to extension from large airway disease or interstitial lung disease, will be presented. Future multi-institutional and multidisciplinary discussions among clinicians, radiologists, and pathologists will help refine and develop this classification scheme to promote early and specific recognition and optimize treatment. Abstract Background: Extracorporeal membrane oxygenation (ECMO) has been successfully used as a method for the interhospital transportation of critically ill patients. In South Korea, a well-established ECMO interhospital transport system is lacking due to limited resources. We developed a simplified ECMO transport system without mechanical ventilation for use by public emergency medical services. Methods: Eighteen patients utilized our ECMO transport system from December 2011 to September 2015. We retrospectively analyzed the indications for ECMO, the patient status during transport, and the patient outcomes. Results: All transport was conducted on the ground by ambulance. The distances covered ranged from 26 to 408 km (mean, 65.9±88.1 km) and the average transport time was 56.1±57.3 minutes (range, 30 to 280 minutes). All patients were transported without adverse events. After transport, 4 patients (22.2%) underwent lung transplantation because of interstitial lung disease. Eight patients who had severe acute respiratory distress syndrome showed recovery of heart and lung function after ECMO therapy. A total of 13 patients (70.6%) were successfully taken off ECMO, and 11 patients (61.1%) survived. Conclusion: Our ECMO transport system without mechanical ventilation can be considered a safe and useful method for interhospital transport and could be a good alternative option for ECMO transport in Korean hospitals with limited resources. Abstract Emerging diseases are increasing burdens on public health, negatively affecting the world economy, causing extinction of species, and disrupting ecological integrity. One Health recognizes that human, domestic animal, and wildlife health are interconnected within ecosystem health and provides a framework for the development of multidisciplinary solutions to global health challenges. To date, most health-promoting interventions have focused largely on single-sector outcomes. For example, risk for transmission of zoonotic pathogens from bush-meat hunting is primarily focused on human hygiene and personal protection. However, bush-meat hunting is a complex issue promoting the need for holistic strategies to reduce transmission of zoonotic disease while addressing food security and wildlife conservation issues. Temporal and spatial separation of humans and wildlife, risk communication, and other preventative strategies should allow wildlife and humans to co-exist. Upstream surveillance, vaccination, and other tools to prevent pathogen spillover are also needed. Clear multi-sector outcomes should be defined, and a systems-based approach is needed to develop interventions that reduce risks and balance the needs of humans, wildlife, and the environment. The ultimate goal is long-term action to reduce forces driving emerging diseases and provide interdisciplinary scientific approaches to management of risks, thereby achieving optimal outcomes for human, animal, and environmental health. Abstract Background: Four sampling techniques commonly are used for antemortem identification of pathogens from cattle with bovine respiratory disease (BRD): the nasal swab (NS), guarded nasopharyngeal swab (NPS), bronchoalveolar lavage (BAL), and transtracheal wash (TTW). Agreement among these methods has not been well characterized.Objective: To evaluate agreement among TTW and NS, NPS, or BAL for identification of viral and bacterial pathogens in dairy calves with BRD.Animals: One hundred dairy calves with naturally acquired BRD.Methods: Calves were sampled by all 4 methods. Viral agents were identified by real-time RT-PCR, bacteria were identified by aerobic culture, and Mycoplasma bovis (M. bovis) isolates were speciated by PCR. Agreement among TTW and NS, NPS, or BAL was evaluated by calculating the kappa statistic and percent positive agreement. McNemar's exact test was used to compare the proportions of positive results.Results: Agreement among TTW and NS, TTW and NPS, and TTW and BAL, was very good for identification of P. multocida, M. haemolytica, and M. bovis. For bovine respiratory syncytial virus (BRSV), agreement with TTW was moderate for NS, good for NPS, and very good for BAL. For bovine coronavirus (BCV), agreement with TTW was moderate for NS and NPS, and good for BAL. McNemar's test was significant only for BCV, indicating that for this pathogen the proportion of positive results from NS and NPS could not be considered comparable to TTW.Conclusions and Clinical Importance: This study provides guidance for veterinarians selecting diagnostic tests for antemortem identification of pathogens associated with BRD. Abstract Production of large quantities of viral vectors is crucial for the success of gene therapy in the clinic. There is a need for higher titers of herpes simplex virus-1 (HSV-1) vectors both for therapeutic use as well as in the manufacturing of clinical grade adenoassociated virus (AAV) vectors. HSV-1 yield increased when primary human fibroblasts were treated with anti-inflammatory drugs like dexamethasone or valproic acid. In our search for compounds that would increase HSV-1 yield, we investigated another anti-inflammatory compound, aurintricarboxylic acid (ATA). Although ATA has been previously shown to have antiviral effects, we find that low (micromolar) concentrations of ATA increased HSV-1 vector production yields. Our results showing the use of ATA to increase HSV-1 titers have important implications for the production of certain HSV-1 vectors as well as recombinant AAV vectors. Abstract Original article Highlights The serial interval of novel coronavirus infections was estimated from a total of 28 infector-infectee pairs. J o u r n a l P r e -p r o o f -2 - The median serial interval is shorter than the median incubation period, suggesting a substantial proportion of pre-symptomatic transmission. A short serial interval makes it difficult to trace contacts due to the rapid turnover of case generations.Objective: To estimate the serial interval of novel coronavirus from information on 28 infector-infectee pairs.We collected dates of illness onset for primary cases (infectors) and secondary cases (infectees) from published research articles and case investigation reports. We subjectively ranked the credibility of the data and performed analyses on both the full dataset (n=28) and a subset of pairs with highest certainty in reporting (n=18). In addition, we adjust for right truncation of the data as the epidemic is still in its growth phase.Results: Accounting for right truncation and analyzing all pairs, we estimated the median serial interval at 4.0 days (95% credible interval [CrI]: 3.1, 4.9).Limiting our data to only the most certain pairs, the median serial interval was estimated at 4.6 days (95% CrI: 3.5, 5.9).The serial interval of COVID-19 is close to or shorter than its median incubation period. This suggests that a substantial proportion of secondary transmission may occur prior to illness onset. The COVID-19 serial interval is also shorter than the serial interval of severe acute respiratory syndrome (SARS), indicating that calculations made using the SARS serial interval may introduce bias.J o u r n a l P r e -p r o o f -3 -If the transmission takes place during the symptomatic period of the primary case, the serial interval is longer than the incubation period. However, this relationship can be reversed when pre-symptomatic transmission takes place.Furthermore, it is possible that the secondary case may even experience illness onset prior to onset in their infector.J o u r n a l P r e -p r o o f Abstract Ebola virus and Marburg virus are members of the family of Filoviridae and are etiological agents of a deadly hemorrhagic fever disease. The clinical symptoms of Ebola and Marburg hemorrhagic fevers are difficult to distinguish and there are currently no specific antiviral therapies against either of the viruses. Therefore, a drug that is safe and effective against both would be an enormous breakthrough. We and others have shown that the folding of the glycoproteins of many enveloped viruses, including the filoviruses, is far more dependent upon the calnexin pathway of protein folding than are most host glycoproteins. Drugs that inhibit this pathway would be expected to be selectively antiviral. Indeed, as we summarize in this review, imino sugars that are competitive inhibitors of the host endoplasmic reticular a-glucosidases I and II, which are enzymes that process N-glycan on nascent glycoproteins and thereby inhibit calnexin binding to the nascent glycoproteins, have been shown to have antiviral activity against a number of enveloped viruses including filoviruses. In this review, we describe the state of development of imino sugars for use against the filoviruses, and provide an explanation for the basis of their antiviral activity as well as limitations. Abstract Background: Patient-centered care (PCC) and integrative care approach are widely advocated. However, their implementation usually requires an extended consultation time. Despite significant advances in medical diagnosis and treatment, no studies have examined consultation time and patient centeredness in Korea. Methods: We conducted a "15-Minute Consultation" for first-time patients in outpatient clinics of 13 departments. A control group was selected from the same physicians' first-time patients, adjusting for age and gender. A total of 275 patients were selected for receiving in-depth consultation and 141 control patients were selected for regular consultation. Data were collected from patients using a questionnaire comprising a patient-centeredness scale and items on potential predictors such as socio-demographic and clinical factors. We also investigated the participating physician's professionalism. Results: As compared to the control group, the in-depth consultation group scored higher on 5 variables associated with PCC, including (patients' perception of ) medical professionals, wait and consultation times, treatment, patient advocacy, and patient satisfaction. While 92.4% of patients in the in-depth consultation group reported that the consultation time was sufficient, only 69.0% of those in the control group reported the same (P < 0.01). In the in-depth consultation group, scores on satisfaction level were the highest for the department of internal medicine, followed by departments of surgery and pediatrics. Participating physicians' improved satisfaction following the intervention proved that in-depth consultation facilitated building a rapport with patients. Conclusion: This study illustrated that the provision of sufficiently long consultation for serious and rare diseases could improve PCC and physicians' professionalism. Health authorities should reshuffle the healthcare delivery system and provide sufficient consultation time to ensure PCC and medical professionalism. Abstract Objective: The aim of this study was to determine the etiology and prevalence of pediatric CAP in Beijing using a real-time polymerase chain reaction (PCR) technique. Methods: Between February 15, 2011 and January 18, 2012, 371 pediatric patients with CAP were enrolled at Beijing Children's Hospital. Sixteen respiratory viruses and two bacteria were detected from tracheal aspirate specimens using commercially available multiplex real-time reverse transcription PCR (RT-PCR) kits. Results: A single viral pathogen was detected in 35.3% of enrolled patients, multiple viruses in 11.6%, and virus/bacteria coinfection in 17.8%. In contrast, only 6.5% of patients had a single bacterial pathogen and 2.2% were infected with multiple bacteria. The etiological agent was unknown for 26.7% of patients. The most common viruses were respiratory syncytial virus (RSV) (43.9%), rhinovirus (14.8%), parainfluenza virus (9.4%), and adenovirus (8.6%). In patients under three years of age, RSV (44.6%), rhinovirus (12.8%), and Streptococcus pneumoniae (9.9%) were the most frequent pathogens. In children aged 3e7 years, S. pneumoniae (38.9%), RSV (30.6%), Haemophilus influenzae (19.4%), and adenovirus (19.4%) were most prevalent. Finally in children over seven years, RSV (47.3%), S. pneumoniae (41.9%), and rhinovirus (21.5%) infections were most frequent. Conclusions: Viral pathogens, specifically RSV, were responsible for the majority of CAP in pediatric patients. However, both S. pneumoniae and H. influenzae contributed as major causes of disease. Commercially available multiplexing real-time PCR allowed for rapid detection of the etiological agent. Abstract The vacuolar aTpase (V-aTpase) plays an important role in tumor progression and metastases. a novel peptide from the a2 isoform of V-aTpase called a2NTD has been shown to exert an immunoregulatory role in the tumor microenvironment by controlling the maturation of monocytes toward a tumor-associated macrophage phenotype. Our data indicate that a2NTD binds to the surface of monocytes. a2NTD was preferentially endocytosed by pro-inflammatory monocytes bearing a cD142 + cD16 + phenotype, which is associated with the monocyte-to-macrophage maturation process. Both a2NTD binding and internalization led to production of the pro-inflammatory cytokines interleukin (IL)-1α and IL-1β by cD142 + cD16 − (classical) and cD142 + cD16 + (intermediate) monocytes. a2NTD was internalized via a macropinocytosis mechanism utilizing scavenger receptors. however, the inhibition of a2NTD endocytosis did not reduce cytokine production by monocytes. This points to the existence of two receptors that respond to a2NTD: scavengers receptors that mediate cellular uptake and an hitherto unidentified receptor stimulating the production of inflammatory cytokines.Abbreviations: a2NTD, N-terminus domain of the a2 isoform vacuolar ATPase; a2V, a2 isoform vacuolar ATPase;CRI, cancer-related inflammation; TAM, tumor-associated macrophage; V-ATPase, vacuolar ATPase Abstract Chronic rhinosinusitis (CRS) is a multifactorial condition in which the microbiota plays a pathogenic role. The nature of the interaction between the microbiota and the local immune system is very complex and has not been fully elucidated. Recent improvements in the microbiological techniques have greatly advanced our understanding of the complex nature of this interaction. This paper summarizes the current state of the rapidly evolving research on this subject. Defining the nature of the role of the microbiota in CRS is important because of the associated therapeutic implications. Abstract To determine whether specific asparaginelinked (N-linked) oligosaccharides present in cell surface glycoproteins are required for cell-cell interactions within the peripheral nervous system, we have used castanospermine to inhibit maturation of N-linked sugars in cell cultures of neurons or neurons plus Schwann cells. Maximally 10-15 % of the N-linked oligosaccharides on neuronal proteins have normal structure when cells are cultured in the presence of 250 lxg/ml castanospermine; the remaining oligosaccharides are present as immature carbohydrate chains not normally found in these glycoproteins. Although cultures were treated for 2 wk with castanospermine, cells always remained viable and appeared healthy. We have analyzed several biological responses of embryonic dorsal root ganglion neurons, with or without Abstract The study of RNA virus populations is a challenging task. Each population of RNA virus is composed of a collection of different, yet related genomes often referred to as mutant spectra or quasispecies. Virologists using deep sequencing technologies face major obstacles when studying virus population dynamics, both experimentally and in natural settings due to the relatively high error rates of these technologies and the lack of high performance pipelines. In order to overcome these hurdles we developed a computational pipeline, termed ViVan (Viral Variance Analysis). ViVan is a complete pipeline facilitating the identification, characterization and comparison of sequence variance in deep sequenced virus populations. Results: Applying ViVan on deep sequenced data obtained from samples that were previously characterized by more classical approaches, we uncovered novel and potentially crucial aspects of virus populations. With our experimental work, we illustrate how ViVan can be used for studies ranging from the more practical, detection of resistant mutations and effects of antiviral treatments, to the more theoretical temporal characterization of the population in evolutionary studies. Availability and implementation: Freely available on the web at http://www.vivanbioinfo.org Contact: nshomron@post.tau.ac.il Supplementary information: Supplementary data are available at Bioinformatics online. Abstract The NIAID-funded Biodefense Proteomics Resource Center (RC) provides storage, dissemination, visualization and analysis capabilities for the experimental data deposited by seven Proteomics Research Centers (PRCs). The data and its publication is to support researchers working to discover candidates for the next generation of vaccines, therapeutics and diagnostics against NIAID's Category A, B and C priority pathogens. The data includes transcriptional profiles, protein profiles, protein structural data and host-pathogen protein interactions, in the context of the pathogen life cycle in vivo and in vitro. The database has stored and supported host or pathogen data derived from Bacillus, Brucella, Cryptosporidium, Salmonella, SARS, Toxoplasma, Vibrio and Yersinia, human tissue libraries, and mouse macrophages. These publicly available data cover diverse data types such as mass spectrometry, yeast two-hybrid (Y2H), gene expression profiles, X-ray and NMR determined protein structures and protein expression clones. The growing database covers over 23 000 unique genes/proteins from different experiments and organisms. All of the genes/proteins are annotated and integrated across experiments using UniProt Knowledgebase (UniProtKB) accession numbers. The web-interface for the database enables searching, querying and downloading at the level of experiment, group and individual gene(s)/ protein(s) via UniProtKB accession numbers or protein function keywords. The system is accessible at http://www.proteomicsresource.org/. Abstract containing 36 ppm germanium. The present study was conducted to better understand the effects of GB on immune responses in a mouse model, and to demonstrate the clearance effects of this mineral against Porcine reproductive and respiratory syndrome virus (PRRSV) in experimentally infected pigs as an initial step towards the development of a feed supplement that would promote immune activity and help prevent diseases. In the mouse model, dietary supplementation with GB enhanced concanavalin A (ConA)-induced lymphocyte proliferation and increased the percentage of CD3+CD8+ T lymphocytes. In pigs experimentally infected with PRRSV, viral titers in lungs and lymphoid tissues from the GB-fed group were significantly decreased compared to those of the control group 12 days post-infection. Corresponding histopathological analyses demonstrated that GB-fed pigs displayed less severe pathological changes associated with PRRSV infection compared to the control group, indicating that GB promotes PRRSV clearance. These antiviral effects in pigs may be related to the ability of GB to increase CD3+CD8+ T lymphocyte production observed in the mice. Hence, this mineral may be an effective feed supplement for increasing immune activity and preventing disease.Keywords: germanium biotite, immune enhancement, porcine reproductive and respiratory syndrome virus 136 Bock-Gie Jung et al. Abstract To analyze the mechanism of integral membrane protein localization in the early Golgi apparatus of Saccharomyces cerevisiae, we have used Ochlp, a cis- Abstract The anti-malaria drug chloroquine is well known as autophagy inhibitor. Chloroquine has also been used as anti-inflammatory drugs to treat inflammatory diseases. We hypothesized that chloroquine could have a dual effect in liver ischemia/reperfusion (I/R) injury: chloroquine on the one hand could protect the liver against I/R injury via inhibition of inflammatory response, but on the other hand could aggravate liver I/R injury through inhibition of autophagy. Rats (n ¼ 6 per group) were pre-treated with chloroquine (60 mg/kg, i.p.) 1 h before warm ischemia, and they were continuously subjected to a daily chloroquine injection for up to 2 days. Rats were killed 0.5, 6, 24 and 48 h after reperfusion. At the early phase (i.e., 0-6 h after reperfusion), chloroquine treatment ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory cytokines and fewer histopathologic changes. In contrast, chloroquine worsened liver injury at the late phase of reperfusion (i.e., 24-48 h after reperfusion). The mechanism of protective action of chloroquine appeared to involve its ability to modulate mitogen-activated protein kinase activation, reduce high-mobility group box 1 release and inflammatory cytokines production, whereas chloroquine worsened liver injury via inhibition of autophagy and induction of hepatic apoptosis at the late phase.In conclusion, chloroquine prevents ischemic liver damage at the early phase, but aggravates liver damage at the late phase in liver I/R injury. This dual role of chloroquine should be considered when using chloroquine as an inhibitor of inflammation or autophagy in I/R injury. Abstract Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine. Abstract Epithelial cells accumulate distinct popula- Abstract Background: Acute respiratory tract infection (ARTI) is one of the most common infections worldwide, causing significant morbidity and mortality.Objectives: This study was conducted to determine the prevalence and seasonal distribution of respiratory viruses in our region, in children and adults with a pre-diagnosis of ARTI.A total of 845 nasopharyngeal swab specimens were analyzed with the RespiFinder Smart 22 kit (PathoFinder BV, Netherlands) and the Rotor-Gene 6000 real-time PCR system.Results: At least one pathogen was detected in 612 (72.4%) of the specimens. Overall, 902 pathogens were detected; 821 (91%) were viruses and 81 (9%) were bacteria. The most commonly detected pathogens were influenza A virus (IFV-A) (n = 219), influenza B virus (IFV-B) (n=157), rhinovirus/enterovirus (n = 107), human bocavirus (HBoV) (n = 91), respiratory syncytial virus (RSV) A/B (n = 64), adenovirus (n = 56), human coronaviruses (n = 51), Mycoplasma pneumoniae (n = 49), parainfluenza viruses (n = 40), human metapneumovirus (n = 36), Bordetella pertussis (n = 15), Legionella pneumophila (n = 11), and Chlamydophila pneumoniae (n = 6), respectively. Among the 215 (25.4%) co-infected cases, IFV-A/HBoV and IFV-A/IFV-B were the most common co-infections. IFV-A was the most prevalent agent in all age groups except for children under 5 years of age, in whom RSV A/B was the most common pathogen. Approximately two thirds of the respiratory viruses were detected in early spring and winter, with peaks in January, March, and April.With regard to the prevalence and seasonal distribution of respiratory viruses, our epidemiological data for the 2014 -2015 season in Istanbul showed a predominance of IFV-A infections with a peak activity in early spring. Enhanced surveillance and early detection of respiratory viral pathogens can be useful in the diagnosis, treatment, and prevention of ARTIs, and for guiding the development of appropriate public health strategies. Abstract We hypothesized that fever in children with viral bronchiolitis indicates the need for consideration of superimposed bacterial pneumonia. We conducted a retrospective study of 349 children aged 2 years and younger with diagnoses of respiratory syncytial virus (RSV) and viral upper respiratory infection. Data were analyzed using Pearson χ 2 test. One hundred seventy-eight children had RSV with no other identified virus. The majority of children (56%) who had only RSV were afebrile. Febrile children with RSV were over twice as likely to be diagnosed with bacterial pneumonia as those who were afebrile (60% vs 27%, P < .001). In the 171 children who had bronchiolitis caused by a virus other than RSV, 51% were afebrile. These children were 8 times more likely to be diagnosed with pneumonia than those who were afebrile (65% vs 8%, P < .001). Evaluation of febrile children with viral bronchiolitis may allow early diagnosis and treatment of secondary bacterial pneumonia. Abstract We developed a Rapid Diagnostic Test (RDT) kit for detecting IgG/IgM antibodies against Zika virus (ZIKV) using monoclonal antibodies to the envelope (E) and non-structural protein 1 (NS1) of ZIKV. These proteins were produced using baculovirus expression vector with Sf9 cells. Monoclonal antibodies J2G7 to NS1 and J5E1 to E protein were selected and conjugated with colloidal gold to produce the Zika IgG/IgM RDT kit (Zika RDT). Comparisons with ELISA, plaque reduction neutralization test (PRNT), and PCR were done to investigate the analytical sensitivity of Zika RDT, which resulted in 100% identical results. Sensitivity and specificity of Zika RDT in a field test was determined using positive and negative samples from Brazil and Korea. The diagnostic accuracy of Zika RDT was fairly high; sensitivity and specificity for IgG was 99.0 and 99.3%, respectively, while for IgM it was 96.7 and 98.7%, respectively. Cross reaction with dengue virus was evaluated using anti-Dengue Mixed Titer Performance Panel (PVD201), in which the Zika RDT showed crossreactions with DENV in 16.7% and 5.6% in IgG and IgM, respectively. Cross reactions were not observed with West Nile, yellow fever, and hepatitis C virus infected sera. Zika RDT kit is very simple to use, rapid to assay, and very sensitive, and highly specific. Therefore, it would serve as a choice of method for point-of-care diagnosis and large scale surveys of ZIKV infection under clinical or field conditions worldwide in endemic areas. Abstract Purpose: The purpose of this study was to evaluate the prognostic effect of patient compliance with supportive periodontal treatment (PC-SPT). Chronic periodontitis patients were classified based on their compliance level, and factors affecting PC-SPT and the prognosis of PC-SPT were investigated. Methods: This study selected 206 patients who started SPT after receiving periodontal treatment between 2010 and 2012. Patients who continued SPT through February 2016 were included. The patients were classified according to whether they exhibited complete compliance (100% of visits), excellent compliance (≥70% of visits), incomplete compliance (<70% of visits), or non-compliance (only 2 visits). Patient characteristics that could affect PC-SPT, such as age, sex, distance of the clinic from their residence, implantation, and periodontal treatment, were investigated. The number of newly decayed and extracted teeth, alveolar bone level changes around the teeth and implants, and implant removal were examined to evaluate the prognosis of PC-SPT. Results: Sex and the presence of an implant significantly affected PC-SPT. Additionally, the number of newly decayed and extracted teeth and changes in alveolar bone levels around the teeth and implants were significant prognostic factors related to PC-SPT. Conclusions: PC-SPT in chronic periodontitis patients will help maintain periodontal health and prevent further periodontal disease. Abstract Paediatric Virology is a bold new scientific field, which combines Paediatrics with Virology, Epidemiology, Molecular Medicine, Evidence-based Medicine, Clinical Governance, Quality Improvement, Pharmacology and Immunology. The Workshop on Paediatric Virology, which took place on Saturday October 10, 2015 in Athens, Greece, provided an overview of recent views and advances on viral infections occurring in neonates and children. It was included in the official programme of the 20th World Congress on Advances in Oncology and the 18th International Symposium on Molecular Medicine, which attracted over 500 delegates from the five continents. During the Workshop, the topics covered included the challenges of vaccine implementation against human papillomaviruses in countries under financial crisis, strategies for eradicating poliomyelitis and its 60th vaccine anniversary, as well as the debate on the association between autism and vaccination against measles, mumps and rubella. Among the non-vaccine related topics, emphasis was given to viral infections in prematurely born infants and their long-term outcomes, new paediatric intensive care management options for bronchiolitis related to respiratory syncytial virus, the clinical implications of hepatitis B virus and cytomegalovirus genotyping, the Ebola virus threat and preparedness in Paediatric Emergency Departments, oral, oropharynx, laryngeal, nasal and ocular viral infections and Merkel cell polyomavirus as a novel emerging virus of infancy and childhood. In this review, we provide selected presentations and reports discussed at the Workshop. Abstract Objectives To conduct on-site assessments of public health implications at key European pet markets.Design Observational study of visitor behaviour at stalls that displayed and sold animals, mainly amphibians and reptiles, to assess potential contamination risk from zoonotic pathogens. We noted initial modes of contact as 'direct' (handling animals) as well as 'indirect' (touching presumed contaminated animal-related sources) and observed whether these visitors subsequently touched their own head or mouth (H1), body (H2) or another person (H3).Setting Publicly accessible exotic animal markets in the UK, Germany and Spain.Participants Anonymous members of the public in a public place.Main outcome measures Occurrence and frequency of public contact (direct, indirect or no contact) with a presumed contaminated source.Results A total of 813 public visitors were observed as they attended vendors. Of these, 29 (3.6%) made direct contact with an animal and 222 (27.3%) made indirect contact with a presumed contaminated source, with subsequent modes of contact being H1 18.7%, H2 52.2% and H3 9.9%.Our observations indicate that opportunities for direct and indirect contact at pet markets with presumed contaminated animals and inanimate items constitute a significant and major concern, and that public attendees are exposed to rapid contamination on their person, whether or not these contaminations become associated with any episode of disease involving themselves or others. These public health risks appear unresolvable given the format of the market environment. Abstract Background Isolation of cases and contact tracing is used to control outbreaks of infectious diseases, and has been used for coronavirus disease 2019 (COVID-19). Whether this strategy will achieve control depends on characteristics of both the pathogen and the response. Here we use a mathematical model to assess if isolation and contact tracing are able to control onwards transmission from imported cases of COVID-19.We developed a stochastic transmission model, parameterised to the COVID-19 outbreak. We used the model to quantify the potential effectiveness of contact tracing and isolation of cases at controlling a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-like pathogen. We considered scenarios that varied in the number of initial cases, the basic reproduction number (R 0 ), the delay from symptom onset to isolation, the probability that contacts were traced, the proportion of transmission that occurred before symptom onset, and the proportion of subclinical infections. We assumed isolation prevented all further transmission in the model. Outbreaks were deemed controlled if transmission ended within 12 weeks or before 5000 cases in total. We measured the success of controlling outbreaks using isolation and contact tracing, and quantified the weekly maximum number of cases traced to measure feasibility of public health effort.Findings Simulated outbreaks starting with five initial cases, an R 0 of 1·5, and 0% transmission before symptom onset could be controlled even with low contact tracing probability; however, the probability of controlling an outbreak decreased with the number of initial cases, when R 0 was 2·5 or 3·5 and with more transmission before symptom onset. Across different initial numbers of cases, the majority of scenarios with an R 0 of 1·5 were controllable with less than 50% of contacts successfully traced. To control the majority of outbreaks, for R 0 of 2·5 more than 70% of contacts had to be traced, and for an R 0 of 3·5 more than 90% of contacts had to be traced. The delay between symptom onset and isolation had the largest role in determining whether an outbreak was controllable when R 0 was 1·5. For R 0 values of 2·5 or 3·5, if there were 40 initial cases, contact tracing and isolation were only potentially feasible when less than 1% of transmission occurred before symptom onset.Interpretation In most scenarios, highly effective contact tracing and case isolation is enough to control a new outbreak of COVID-19 within 3 months. The probability of control decreases with long delays from symptom onset to isolation, fewer cases ascertained by contact tracing, and increasing transmission before symptoms. This model can be modified to reflect updated transmission characteristics and more specific definitions of outbreak control to assess the potential success of local response efforts. Abstract Objective To systematically review the literature exploring impact of isolation on hospitalised patients who are infectious: psychological and non-psychological outcomes Design Systematic review with quantification Data Sources Embase, Medline and Psychinfo were searched from inception until December 2018.Reference lists and Google Scholar were also handsearched.Twenty seven papers published from database inception until December 2018 were reviewed. A wide range of psychological and non-psychological outcomes were reported. There was a marked trend for isolated patients to exhibit higher risk of depression, anxiety and worse outcomes for a range of care-related factors but with significant variation. 5 Wigglesworth N, Wilcox MH. Prospective evaluation of hospital isolation room capacity. Zingg W, Holmes A, Dettenkofer M, et al. Hospital organisation, management, and structure for prevention of health-care-associated infection: a systematic review and expert consensus. Lancet Infect Dis 2015;15:212-24. 9 Simon M, Maben J, Murrells T, et al. Is single room hospital accommodation associated with differences in healthcare-associated infection, falls, pressure ulcers or medication errors? A natural experiment with non-equivalent controls. 12 Moher D, Liberati A, Tetzlaff J, et al. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement.ABSTRACT2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number. Abstract Physicians play multiple roles in a health system. They typically serve simultaneously as the agent for patients, for insurers, for their own medical practices, and for the hospital facilities where they practice. Theoretical and empirical results have demonstrated that financial relations among these different stakeholders can affect clinical outcomes as well as the efficiency and quality of care. What are the physicians' roles as the agents of Chinese patients? The marketization approach of China's economic reforms since 1978 has made hospitals and physicians profit-driven. Such profit-driven behavior and the financial tie between hospitals and physicians have in turn made physicians more the agents of hospitals rather than of their patients. While this commentary acknowledges physicians' ethics and their dedication to their patients, it argues that the current physician agency relation in China has created barriers to achieving some of the central goals of current provider-side health care reform efforts. In addition to eliminating existing perverse financial incentives for both hospitals and physicians, the need for which is already agreed upon by numerous scholars, we argue that the success of the ongoing Chinese public hospital reform and of overall health care reform also relies on establishing appropriate physician-hospital agency relations. This commentary proposes 2 essential steps to establish such physician-hospital agency relations: (1) minimize financial ties between senior physicians and tertiary-level public hospitals by establishing a separate reimbursement system for senior physicians, and (2) establishing a comprehensive physician professionalism system underwritten by the Chinese government, professional physician associations, and major health care facilities as well as by physician leadership representatives. Neither of these suggestions is addressed adequately in current health care reform activities.Keywords physician payment, physician professionalism, China health care system, Chinese health care reform, China health policyCommentaryThe need for eliminating existing perverse financial incentives for both hospitals and physicians was already agreed upon by numerous scholars. How does your research contribute to the field? We argue that the success of the ongoing Chinese public hospital reform and of overall health care reform also relies on establishing appropriate physician-hospital agency relations. What are your research's implications toward theory, practice, or policy? This commentary proposes 2 essential steps to establish such physician-hospital agency relations, and neither of these suggestions is addressed adequately in current health care reform activities. Abstract Backgrounds: An ongoing outbreak of a novel coronavirus (2019-nCoV) pneumonia hit a major city in China, Wuhan, December 2019 and subsequently reached other provinces/regions of China and other countries. We present estimates of the basic reproduction number, R 0 , of 2019-nCoV in the early phase of the outbreak. Methods: Accounting for the impact of the variations in disease reporting rate, we modelled the epidemic curve of 2019-nCoV cases time series, in mainland China from January 10 to January 24, 2020, through the exponential growth. With the estimated intrinsic growth rate (g), we estimated R 0 by using the serial intervals (SI) of two other well-known coronavirus diseases, MERS and SARS, as approximations for the true unknown SI. Findings: The early outbreak data largely follows the exponential growth. We estimated that the mean R 0 ranges from 2.24 (95%CI: 1.96-2.55) to 3.58 (95%CI: 2.89-4.39) associated with 8-fold to 2-fold increase in the reporting rate. We demonstrated that changes in reporting rate substantially affect estimates of R 0 . Conclusion: The mean estimate of R 0 for the 2019-nCoV ranges from 2.24 to 3.58, and is significantly larger than 1. Our findings indicate the potential of 2019-nCoV to cause outbreaks. Abstract This study was aiming to investigate the knowledge, practice and attitudes of secondary school and university students toward MERS-CoV infection. This is a cross-sectional study conducted in Riyadh, Saudi Arabia. Study participants were recruited from several constituent colleges of King Saud University and secondary schools in Riyadh. Data were collected using selfadministered, closed-ended questionnaires. Frequencies and proportions were computed for descriptive purposes. Chi square test was utilized to depict statistical difference between groups. Among the 1109 students who answered the questionnaires, 53.1% were male, and 46.9% were female. Level of knowledge about clinical presentation of MERS is generally similar among university and school students. The most frequently reported source of transmission is entering crowded spaces and being exposed to coughing and sneezing. Additionally, hand washing was the most commonly reported method of protection against the infection. The localized spread of MERS in Saudi Arabia and the number of fatalities associated with it might have increased public interest in understanding how to maintain proper precautionary measures both on a community and on an Abstract Background: Osteonecrosis is an incapacitating disorder with high morbidity. Though extracorporeal shockwave therapy (ESWT) provides a noninvasive treatment option, controversial subjects still exist about its effectiveness, indications, and mechanism of action.Methods: An electronic databases search was performed using PubMed, Embase, and the Cochrane library to collect clinical trials, case reports, and cases series on this topic and then useful data were extracted and appraised by experienced clinicians. We evaluated the quality of included evidences by using the Oxford Centre for evidence-based medicine (EBM) Levels of Evidence.Results: A total of 17 articles including 2 case reports, 9 open label trials, 2 cohorts, and 6 randomized controlled trials were considered to be eligible for this systematic review. Visual analog scale (VAS), Harris hip scores, and the imaging results were the frequently-used outcome estimates of included studies.Conclusion: By systematically analyzing these evidences, we could conclude that ESWT could act as a safe and effective method to improve the motor function and relieve the pain of patients with osteonecrosis of femoral hip, especially those at early stage. Imaging revealed that bone marrow edema was significantly relieved, but the necrotic bone could not be reversed after ESWT. This technique could slow or even block the progression of ONFH and therefore reduce the demand for surgery. Collaboration with other conservative modalities would not improve the curative benefits of ESWT. Meanwhile, ONFH with various risk factors showed similar reaction to this noninvasive treatment method. However, these conclusions should be interpreted carefully for the low-quality of included publications and further studies are requisite to validate the effect of ESWT in ONFH.Abbreviations: allo-HSCT = allogeneic hematopoietic stem cell transplantation, ARCO = association research circulation osseous, BMP = bone morphogenic protein, CD = core decompression, EBM = evidence-based medicine, ESWT = extracorporeal shockwave therapy, ESWT = extracorporeal shockwave therapy, HBO = hyperbaric oxygen therapy, ONFH = osteonecrosis of femoral head, RUNX2 = Runt-related transcription factor 2, SARS = severe acute respiratory syndrome, SF-12 = 12-item Short Form, SLE = systemic lupus erythematosus, SR = systematic review, THA = total hip arthroplasty, VAS = visual analog scale, VEGF = vessel endothelial growth factor, WOMAC = Western Ontario and McMaster Universities Arthritis Index. Abstract In the face of the worldwide threat of severe acute respiratory syndrome (SARS) to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA), these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potential epitope regions were located at Codons 469-882 in the S protein, and one epitope site was located at Codons 599-620. Identification of antigenic regions in the SARS-CoV S protein may be important for the functional studies of this virus or the development of clinical diagnosis. Abstract Background: Bacterial pneumonia occurring after respiratory viral infection is common. However, the predominant bacterial species causing pneumonia secondary to respiratory viral infections other than influenza remain unknown. The purpose of this study was to know whether the pathogens causing post-viral bacterial pneumonia vary according to the type of respiratory virus. Methods: Study subjects were 5,298 patients, who underwent multiplex real-time polymerase chain reaction for simultaneous detection of respiratory viruses, among who visited the emergency department or outpatient clinic with respiratory symptoms at Ulsan University Hospital between April 2013 and March 2016. The patients' medical records were retrospectively reviewed. Results: A total of 251 clinically significant bacteria were identified in 233 patients with post-viral bacterial pneumonia. Mycoplasma pneumoniae was the most frequent bacterium in patients aged <16 years, regardless of the preceding virus type (p=0.630). In patients aged ≥16 years, the isolated bacteria varied according to the preceding virus type. The major results were as follows (p<0.001): pneumonia in patients with influenza virus (type A/B), rhinovirus, and human metapneumovirus infections was caused by similar bacteria, and the findings indicated that Staphylococcus aureus pneumonia was very common in these patients. In contrast, coronavirus, parainfluenza virus, and respiratory syncytial virus infections were associated with pneumonia caused by gram-negative bacteria. Conclusion: The pathogens causing post-viral bacterial pneumonia vary according to the type of preceding respiratory virus. This information could help in selecting empirical antibiotics in patients with post-viral pneumonia. Abstract Case summary This work describes the diagnosis and successful treatment of a 2-year-old domestic cat infected with Leishmania species and presenting fever, and ulcerative and nodular skin lesions after being treated for pyodermatitis for 1 year without clinical improvement. After anamnesis the cat was submitted to a complete clinical examination. Blood was collected for determination of haematological and biochemical parameters, detection of feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), feline coronavirus (FCoV) and Leishmania amastigotes. Fine-needle aspiration puncture from the skin nodules was also performed. After definitive diagnosis the animal was treated and followed up over a 2 year period. The animal tested negative for FIV-specific antibodies, FeLV antigen and feline coronavirus RNA. Leishmania amastigotes in the skin nodules were confirmed by cytology and molecular diagnosis. Treatment was initiated with allopurinol, resulting in a slight clinical improvement. Thus, N-methyl-glucamine antimoniate was added and administered for 30 days, with complete closure of the ulcerative lesions in the hindlimbs requiring a surgical approach. Close monitoring of the patient in the following 24 months indicated that combined therapy was safe and clinical cure was achieved without further relapses or side effects. Relevance and novel information Considering the increasing number of feline leishmaniosis cases and the inconsistent results of most therapeutic protocols described in the literature, the use of new approaches, especially in refractory cases, is essential. Although the use of allopurinol and N-methyl-glucamine antimoniate is off-label in cats, in this case the combination treatment was followed by an extensive analytical monitoring, supporting their safety and effectiveness. Abstract These authors contributed equally to this work.Genome sequencing technologies continue to develop with remarkable pace, yet analytical approaches for reconstructing and classifying viral genomes from mixed samples remain limited in their performance and usability. Existing solutions generally target expert users and often have unclear scope, making it challenging to critically evaluate their performance. There is a growing need for intuitive analytical tooling for researchers lacking specialist computing expertise and that is applicable in diverse experimental circumstances. Notable technical challenges have impeded progress; for example, fragments of viral genomes are typically orders of magnitude less abundant than those of host, bacteria, and/or other organisms in clinical and environmental metagenomes; observed viral genomes often deviate considerably from reference genomes demanding use of exhaustive alignment approaches; high intrapopulation viral diversity can lead to ambiguous sequence reconstruction; and finally, the relatively few documented viral reference genomes compared to the estimated number of distinct viral taxa renders classification problematic. Various software tools have been developed to accommodate the unique challenges and use cases associated with characterizing viral sequences; however, the quality of these tools varies, and their use often necessitates computing expertise or access to powerful computers, thus limiting their usefulness to many researchers. In this review, we consider the general and application-specific challenges posed by viral sequencing and analysis, outline the landscape of available tools and methodologies, and propose ways of overcoming the current barriers to effective analysis. Abstract The leaves of Lagerstroemia speciosa (Lythraceae), a Southeast Asian tree more commonly known as banaba, have been traditionally consumed in various forms by Philippinos for treatment of diabetes and kidney related diseases. In the 1990s, the popularity of this herbal medicine began to attract the attention of scientists worldwide. Since then, researchers have conducted numerous in vitro and in vivo studies that consistently confirmed the antidiabetic activity of banaba. Scientists have identified different components of banaba to be responsible for its activity. Using tumor cells as a cell model, corosolic acid was isolated from the methanol extract of banaba and shown to be an active compound. More recently, a different cell model and the focus on the water soluble fraction of the extract led to the discovery of other compounds. The ellagitannin Lagerstroemin was identified as an effective component of the banaba extract responsible for the activity. In a different approach, using 3T3-L1 adipocytes as a cell model and a glucose uptake assay as the functional screening method, Chen et al. showed that the banaba water extract exhibited an insulin-like glucose transport inducing activity. Coupling HPLC fractionation with a glucose uptake assay, gallotannins were identified in the banaba extract as components responsible for the activity, not corosolic acid. Penta-O-galloyl-glucopyranose (PGG) was identified as the most potent gallotannin. A comparison of published data with results obtained for PGG indicates that PGG has a significantly higher glucose transport stimulatory activity than Lagerstroemin. Chen et al. have also shown that PGG exhibits anti-adipogenic properties in addition to stimulating the glucose uptake in adipocytes. The combination of glucose uptake and anti-adipogenesis activity is not found in the current insulin mimetic drugs and may indicate a great therapeutic potential of PGG. Abstract Supplementary Text 1: Details of specimen collection, processing, polymerase chain reaction 34 assays, quality control measures, and assay parameters used to identify respiratory viruses.Specimen collection 36 All swabs were collected using a plastic-shaft, rayon-budded swab in a transport tube with a foam 37 pad reservoir soaked with viral transport medium (Virocult MW950, Medical Wire & Equipment, 38 Wiltshire, England). A single swab was used to sample both nostrils. 39 Quality Control measures 40 A known amount of whole equine herpesvirus (EHV) spiked into each sample assessed nucleic 41 acid extraction quality and presence of polymerase chain reaction (PCR) inhibitors. 1 Any extract 42having a >3 cycle-threshold (Ct) difference to that of the expected value by EHV real-time PCR 43 assays was considered to have failed quality control and the sample was re-extracted.Specimen quality was assessed by testing for a marker of human genomic DNA, endogenous 46 retrovirus-3 (ERV-3). 2 We have demonstrated previously that among specimens with an ERV-3 Ct a adjusted for clustering using sandwich estimators to account for within-infant correlation between observations; b Individual rhinovirus species do not add up to 'All rhinovirus' detections because of a combination of more than one rhinovirus species detected within the same ARI episode, or single new virus detection episodes where HRV positive specimens could not be sequenced. Abstract Composition vector trees (CVTrees) are inferred from whole-genome data by an alignment-free and parameter-free method. The agreement of these trees with the corresponding taxonomy provides an objective justification of the inferred phylogeny. In this work, we show the stability and self-consistency of CVTrees by performing bootstrap and jackknife re-sampling tests adapted to this alignment-free approach. Our ultimate goal is to advocate the viewpoint that time-consuming statistical re-sampling tests can be avoided at all in using this alignment-free approach. Agreement with taxonomy should be taken as a major criterion to estimate prokaryotic phylogenetic trees. Abstract Schmallenberg virus (SBV), discovered in 2011 in Germany, is associated with clinical manifestations of fever, diarrhea, reduced milk yield, abortions and congenital malformations in ruminants. Despite many studies performed for SBV, there is no detailed research on in vitro apoptotic effect of SBV. This study is aimed to determine apoptosis pathways and role of proapoptotic and anti-apoptotic molecules in Vero cells infected with SBV. The study results showed that SBV induced apoptosis via both extrinsic and intrinsic pathways by activating both caspase-8 and caspase-9, respectively. Expression analyses of pro-apoptotic (Bax, Bak and Puma) and anti-apoptotic (Bcl-2 and Bcl-XL) genes revealed that SBV-induced apoptosis causes upregulation of pro-apoptotic genes, dominantly via Puma gene, whereas Bcl-2 and Bcl-XL genes were downregulated. In conclusion, this is the first detailed report about SBV induced apoptosis in the Vero cells via both extrinsic and intrinsic cascades and apoptosis induction is seem to be regulated by Puma. Abstract Herein we describe the discovery and functional characterization of a steroidal glycosyltransferase (SGT) from Ornithogalum saundersiae and a steroidal glycoside acyltransferase (SGA) from Escherichia coli and their application in the biosynthesis of acylated steroidal glycosides (ASGs). Initially, an SGT gene, designated as OsSGT1, was isolated from O. saundersiae. OsSGT1-containing cell free extract was then used as the biocatalyst to react with 49 structurally diverse drug-like compounds. The recombinant OsSGT1 was shown to be active against both 3β-and 17β-hydroxyl steroids. Unexpectedly, in an effort to identify OsSGT1, we found the bacteria lacA gene in lac operon actually encoded an SGA, specifically catalyzing the acetylations of sugar moieties of steroid 17β-glucosides. Finally, a novel enzymatic two-step synthesis of two ASGs, acetylated testosterone-17-O-β-glucosides (AT-17β-Gs) and acetylated estradiol-17-O-β-glucosides (AE-17β-Gs), from the abundantly available free steroids using OsSGT1 and EcSGA1 as the biocatalysts was developed. The two-step process is characterized by EcSGA1-catalyzed regioselective acylations of all hydroxyl groups on the sugar unit of unprotected steroidal glycosides (SGs) in the late stage, thereby significantly streamlining the synthetic route towards ASGs and thus forming four monoacylates. The improved cytotoxic activities of 3 0 -acetylated testosterone17-O-β-glucoside towards seven human tumor cell lines were thus observable. Abstract Coronaviruses (CoVs) are a large group of enveloped viruses with a single-strand RNA genome, which continuously circulate in mammals and birds and pose a threat to livestock, companion animals, and humans. CoVs harboured by avian species are classified to the genera gamma-and deltacoronaviruses. Within the gamma-CoVs the main representative is avian coronavirus, a taxonomic name which includes the highly contagious infectious bronchitis viruses (IBVs) in chickens and similar viruses infecting other domestic birds such as turkeys, guinea fowls, or quails. Additionally, IBVs have been detected in healthy wild birds, demonstrating that they may act as the vector between domestic and free-living birds. Moreover, CoVs other than IBVs, are identified in wild birds, which suggests that wild birds play a key role in the epidemiology of other gammaCoVs and deltaCoVs. Development of molecular techniques has significantly improved knowledge of the prevalence of CoVs in avian species. The methods adopted in monitoring studies of CoVs in different avian species are mainly based on detection of conservative regions within the viral replicase, nucleocapsid genes, and 3'UTR or 5'UTR. The purpose of this review is to summarise recent discoveries in the areas of epidemiology and diagnosis of CoVs in avian species and to understand the role of wild birds in the virus distribution. Abstract Objective To investigate the effects of the newly developed Spine Balance 3D system on the balance and gait abilities of hemiplegic stroke patients. Methods Twenty-eight hemiplegic patients with chronic stroke were randomly assigned to an experimental (n=14) or control group (n=14). The experimental and control groups performed balance training by using the newly developed Spine Balance 3D system and the well-known Biodex Balance System 30 minutes per day, three times a week for 7 weeks. The Berg Balance Scale (BBS), 10-m walking test (10mWT), Timed Up and Go Test (TUG), Functional Reach Test (FRT), the Korean version of the Fall Efficacy Scale-International (KFES-I), trunk muscle strength and stability were evaluated before and after 7 weeks of intervention. Results The 10mWT improved significantly (p=0.001) in the experimental group (using the Spine Balance 3D system) but not in the control group, and core muscle strength, which we checked using Spine Balance 3D system evaluation program, improved more in the experimental group as well. The results of the BBS, FRT, TUG, KFES-I, and Biodex Balance System evaluation program improved in both groups after 7 weeks of balance training. Conclusion We suggest that the newly-developed Spine Balance 3D system can be a more useful therapeutic tool for gait and dynamic balance rehabilitation in hemiplegic patients than a conventional 2D-based balance training system. A large-scale randomized controlled study is needed to prove the effect of this system. Abstract Objectives: The present study aimed to investigate the prevalence of and factors associated with H1N1 preventive behaviors in a community-based population. Methods: A cross-sectional study was conducted in three urban and two rural communities in Korea. Interviews were conducted with 3462 individuals (1608 men and 1854 women) aged ! 19 years during FebruaryeMarch 2010. Influenzarelated information including anxiety, preventive behaviors and their perceived effectiveness, vaccination status, past influenza-like illness symptoms, and sources of and trust in information was obtained. Results: Among 3462 participants, 173 reported experiencing influenza-like illness symptoms within the past 12 months. The mean H1N1 preventive behavior score was 25.5 AE 5.5 (out of a possible 40). The percent of participants reporting high perceived effectiveness and high anxiety was 46.2% and 21.4%, respectively. After controlling for potential confounders, H1N1 preventive behavior scores were predicted by a high (b Z 3.577, p < 0.001) or moderate (b Z 2.529, p < 0.001) perception of their effectiveness. Similarly, moderate (b Z 1.516, p < 0.001) and high (b Z 4.103, p < 0.001) anxiety scores predicted high preventive behavior scores. Conclusion: Effective methods of promoting population behavior change may be nationwide campaigns through mass media, as well as education and promotion by health care providers and broadcasters. Abstract Little is known about platelet dynamics and the effect of antiplatelet therapy in Kawasaki disease (KD). This study sought to define platelet activation dynamics in KD patients by assaying platelet-derived microparticles (PDMPs). We measured plasma PDMPs levels in 46 patients with KD using an enzyme-linked immunosorbent assay (ELISA). Blood samples were collected before, at 2-5 days, and 9-15 days after intravenous immunoglobulin (IVIG) infusion, 2 months and 4-5 months after the onset of KD. We measured PDMP levels in 23 febrile and 10 afebrile control patients. In the acute phase of KD patients, PDMP levels increased significantly after IVIG treatment (12.04 ± 5.58 nmol before IVIG infusion vs. 19.81 ± 13.21 nmol at 2-5 days after IVIG infusion, P = 0.006). PDMP levels were negatively correlated with age and positively correlated with procalcitonin levels in the acute phase of KD. No significant difference was found in PDMP levels between KD patients with and without coronary artery lesion (CAL). Elevated PDMP levels after IVIG therapy significantly decreased below the pre-IVIG level in subacute phase (19.81 ± 13.21 nmol at 2-5 days after IVIG infusion vs. 8.33 ± 2.02 nmol at 9-15 days after IVIG infusion, P < 0.001), and PDMP levels stayed below the pre-IVIG level in the convalescent phase, during which antiplatelet therapy was given. However, PDMP levels rebounded after discontinuing aspirin in 17 patients. In conclusion, enhanced platelet activation was noted before treatment of KD and peaked immediately after IVIG treatment. Recurrent rising of PDMP levels was observed after discontinuing aspirin, although there were no significant differences between the PDMP levels at 2 months after the onset of KD and those at 4-5 months after the onset of the disease. Abstract This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Macrophage-derived chemokine, C-C motif chemokine 22 (MDC/CCL22), is one of the inflammatory chemokines that controls the movement of monocytes, monocyte-derived dendritic cells, and natural killer cells. Serum and skin MDC/CCL22 levels are elevated in atopic dermatitis, which suggests that the chemokines produced from keratinocytes are responsible for attracting inflammatory lymphocytes to the skin. A major signaling pathway in the interferon-γ (IFN-γ)-stimulated inflammation response involves the signal transducers and activators of transcription 1 (STAT1). In the present study, we investigated the anti-inflammatory effect of dieckol and its possible action mechanisms in the category of skin inflammation including atopic dermatitis. Dieckol inhibited MDC/CCL22 production induced by IFN-γ (10 ng/mL) in a dose dependent manner. Dieckol (5 and 10 mM) suppressed the phosphorylation and the nuclear translocation of STAT1. These results suggest that dieckol exhibits anti-inflammatory effect via the down-regulation of STAT1 activation.Abstract Abstract To rapidly distinguish Canine distemper virus (CDV), canine parvovirus (CPV), and canine kobuvirus (CaKoV) in practice, a one-step multiplex PCR/RT-PCR assay was developed, with detection limits of 10 2.1 TCID 50 for CDV, 10 1.9 TCID 50 for CPV and 10 3 copies for CaKoV. This method did not amplify nonspecific DNA or RNA from other canine viruses. Therefore, the assay provides a sensitive tool for the rapid clinical detection and epidemiological surveillance of CDV, CPV and CaKoV in dogs. Abstract Memory and learning declines are consequences of normal aging. Since those functions are associated with the hippocampus, I analyzed the global gene expression data from post-mortem hippocampal tissue of 25 old (age $ 60 yrs) and 15 young (age # 45 yrs) cognitively intact human subjects. By employing a rigorous, multi-method bioinformatic approach, I identified 36 genes that were the most significant in terms of differential expression; and by employing mathematical modeling, I demonstrated that 7 of the 36 genes were able to discriminate between the old and young subjects with high accuracy. Remarkably, 90% of the known genes from those 36 most significant genes are associated with either inflammation or immune system activation. This suggests that chronic inflammation and immune system over-activity may underlie the aging process of the human brain, and that potential anti-inflammatory treatments targeting those genes may slow down this process and alleviate its symptoms.Mathematical modeling of aging. Given the aforementioned biovariability in connection with hippocampal gene expression, I wanted to explore whether, via mathematical modeling, I could generate a function that could Abstract Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory. Abstract Oral corticosteroids are the cornerstone of acute asthma management in the emergency department.Recent evidence has raised doubt about the efficacy of this treatment in preschool-aged children with viral-induced wheezing and in smoking adults. The aims of the study are to: (1) document the magnitude of response to oral corticosteroids in children presenting to the emergency department with moderate or severe asthma; (2) quantify potential determinants of response to corticosteroids, and (3) explore the role of gene polymorphisms associated with the responsiveness to corticosteroidsThe design is a prospective cohort study of 1008 children aged 1-17 years meeting a strict definition of asthma and presenting with a clinical score of ≥4 on the validated Pediatric Respiratory Assessment Measure (PRAM). All children will receive standardized severity-specific treatment with prednisone/prednisolone and co-interventions (salbutamol with/without ipratropium bromide).Determinants, namely viral aetiology, environmental tobacco smoke, and single nucleotide polymorphism will be objectively documented. The primary efficacy endpoint is hospital admission within 72 hours. Secondary endpoints include other measures of asthma severity and time to recovery within 7 days of the index visit. The study has 80% power for detecting a risk difference of 7.5% associated with each determinant from a baseline risk of 21%, at an α of 0.05.Ethical approval has been obtained from all participating institutions. An impaired response to systemic steroids in certain subgroups will challenge the current standard of practice and call for the immediate search for better approaches. A potential host-environment interaction will broaden our understanding Abstract We report a case of acute disseminated encephalomyelitis (ADEM) secondary to Mycoplasma pneumoniae infection that failed to improve with methylprednisolone and intravenous immunoglobulin (IVIG); who responded with plasmapheresis. A 21-year-old female with an unremarkable medical history, initially presented to an outside hospital with fever and an influenza-like illness and was subsequently intubated for worsening sensorium. Brain magnetic resonance imaging was suggestive of ADEM or vasculitis for which she received five days of pulse steroids and IVIG. She showed no signs of improvement and was transferred to our hospital for plasmapheresis. Her work up revealed an elevated IgM antibody and positive sputum for Mycoplasma pneumonia by polymerase chain reaction, suggesting the pathogen as the culprit for her ADEM. Intravenous azithromycin and daily plasmapheresis were initiated for seven consecutive days. Following commencement of her treatment, the patient experienced good recovery and was subsequently extubated. She continued to improve with physical therapy and gained mobility, with the help of a walker. Patients commonly present with ADEM following viral infection or vaccination and less frequently post bacterial infection. The current treatment of ADEM due to Mycoplasma pneumoniae is based on limited case reports. Our patient poorly responded to pulse steroids and IVIG, while she markedly improved on azithromycin and plasmapheresis. In patients presenting with encephalopathic signs and neurological manifestations following pneumonia; Mycoplasma pneumoniae infection and subsequent immune-mediated demyelination should be considered. (A. Laila), ellabar@clevelandclinicabudhabi.ae (R.M. El-Lababidi), hishamm@clevelandclinicabudhabi.ae (M. Hisham), MootyM@ClevelandClinicAbuDhabi.ae (M. Mooty). IDCases 12 (2018) 41-43 2214-2509/ Abstract One case report identified a cat with visceral leishmaniosis and concurrent pancytopenia thought to be of immune-mediated origin, 1 yielding an IME value of 2.62. The cat had persistent autoagglutination, and Leishmania amastigotes were identified in the bone marrow, peripheral blood, and other body tissues. Abstract Most acute respiratory infections (ARIs) in children are due to viral etiology, and represent an important cause of mortality and morbidity in children <5 years old in developing countries. The pathogens that cause ARIs vary geographically and by season, and viruses serve a major role. In the present study, the distribution of the seven respiratory viruses that are more prevalent in Southern European countries were retrospectively analyzed in a Southern Italy Hospital, that centralizes pediatric diseases from the Naples province. Viruses were categorized by a FilmArray Respiratory Panel, and demonstrated no substantial differences in sex, age and seasonal viruses distribution. However, all the investigated viruses had a higher detection rate in the surrounding municipalities than in the metropolitan area of Naples. In recent years, the association between air pollution and respiratory infections has become an increasing public health concern. The data in this study support this association in the surrounding areas of Naples extensively contaminated by environmental toxic agents. In these areas, characterization of the epidemiology of ARIs is required to implement a prevention and control program. Abstract Members of the rab/YPTI/SEC4 gene family Abstract The need for multidisciplinary research to address today's complex health and environmental challenges has never been greater. The One Health (OH) approach to research ensures that human, animal, and environmental health questions are evaluated in an integrated and holistic manner to provide a more comprehensive understanding of the problem and potential solutions than would be possible with siloed approaches. However, the OH approach is complex, and there is limited guidance available for investigators regarding the practical design and implementation of OH research. In this paper we provide a framework to guide researchers through conceptualizing and planning an OH study. We discuss key steps in designing an OH study, including conceptualization of hypotheses and study aims, identification of collaborators for a multi-disciplinary research team, study design options, data sources and collection methods, and analytical methods. We illustrate these concepts through the presentation of a case study of health impacts associated with land application of biosolids. Finally, we discuss opportunities for applying an OH approach to identify solutions to current global health issues, and the need for cross-disciplinary funding sources to foster an OH approach to research. Abstract Epidemics such as viral haemorrhagic fevers, severe acute respiratory syndrome, Middle East respiratory syndrome coronavirus or yet unknown ones have few chances of disappearing. Globalization, worldwide travel, climate change, social conflicts and wars, among others, are likely to favor the emergence of epidemics. Preparedness of hospitals to prevent the spread of these outbreaks is among the prioritized political programmes of many countries. The EuroNHID network has in the past drawn a map of features and equipment of hospitals across Europe to take care of highly contagious patients. We update the data regarding isolation capabilities and recommendations, with an emphasis on Mediterranean countries. Abstract Bovine norovirus (BNoV) has emerged as a viral pathogen that causes a gastrointestinal illness and diarrhea in cattle. Despite its worldwide distribution, very little information is known about BNoV in Africa. In this study, BNoV was detected in 27.6% (8/29) of tested fecal materials, collected from sporadic cases of diarrheic calves, using the reverse transcription-polymerase chain reaction (RT-PCR) and primers that target RNA dependent RNA polymerase gene. Additionally, one primer pair was designed to flank the BNoV-VP2 (small capsid protein) gene for molecular analysis. Study VP2 sequences were phylogenetically-related to BNoV-GIII.2 (Newbury2-like) genotype, which is highly prevalent all over the world. However, they were separated within the cluster and one strain (41FR) grouped with recombinant GIII.P1/GIII.2 strains. Compared to reference VP2 sequences, 14 amino acid substitution mutations were found to be unique to our strains. The study confirms that BNoV is currently circulating among diarrheic calves of Egypt and also characterizes its ORF3 (VP2) genetically. The status of BNoV should be continuously evaluated in Egypt for effective prevention and control.T 1 The BNoV-RdRp targeting primers were used in one step RT-PCR for the purpose of BNoV detection. 2 The BNoV-VP2 targeting primers were used in one step RT-PCR for the purpose of sequencing and molecular analysis. 3 F; forward (sense) primer, R; reverse (antisense) primer. 4 The VP2 specific primers were designed based on the GenBank reference sequence (Bo/Newbury2/1976/UK/ AF097917). Abstract Background: Live poultry markets (LPMs) pose a threat to public health by promoting the amplification and dissemination of avian influenza viruses (AIVs) and by providing the ideal setting for zoonotic influenza transmission. Objective: This review assessed the impact of different interventions implemented in LPMs to control the emergence of zoonotic influenza. Methods: Publications were identified through a systematic literature search in the PubMed, MEDLINE and Web of Science databases. Eligible studies assessed the impact of different interventions, such as temporary market closure or a ban on holding poultry overnight, in reducing i) AIV-detection rates in birds and the market environment or ii) influenza incidence in humans. Unpublished literature, reviews, editorials, cross-sectional studies, theoretical models and publications in languages other than English were excluded. Relevant findings were extracted and critically evaluated. For the comparative analysis of findings across studies, standardized outcome measures were computed as i) the relative risk reduction (RRR) of AIV-detection in LPMs and ii) incidence rate ratios (IRRs) of H7N9incidence in humans. Results: A total of 16 publications were identified and reviewed. Collectively, the data suggest that AIV-circulation can be significantly reduced in the LPM-environment and among market-birds through (i) temporary LPM closure, (ii) periodic rest days (iii) market depopulation overnight and (iv) improved hygiene and disinfection. Overall, the findings indicate that the length of stay of poultry in the market is a critical control point to interrupt the AIV-replication cycle within LPMs. In addition, temporary LPM closure was associated with a significant reduction of the incidence of zoonotic influenza. The interpretation of these findings is limited by variations in the implementation of interventions. In addition, some of the included studies were of ecologic nature or lacked an inferential framework, which might have lead to cosiderable confounding and bias. Conclusions: The evidence collected in this review endorses permanent LPM-closure as a long-term objective to reduce the zoonotic risk of avian influenza, although its economic and socio-political implications favour less drastic interventions, e.g. weekly rest days, for implementation in the short-term. Abstract The FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18-and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans which might play an important role on lipometabolism. To reveal the function of fat1 gene in bovine fetal fibroblast cells and gain the best cell nuclear donor for transgenic bovines, the codon of fat1 sequence was optimized based on the codon usage frequency preference of bovine muscle protein, and directionally cloned into the eukaryotic expression vector pEF-GFP. After identifying by restrictive enzyme digests with AatII/XbaI and sequencing, the fusion plasmid pEF-GFP-fat1 was identified successfully. The pEF-GFP-fat1 vector was transfected into bovine fetal fibroblast cells mediated by Lipofectamine2000 TM . The positive bovine fetal fibroblast cells were selected by G418 and detected by RT-PCR. The results showed that a 1,234 bp transcription was amplified by reverse transcription PCR and the positive transgenic fat1 cell line was successfully established. Then the expression level of fat1 gene in positive cells was detected using quantitative PCR, and the catalysis efficiency was detected by gas chromatography. The results demonstrated that the catalysis efficiency of fat1 was significantly high, which can improve the total PUFAs rich in EPA, DHA and DPA. Construction and expression of pEF-GFP-fat1 vector should be helpful for further understanding the mechanism of regulation of fat1 in vitro. It could also be the first step in the production of fat1 transgenic cattle. ( Abstract Lipid rafts are membrane domains, more ordered than the bulk membrane and enriched in cholesterol and sphingolipids. They represent a platform for protein-lipid and proteinprotein interactions and for cellular signaling events. In addition to their normal functions, including membrane trafficking, ligand binding (including viruses), axonal development and maintenance of synaptic integrity, rafts have also been implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD). Lipid rafts promote interaction of the amyloid precursor protein (APP) with the secretase (BACE-1) responsible for generation of the amyloid β peptide, Aβ. Rafts also regulate cholinergic signaling as well as acetylcholinesterase and Aβ interaction. In addition, such major lipid raft components as cholesterol and GM1 ganglioside have been directly implicated in pathogenesis of the disease. Perturbation of lipid raft integrity can also affect various signaling pathways leading to cellular death and AD. In this review, we discuss modulation of APP cleavage by lipid rafts and their components, while also looking at more recent findings on the role of lipid rafts in signaling events. Abstract Ebolaviruses constitute a public health threat, particularly in Central and Western Africa. Host cell factors required for spread of ebolaviruses may serve as targets for antiviral intervention. Lectins, TAM receptor tyrosine kinases (Tyro3, Axl, Mer), T cell immunoglobulin and mucin domain (TIM) proteins, integrins, and Niemann-Pick C1 (NPC1) have been reported to promote entry of ebolaviruses into certain cellular systems. However, the factors used by ebolaviruses to invade macrophages, major viral targets, are poorly defined. Here, we show that mannose-specific lectins, TIM-1 and Axl augment entry into certain cell lines but do not contribute to Ebola virus (EBOV)-glycoprotein (GP)-driven transduction of macrophages. In contrast, expression of Mer, integrin αV, and NPC1 was required for efficient GP-mediated transduction and EBOV infection of macrophages. These results define cellular factors hijacked by EBOV for entry into macrophages and, considering that Mer and integrin αV promote phagocytosis of apoptotic cells, support the concept that EBOV relies on apoptotic mimicry to invade target cells. Abstract The Sabin Vaccine Institute Product Development Partnership is developing a Pan-anthelmintic vaccine that simultaneously targets the major soil-transmitted nematode infections, in other words, ascariasis, trichuriasis and hookworm infection. The approach builds off the current bivalent Human Hookworm Vaccine now in clinical development and would ultimately add both a larval Ascaris lumbricoides antigen and an adult-stage Trichuris trichiura antigen from the parasite stichosome. Each selected antigen would partially reproduce the protective immunity afforded by UV-attenuated Ascaris eggs and Trichuris stichosome extracts, respectively. Final antigen selection will apply a ranking system that includes the evaluation of expression yields and solubility, feasibility of process development and the absence of circulating antigen-specific IgE among populations living in helminth-endemic regions. Here we describe a five year roadmap for the antigen discovery, feasibility and antigen selection, which will ultimately lead to the scale-up expression, process development, manufacture, good laboratory practices toxicology and preclinical evaluation, ultimately leading to Phase 1 clinical testing. Abstract Viral hepatitis remains a significant worldwide threat, in spite of the availability of several successful therapeutic and vaccination strategies. Complications associated with acute and chronic infections, such as liver failure, cirrhosis and hepatocellular carcinoma, are the cause of considerable morbidity and mortality. Given the significant burden on the healthcare system caused by viral hepatitis, it is essential that novel, more effective therapeutics be developed. The present review attempts to summarize the current treatments against viral hepatitis, and provides an outline for upcoming, promising new therapeutics. Development of novel therapeutics requires an understanding of the viral life cycles and viral effectors in molecular detail. As such, this review also discusses virallyencoded effectors, found to be essential for virus survival and replication in the host milieu, which may be utilized as potential candidates for development of alternative therapies in the future. Abstract Angiotensin II (Ang II) plays an important role in cardiomyocyte hypertrophy. The combined effect of hepatocyte growth factor (HGF) and Ang II on cardiomyocytes is unknown. The present study was designed to determine the effect of HGF on cardiomyocyte hypertrophy and to explore the combined effect of HGF and Ang II on cardiomyocyte hypertrophy. Primary cardiomyocytes were isolated from neonatal rat hearts and cultured in vitro. Cells were treated with Ang II (1 µM) alone, HGF (10 ng/mL) alone, and Ang II (1 µM) plus HGF (10 ng/mL) for 24, 48, and 72 h. The amount of [ 3 H]-leucine incorporation was then measured to evaluate protein synthesis. The mRNA levels of β-myosin heavy chain and atrial natriuretic factor were determined by real-time PCR to evaluate the presence of fetal phenotypes of gene expression. The cell size of cardiomyocytes was also studied. Ang II (1 µM) increased cardiomyocyte hypertrophy. Similar to Ang II, treatment with 1 µM HGF promoted cardiomyocyte hypertrophy. Moreover, the combination of 1 µM Ang II and 10 ng/mL HGF clearly induced a combined pro-hypertrophy effect on cardiomyocytes. The present study demonstrates for the first time a novel, combined effect of HGF and Ang II in promoting cardiomyocyte hypertrophy. Abstract Background: Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in humans in 2012. A systematic literature review was conducted to synthesize current knowledge and identify critical knowledge gaps. Materials and Methods: We conducted a systematic review on MERS-CoV using PRISMA guidelines. We identified 407 relevant, peer-reviewed publications and selected 208 of these based on their contributions to four key areas: virology; clinical characteristics, outcomes, therapeutic and preventive options; epidemiology and transmission; and animal interface and the search for natural hosts of MERS-CoV. Results: Dipeptidyl peptidase 4 (DPP4/CD26) was identified as the human receptor for MERS-CoV, and a variety of molecular and serological assays developed. Dromedary camels remain the only documented zoonotic source of human infection, but MERS-like CoVs have been detected in bat species globally, as well as in dromedary camels throughout the Middle East and Africa. However, despite evidence of camel-to-human MERS-CoV transmission and cases apparently related to camel contact, the source of many primary cases remains unknown. There have been sustained health care-associated human outbreaks in Saudi Arabia and South Korea, the latter originating from one traveler returning from the Middle East. Transmission mechanisms are poorly understood; for health care, this may include environmental contamination. Various potential therapeutics have been identified, but not yet evaluated in human clinical trials. At least one candidate vaccine has progressed to Phase I trials.Conclusions: There has been substantial MERS-CoV research since 2012, but significant knowledge gaps persist, especially in epidemiology and natural history of the infection. There have been few rigorous studies of baseline prevalence, transmission, and spectrum of disease. Terms such as ''camel exposure'' and the epidemiological relationships of cases should be clearly defined and standardized. We strongly recommend a shared and accessible registry or database. Coronaviruses will likely continue to emerge, arguing for a unified ''One Health'' approach. Abstract Introduction Viral acute respiratory infections (ARIs) cause substantial child morbidity. Sensitive molecularbased assays aid virus detection, but the clinical significance of positive tests remains uncertain as some viruses may be found in both acutely ill and healthy children. We describe disease-pathogen associations of respiratory viruses and quantify virus-specific attributable risk of ARIs in healthy children during the first 2 years of life. Methods One hundred fifty-eight term newborn babies in Brisbane, Australia, were recruited progressively into a longitudinal, community-based, birth cohort study conducted between September 2010 and October 2014. A daily tick-box diary captured predefined respiratory symptoms from birth until their second birthday. Weekly parent-collected nasal swabs were batch-tested for 17 respiratory viruses by PCR assays, allowing calculation of virus-specific attributable fractions in the exposed (AFE) to determine the proportion of virus-positive children whose ARI symptoms could be attributed to that particular virus. Results Of 8100 nasal swabs analysed, 2646 (32.7%) were virus-positive (275 virus codetections, 3.4%), with human rhinoviruses accounting for 2058/2646 (77.8%) positive swabs. Viruses were detected in 1154/1530 (75.4%) ARI episodes and in 984/4308 (22.8%) swabs from asymptomatic periods. Respiratory syncytial virus (AFE: 68% (95% CI 45% to 82%)) and human metapneumovirus (AFE: 69% (95% CI 43% to 83%)) were strongly associated with higher risk of lower respiratory symptoms. Discussion The strong association of respiratory syncytial virus and human metapneumovirus with ARIs and lower respiratory symptoms in young children managed within the community indicates successful development of vaccines against these two viruses should provide substantial health benefits. Abstract Small-cell lung cancer (SCLC) is a lung cancer histological subtype unusual in its favorable response to cytotoxic chemotherapy. Life-threatening manifestations at presentation are rarely reported and should be an important clinical concern. We report a case of a 63-yearold man presenting with rapid-onset refractory severe thrombocytopenia, development of massive hemoptysis, and death from respiratory failure. This case provides clinicians a reference for this unusual presentation and carries clinical implications for managing SCLC patients. Abstract Chikungunya fever is caused by Chikungunya virus (CHIKV) and is generally considered a self-limiting disease. However, severe clinical presentations with a high mortality rate have been reported in association with underlying medical conditions. This study reports the molecular characterization of the virus and an abnormal pattern of circulating cytokines in a unique lethal CHIKV case during the 2017 outbreak in Italy, which involved an elderly patient with underlying cardiac disease. Analysis of inflammatory cytokines revealed a strong increase of interferon (IFN)-α and IFN-β, as well as interleukin-6, suggesting a possible role of type-I IFN in the cytokine storm, which may be correlated with unfavorable prognosis of CHIKV infection. Abstract Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv). Here, we have analyzed the disulfide bonding of vv proteins in detail. In vv-infected cells cytoplasmically synthesized vv core proteins became disulfide bonded in the newly assembled intracellular mature viruses (IMVs). vv membrane proteins also assembled disulfide bonds, but independent of IMV formation and to a large extent on their cytoplasmic domains. If disulfide bonding was prevented, virus assembly was only partially impaired as shown by electron microscopy as well as a biochemical assay of IMV formation. Under these con-ditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core. During the viral infection process the membrane proteins remained disulfide bonded, whereas the core proteins were reduced, concomitant with delivery of the cores into the cytoplasm. Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV. Abstract The rate-limiting enzyme in the mevalonic acid (MVA) pathway which can lead to triterpenoid saponin glycyrrhizic acid (GA) is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway, the HMGR gene from Glycyrrhiza uralensis Fisch. (GuHMGR) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC. The results showed that all the recombinant P. pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains. However, as the copy number increased, the content of ergosterol showed an increasing-decreasing-increasing pattern. This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G. uralensis. Abstract Background. Parainfluenza virus (PIV) is a common cause of acute respiratory tract infections, but little is known about PIV infection in children and adults in Africa, especially in settings where human immunodeficiency virus (HIV) prevalence is high.Methods. We conducted active, prospective sentinel surveillance for children and adults hospitalized with severe acute respiratory illness (SARI) from 2009 to 2014 in South Africa. We enrolled controls (outpatients without febrile or respiratory illness) to calculate the attributable fraction for PIV infection. Respiratory specimens were tested by multiplex real-time reverse-transcription polymerase chain reaction assay for parainfluenza types 1, 2, and 3.Results. Of 18 282 SARI cases enrolled, 1188 (6.5%) tested positive for any PIV type: 230 (19.4%) were type 1; 168 (14.1%) were type 2; 762 (64.1%) were type 3; and 28 (2.4%) had coinfection with 2 PIV types. After adjusting for age, HIV serostatus, and respiratory viral coinfection, the attributable fraction for PIV was 65.6% (95% CI [confidence interval], 47.1-77.7); PIV contributed to SARI among HIV-infected and -uninfected children <5 years of age and among individuals infected with PIV types 1 and 3. The observed overall incidence of PIV-associated SARI was 38 (95% CI, 36-39) cases per 100 000 population and was highest in children <1 year of age (925 [95% CI, 864-989] cases per 100 000 population). Compared with persons without HIV, persons with HIV had an increased relative risk of PIV hospitalization (9.4; 95% CI, 8.5-10.3).Conclusions. Parainfluenza virus causes substantial severe respiratory disease in South Africa among children <5 years of age, especially those that are infected with HIV. Abstract Biomarkers enable early diagnosis, guide molecularly targeted therapy and monitor the activity and therapeutic responses across a variety of diseases. Despite intensified interest and research, however, the overall rate of development of novel biomarkers has been falling. Moreover, no solution is yet available that efficiently retrieves and processes biomarker information pertaining to infectious diseases. Infectious Disease Biomarker Database (IDBD) is one of the first efforts to build an easily accessible and comprehensive literature-derived database covering known infectious disease biomarkers. IDBD is a community annotation database, utilizing collaborative Web 2.0 features, providing a convenient user interface to input and revise data online. It allows users to link infectious diseases or pathogens to protein, gene or carbohydrate biomarkers through the use of search tools. It supports various types of data searches and application tools to analyze sequence and structure features of potential and validated biomarkers. Currently, IDBD integrates 611 biomarkers for 66 infectious diseases and 70 pathogens. It is publicly accessible at Abstract Context: Evidence of the effect of continuous quality improvement (CQI) in public health and valid tools to judge that such effects are not fully formed. Objective: The objective was to adapt and apply Shortell et al.' s (1998) four dimensions of CQI in an examination of a public health accountability and performance management initiative in Ontario, Canada. Methods: In total, 24 semi-structured, in-depth interviews were conducted with informants from public health units and the Ministry of Health and Long-Term Care. A web survey of public health managers in the province was also carried out. Results: A mix of facilitators and barriers was identified. Leadership and organizational cultures, conducive to CQI success were evident. However, limitations in performance measurement and managerial discretion were key barriers. Conclusion: The four dimensions of CQI provided insight into both facilitators and barriers of CQI adoption in public health. Future research should compare the outcomes of public health CQI initiatives to the framework' s stated facilitators and barriers. Abstract The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The results indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully. Abstract Objective: The analysis of epidemiology, risk factors and outcome of viral infections in children and adolescents after hematopoietic cell transplantation (HCT). Methods: In this multicenter nationwide study a total of 971 HCT procedures (741 allo-HCT; 230 auto-HCT) over a period of 6 years were analyzed. Results: During this period 801 episodes of viral infections were diagnosed in 442 patients. The incidence of viral infections was 57.9% in allo-HCT and 4.8% in auto-HCT patients. The most frequent infections after allo-HCT were caused by cytomegalovirus (CMV), polyoma BK virus (BKV) and Epstein-Barr virus (EBV). The majority of infections occurred within the first 4 months after allo-HCT and over 80% required pharmacotherapy or symptomatic therapy. The median time of treatment of specific viral infection ranged from 7 (for EBV) to 24 (for CMV) days. The highest mortality was observed in case of CMV infection. The risk factors for viral infections were allo-HCT, acute leukemia, acute and chronic graft versus host disease (a/cGVHD), and matched unrelated donor (MUD)/mismatched unrelated donor (MMUD)-HCT. The risk factor for death from viral infection were CMV-IgG seropositivity in acute lymphoblastic leukemia recipient, and MUD/MMUD-HCT. The incidence of EBV infection requiring pre-emptive treatment with rituximab in allo-HCT children was 19.3%. In 30.8% cases of EBV infection, these episodes were preceded by other viral infection and treated with antivirals, which did not prevent development of EBV-DNA-emia with need of rituximab treatment in 81.5% cases. In 47.7% of these cases, GVHD was a factor enabling development of significant EBV-DNA-emia during antiviral therapy of other infection. Conclusion: We have shown that antiviral drugs do not prevent EBV reactivation in allo-HCT pediatric patients. Abstract We report a case of acute disseminated encephalomyelitis (ADEM) secondary to Mycoplasma pneumoniae infection that failed to improve with methylprednisolone and intravenous immunoglobulin (IVIG); who responded with plasmapheresis. A 21-year-old female with an unremarkable medical history, initially presented to an outside hospital with fever and an influenza-like illness and was subsequently intubated for worsening sensorium. Brain magnetic resonance imaging was suggestive of ADEM or vasculitis for which she received five days of pulse steroids and IVIG. She showed no signs of improvement and was transferred to our hospital for plasmapheresis. Her work up revealed an elevated IgM antibody and positive sputum for Mycoplasma pneumonia by polymerase chain reaction, suggesting the pathogen as the culprit for her ADEM. Intravenous azithromycin and daily plasmapheresis were initiated for seven consecutive days. Following commencement of her treatment, the patient experienced good recovery and was subsequently extubated. She continued to improve with physical therapy and gained mobility, with the help of a walker. Patients commonly present with ADEM following viral infection or vaccination and less frequently post bacterial infection. The current treatment of ADEM due to Mycoplasma pneumoniae is based on limited case reports. Our patient poorly responded to pulse steroids and IVIG, while she markedly improved on azithromycin and plasmapheresis. In patients presenting with encephalopathic signs and neurological manifestations following pneumonia; Mycoplasma pneumoniae infection and subsequent immune-mediated demyelination should be considered. (A. Laila), ellabar@clevelandclinicabudhabi.ae (R.M. El-Lababidi), hishamm@clevelandclinicabudhabi.ae (M. Hisham), MootyM@ClevelandClinicAbuDhabi.ae (M. Mooty). IDCases 12 (2018) 41-43 2214-2509/ Abstract A 12-year-old, 3.6-kg, spayed female domestic shorthaired cat had a 2-month history of anorexia and weight loss. Abdominal ultrasonography and computed tomography revealed an exophytic mass originating from the jejunum with very poor central and poor peripheral contrast enhancement. On day 14, surgical resection of the jejunum and mass with 5-cm margins and an end-to-end anastomosis were performed. Histopathological examination revealed the mass was a transmural, invasive cancer showing exophytic growth and originating from the small intestinal muscle layer. Immunohistochemical analysis of tumor cells revealed diffuse positivity for KIT protein and negativity for desmin and S-100. The mass was diagnosed as a gastrointestinal stromal tumor (GIST). Ultrasonographic findings indicated the tumor probably metastasized to the liver and omentum, as seen in humans and dogs. The owner rejected further treatment at the last visit on day 192. To our knowledge, this is the first report of intestinal tumor and metastasis in feline GIST and its imaging features.Intestinal tumors are generally rare in cats. Lymphomas account for nearly 30% of all feline tumors and are the most common intestinal tumors [20] . Adenocarcinomas are the second most frequent tumors in cats, followed by mast cell tumors. Gastrointestinal stromal tumors (GISTs) are thought to originate from the interstitial cells of Cajal (ICCs), which form a network that coordinates peristalsis in the gastrointestinal tract [11] . Many of the smooth muscle tumors in human and canine patients have now been reclassified as GISTs, which by definition express CD117 (KIT) [13, 15] . GISTs have been described in humans, dogs, horses, Spanish ibex, a ferret, a rat, and nonhuman primates [5, 18, 19] . These occur most frequently in the jejunum, cecum and colon [16] . To the authors' knowledge, only one report has described "a feline GIST" in the stomach [17] .A 12-year-old, 3.6-kg, spayed female domestic shorthaired cat with a body condition score of 2/5 presented with a 2-month history of anorexia and weight loss. On physical examination, the animal had a palpable large mass (about 5.0 cm) in the abdomen. No lymphadenopathies were noted at the superficial lymph nodes. A complete blood count test revealed a low packed cell volume (PCV, 24.0%; reference range, 30.3-52.3%), neutrophilia (21,141/µl; reference range, 2,500-12,500/µl) and monocytosis (1,292/µl; reference range, 0-850/µl). A serum biochemical profile revealed hypoalbuminemia (2.6 g/dl; reference range, 2.7-3.8 g/dl) and hyperglobulinemia (5.4 g/dl; reference range, 2.8-5.1 g/dl). The serum protein fraction was normal. Enzyme-linked immunosorbent assays for feline leukemia virus and feline immunodeficiency virus yielded negative results. Radiography indicated a large mass in the abdomen. Abdominal ultrasonography revealed a hypoechoic mass (approximately 41.8 × 60.6 mm) containing hyperechoic and hypoechoic areas of unknown origin and a nodule (approximately 8.4 × 9.5 mm) containing a hypoechoic rim and an isoechoic center in the left lateral lobe of the liver (Fig. 1A and 1B) . Moreover, ultrasound-guided fine-needle aspiration of the large mass showed mostly macrophages and neutrophils. Polymerase chain reaction analysis of the abdominal mass sample was negative for feline coronavirus. On day 7, contrast-enhanced computed tomography (CT) was performed under anesthesia. Iopamidol (300 mg I/ml, Oypalomin injection 300; Fuji Pharma Co., Ltd., Toyama, Japan) was used as the contrast medium at a dose of 2 ml/ kg and was injected using a power injector over 15 to 20 sec via the jugular vein. Images were acquired before contrast medium injection, in an arterial phase (20 sec), in a portal phase (40 sec) and at equilibrium (180 sec) after contrast medium injection. CT examination revealed the exophytic mass originating from the jejunum. It had very poor contrast enhancement centrally and poor contrast enhancement peripherally (Fig. 1C) . It was assumed to reflect a cavity of necrotic tissue. In the left lateral lobe of the liver, the hypoattenuating nodule was observed in all the phases (Fig. 1D) . We concluded that the mass originated from the jejunum and could be surgically resected. On day 14, laparotomy was used to expose the mass originating from the jejunum that had an irregular surface and adhesive omentum. Surgical resection of the jejunum including the mass with 5-cm margins and an end-to-end anastomosis were performed ( Fig. 2A) . During surgery, we found enlargement of mesenteric lymph nodes Abstract A B S T R A C T Cytomegalovirus (CMV) pneumonia is a rare opportunistic infection in the setting of HIV (Human Immunodeficiency Virus)-infection. Establishing accurate diagnosis of CMV pneumonia in HIV-infection can be challenging. Co-infections by multiple opportunistic pathogens are common and a high degree of clinical vigilance to evaluate for multiple infections, including CMV pneumonia, should be maintained. As there can be a degree of overlap in clinical and radiological features amongst different opportunistic infections affecting the lungs, definitive microbiological and cytohistologic evidences are needed. Reliance on microbiological evidence of CMV in respiratory specimens alone for the diagnosis of CMV pneumonia will lead to an over-diagnosis of the condition and unnecessary treatment.In our case report, we describe a 53-year-old man with recently diagnosed HIV-infection who presented with non-resolving pneumonia. A diagnosis of CMV pneumonia was reached through consistent clinical, radiological, microbiological and cytologic investigations. The patient made a full clinical recovery after being started on anti-CMV treatment. Abstract Meningoencephalitis (ME) is a common inflammatory disorder of the central nervous system in dogs. Clinically, ME has both infectious and non-infectious causes. In the present study, a multiplex quantitative real-time polymerase chain reaction (mqPCR) panel was optimized for the detection of eight canine neurologic pathogens (Blastomyces dermatitidis, Cryptococcus spp., Neospora caninum, Borrelia burgdorferi, Bartonella spp., Toxoplasma gondii, Ehrlichia canis, and canine distemper virus [CDV]). The mqPCR panel was subsequently applied to 53 cerebrospinal fluid (CSF) samples collected from dogs with ME. The analytic sensitivity (i.e., limit of detection, expressed as molecules per 1 L of recombinant vector) was 3.8 for CDV, 3.7 for Ehrlichia canis, 3.7 for Bartonella spp., 3.8 for Borrelia burgdorferi, 3.7 for Blastomyces dermatitidis, 3.7 for Cryptococcus spp., 38 for Neospora caninum, and 3.7 for Toxoplasma gondii. Among the tested CSF samples, seven (15%) were positive for the following pathogens in decreasing order of frequency: Cryptococcus spp. (3/7), Blastomyces dermatitidis (2/7), and Borrelia burgdorferi (2/7). In summary, use of an mqPCR panel with high analytic sensitivity as an initial screen for infectious agents in dogs with ME could facilitate the selection of early treatment strategies and improve outcomes. Abstract Here we use novel methods of phylogenetic transmission graph analysis to reconstruct the geographic spread of MERS-CoV. We compare these results to those derived from text mining and visualization of the World Health Organization's (WHO) Disease Outbreak News. Abstract Objective: To describe detailed clinical and radiological features of the pandemic H1N1 2009 influenza viral infection among healthy young males in a semiclosed institutionalized setting.A total of 18 patients confirmed with the pandemic H1N1 Abstract Prion diseases, also called transmissible spongiform encephalopathies (TSEs), lead to neurological dysfunction in animals and are fatal. Infectious prion proteins are causative agents of many mammalian TSEs, including scrapie (in sheep), chronic wasting disease (in deer and elk), bovine spongiform encephalopathy (BSE; in cattle), and CreutzfeldteJakob disease (CJD; in humans). BSE, better known as mad cow disease, is among the many recently discovered zoonotic diseases. BSE cases were first reported in the United Kingdom in 1986. Variant CJD (vCJD) is a disease that was first detected in 1996, which affects humans and is linked to the BSE epidemic in cattle. vCJD is presumed to be caused by consumption of contaminated meat and other food products derived from affected cattle. The BSE epidemic peaked in 1992 and decreased thereafter; this decline is continuing sharply owing to intensive surveillance and screening programs in the Western world. However, there are still new outbreaks and/or progression of prion diseases, including atypical BSE, and iatrogenic CJD and vCJD via organ transplantation and blood transfusion. This paper summarizes studies on prions, particularly on prion molecular mechanisms, BSE, vCJD, and diagnostic procedures. Risk perception and communication policies of the European Union for the prevention of prion diseases are also addressed to provide recommendations for appropriate government policies in Korea. Abstract A novel bat-origin coronavirus emerged in Wuhan, China in December 2019 and continues to spread across China and the world. At the time of writing, a massive global response has been implemented to control the disease as it spreads from person to person. Yet the high-risk human-wildlife interactions and interfaces that led to the emergence of SARS-CoV and of 2019-nCoV continue to exist in emerging disease hotspots globally. To prevent the next epidemic and pandemic related to these interfaces, we call for research and investment in three areas: 1) surveillance among wildlife to identify the high-risk pathogens they carry; 2) surveillance among people who have contact with wildlife to identify early spillover events; and 3) improvement of market biosecurity regarding the wildlife trade. As the emergence of a novel virus anywhere can impact the furthest reaches of our connected world, international collaboration among scientists is essential to address these risks and prevent the next pandemic. Abstract Osteoporosis is a common skeletal disorder characterized by a decrease in bone mass and density. The peak bone mass (PBM) is a significant determinant of osteoporosis. To gain insights into the indicating effect of PBM to osteoporosis, this study focused on characterizing the PBM networks and identifying key genes. One biological data set with 12 monocyte low PBM samples and 11 high PBM samples was derived to construct protein-protein interaction networks (PPINs). Based on clique-merging, module-identification algorithm was used to identify modules from PPINs. The systematic calculation and comparison were performed to test whether the network entropy can discriminate the low PBM network from high PBM network. We constructed 32 destination networks with 66 modules divided from monocyte low and high PBM networks. Among them, network 11 was the only significantly differential one (P<0.05) with 8 nodes and 28 edges. All genes belonged to precursors of osteoclasts, which were related to calcium transport as well as blood monocytes. In conclusion, based on the entropy in PBM PPINs, the differential network appears to be a novel therapeutic indicator for osteoporosis during the bone monocyte progression; these findings are helpful in disclosing the pathogenetic mechanisms of osteoporosis. Abstract We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem-loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents. Abstract Ribophorins I and II are type I transmembrane glycoproteins of the ER that are segregated to the rough domains of this organelle. Both ribophorins appear to be part of the translocation apparatus for nascent polypeptides that is associated with membranebound ribosomes and participate in the formation of a proteinaceous network within the ER membrane that also includes other components of the translocation apparatus. The ribophorins are both highly stable proteins that lack O-linked sugars but each contains one high mannose N-linked oligosaccharide that remains endo H sensitive throughout their lifetimes.We have previously shown (Tsao, Y. S., N. E. Ivessa, M. Adesnik, D. D. Sabatini, and G. Kreibich. 1992. J. Cell Biol. 116:57-67) that a COOHterminally truncated variant of ribophorin I that contains only the first 332 amino acids of the luminal domain (RI3a2), when synthesized in permanent transformants of HeLa cells, undergoes a rapid degradation with biphasic kinetics in the ER itself and in a second, as yet unidentified nonlysosomal pre-Golgi compartment. We now show that in cells treated with brefeldin A (BFA) RI332 molecules undergo rapid O-glycosylation in a multistep process that involves the 9 The Rockefeller University Press, Cultures of HeLa-RI332 cells were fixed first in the dishes with 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, and after scraping the cells were left in this fixative for a total of 2 h. Samples were stored overnight in 0.1 M sodium caeodylatr buffer and processed for cryomicrotomy and immunolabeling, essentially as described by Tokuyasu (1980) with minor modifications (Ivanov et al., 1984; De Lemos-Chiarandini et al., 1987) . Lectin labeling with WGA or RCA was carried out as described by Caiffiths et al. (1982), using anti-lectin antibodies and protein A-gold (5-ram-diameter). Sections infused with LR White acrylic resin (Keller et al., 1984) were examined with a Philips 301 electron microscope operated at 60 kV. Abstract b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 769-773 h t t p : / / w w w . b j m i c r o b i o l . c o m . b r / Mexico Parvovirus Rotavirus aThis is the first report on circulating canine rotavirus in Mexico. Fifty samples from dogs with gastroenteritis were analyzed used polymerase chain reaction and reverse transcription polymerase chain reaction in order to identify parvovirus and rotavirus, respectively; 7% of dogs were infected with rotavirus exclusively, while 14% were co-infected with both rotavirus and parvovirus; clinical signs in co-infected dogs were more severe. Abstract Insensitivity and technical complexity have impeded the implementation of high-throughput nucleic acid sequencing in differential diagnosis of viral infections in clinical laboratories. Here, we describe the development of a virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) that increases the sensitivity of sequence-based virus detection and characterization. The system uses~2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates, including humans. A biotinylated oligonucleotide library was synthesized on the NimbleGen cleavable array platform and used for solution-based capture of viral nucleic acids present in complex samples containing variable proportions of viral and host nucleic acids. The use of VirCapSeq-VERT resulted in a 100-to 10,000-fold increase in viral reads from blood and tissue homogenates compared to conventional Illumina sequencing using established virus enrichment procedures, including filtration, nuclease treatments, and RiboZero rRNA subtraction. VirCapSeq-VERT had a limit of detection comparable to that of agent-specific real-time PCR in serum, blood, and tissue extracts. Furthermore, the method identified novel viruses whose genomes were approximately 40% different from the known virus genomes used for designing the probe library. The VirCapSeq-VERT platform is ideally suited for analyses of virome composition and dynamics. IMPORTANCE VirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications.Citation Briese T, Kapoor A, Mishra N, Jain K, Kumar A, Jabado OJ, Lipkin WI. 2015. Virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis. mBio 6(5):e01491-15. Abstract Middle east respiratory syndrome coronavirus. N Engl J Med. 2013; 369:407-16.ReFeRenCes Abstract Breathe | December 2017 | Volume 13 | No 4 317 Cite as: Castellana G, Liotino V, Vulpi MR, et al. Bilateral pulmonary nodules and acute respiratory failure in a 22-year-old man with dyspnoea and fever. Breathe 2017; 13: 317-322.Bilateral pulmonary nodules and acute respiratory failure in a 22-year-old man with dyspnoea and fever A 22-year-old white male presented complaining of a 3-week history of fatigue, cough and haemoptysis, accompanied by fever (38°C max), dyspnoea and mucopurulent sputum in the last few days. He denied nausea, vomiting, diarrhoea, dysuria and weight loss. He reported being a smoker (10 pack-years) and a cannabis user, and had a history of allergic rhinitis. He denied occupational exposures.Before our evaluation, he received empiric antibiotic therapy for 5 days, which resulted in the abatement of fever. Following the persistence of dyspnoea and haemoptysis, he had chest radiography with multiple nodular opacities associated with pleural effusion on the left (figure 1) and lung ultrasound (US) with a convex probe showing multiple hypoechoic, bilateral foci associated with B-lines and bilateral pleural effusion, mainly on the left. Abstract Fast viral adaptation and the implication of this rapid evolution in the emergence of several new infectious diseases have turned this issue into a major challenge for various research domains. Indeed, viruses are involved in the development of a wide range of pathologies and understanding how viruses and host cells interact in the context of adaptation remains an open question. In order to provide insights into the complex interactions between viruses and their host organisms and namely in the acquisition of novel functions through exchanges of genetic material, we developed the PhEVER database. This database aims at providing accurate evolutionary and phylogenetic information to analyse the nature of virus-virus and virus-host lateral gene transfers. PhEVER (http:// pbil.univ-lyon1.fr/databases/phever) is a unique database of homologous families both (i) between sequences from different viruses and (ii) between viral sequences and sequences from cellular organisms. PhEVER integrates extensive data from up-to-date completely sequenced genomes (2426 non-redundant viral genomes, 1007 non-redundant prokaryotic genomes, 43 eukaryotic genomes ranging from plants to vertebrates) and offers a clustering of proteins into homologous families containing at least one viral sequences, as well as alignments and phylogenies for each of these families. Public access to PhEVER is available through its webpage and through all dedicated ACNUC retrieval systems. Abstract Objective To examine recent time trends in the incidence of osteonecrosis in Denmark and to investigate different common comorbidities association with osteonecrosis in a population* based setting. Abstract Purpose: Respiratory syncytial virus (RSV) infection can cause various neurological complications. This study aimed to investigate the RSV-associated neurologic manifestations that present with seizures. Methods: We retrospectively reviewed the medical records of patients aged less than 15 years with laboratory-confirmed RSV infections and seizures between January 2011 and December 2016 in a regional hospital in South Korea. Results: During this period, 1,193 patients with laboratory-confirmed RSV infection were identified. Of these, 35 (35 of 1,193, 2.93%; boys, 19; girls, 16; mean age: 20.8 ± 16.6 months) presented with sei zure. Febrile seizure was the most common diagnosis (27 of 35, 77.1%); simple febrile seizures in 13 patients (13 of 27, 48.1%) and complex febrile seizures in 14 (14 of 27, 51.9%). Afebrile seizures without meningitis or encephalo pathy were observed in 5 patients (5 of 35, 14.3%), seizures with meningitis in 2 (2 of 35, 5.7%), and seizure with encephalopathy in 1 (1 of 35, 2.9%) patient. Lower respiratory symptoms were not observed in 8 patients. In a patient with encephalopathy, brain diffusion-weighted magnetic resonance imaging revealed transient changes in white matter, suggesting cytotoxic edema as the mechanism underlying encephalopathy. Most patients recovered with general management, and progression to epilepsy was noted in only 1 patient. Conclusion: Although febrile seizures are the most common type of seizure associated with RSV infection, the propor tion of patients with complex febrile seizures was higher than that of those with general febrile seizures. Transient cytotoxic edema may be a pathogenic mechanism in RSV-related encephalopathy with seizures. Abstract Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves.Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs. Abstract Influenza is a viral infection of the respiratory tract. Infection is normally confined to the upper respiratory tract but certain viral strains have evolved the ability to infect the lower respiratory tract, including the alveoli, leading to inflammation and a disease pattern of diffuse alveolar damage. Factors leading to this sequence of events are novel influenza strains, or strains that have viral proteins, in particular the NS1 protein that allow it to escape the innate immune system. There are three main barriers that prevent infection of pneumocytes -mucin, host defence lectins and cells such as macrophages. Viruses have developed strategies such as neuraminidase and glycosylation patterns that allow this evasion. Though there has been much investment in antiviral drugs, it is proposed that more attention should be directed towards developing or utilizing compounds that enhance the ability of the innate immune system to combat viral infection. Abstract Canine infectious respiratory disease complex (CIRDC) is associated with multiple factors. The possible transmission source can be via community-acquired infection (CAI) or hospital-associated infection (HAI), but the variable factors within these two routes are not well described. This study aimed to (i) investigate a crosssectional incidence of canine respiratory viruses, including influenza (CIV), parainfluenza, distemper (CDV), respiratory coronavirus (CRCoV), adenovirus-2, and herpesvirus, in respiratory-diseased dogs, and (ii) analyze the possibly related risk factors. In total 209 dogs with respiratory illness, consisting of 133 CAI and 76 HAI dogs, were studied. Both nasal and oropharyngeal swabs were sampled from each dog and subjected for CIRDC virus detection using multiplex PCRs. Common six viruses associated with CIRDC were detected in both groups with CIV and CRCoV being predominantly found. Only CDV was significantly more prevalent in CAI than HAI dogs. Multiple virus detections were found in 81.2% and 78.9% of CAI and HAI dogs, respectively. Co-detection of CIV and CRCoV was represented the highest proportion and most often found with other CIRD viruses. Moreover, the clinical severity level was notably related to the age of infected dogs, but not to the vaccination status, sex and transmission route. Since healthy or control dogs were not included in this study, the prevalence of the CIRD virus infections could not be assessed. Abstract Asthma is a common disorder of the airways characterized by airway inflammation and by decline in lung function and airway remodeling in a subset of asthmatics. Airway remodeling is characterized by structural changes which include airway smooth muscle hypertrophy/ hyperplasia, subepithelial fibrosis due to thickening of the reticular basement membrane, mucus metaplasia of the epithelium, and angiogenesis. Epidemiologic studies suggest that both genetic and environmental factors may contribute to decline in lung function and airway remodeling in a subset of asthmatics. Environmental factors include respiratory viral infection-triggered asthma exacerbations, and tobacco smoke. There is also evidence that several asthma candidate genes may contribute to decline in lung function, including ADAM33, PLAUR, VEGF, IL13, CHI3L1, TSLP, GSDMB, TGFB1, POSTN, ESR1 and ARG2. In addition, mediators or cytokines, including cysteinyl leukotrienes, matrix metallopeptidase-9, interleukin-33 and eosinophil expression of transforming growth factor-β, may contribute to airway remodeling in asthma. Although increased airway smooth muscle is associated with reduced lung function (i.e. forced expiratory volume in 1 second) in asthma, there have been few long-term studies to determine how individual pathologic features of airway remodeling contribute to decline in lung function in asthma. Clinical studies with inhibitors of individual gene products, cytokines or mediators are needed in asthmatic patients to identify their individual role in decline in lung function and/or airway remodeling. Abstract Cannabis sativa cannabinoids cannabidiol CBD toxicology olive oil hemp extract CYP liver mutagenicity CW hemp Charlotte's Web, Inc.Cannabinoids are extracted from Cannabis sativa L. and are used for a variety of medicinal purposes. Recently, there has been a focus on the cannabinoid Cannabidiol (CBD) and its potential benefits. This study investigated the safety of a proprietary extract of C. sativa, consisting of 9% hemp extract (of which 6.27% is CBD) and 91% olive oil. The mutagenic potential of the hemp extract was evaluated with the AMES assay inclusive of a hepatic drug metabolizing mix (S9) rich in CYP enzymes. The test article did not elicit evidence of bacterial mutagenicity. GLP compliant 14-day and a 90-day toxicity study were conducted. Olive oil was used as a control. The 90-day study had a 28-day recovery period. Treatments for the 14-day non-recovery range-finding study were 0, 1000, 2000 and 4000 mg test article/kg body weight (bw)/day for 14 days. There was a non-statistically significant (p > 0.05) decrease in body weights for the male and female rats receiving the test article. Hypoactivity, hyperactivity, reduced food consumption and piloerection were observed in the rats receiving 4000 mg test article/kg bw. Histopathology showed an increase in the size of liver cells (hypertrophy) around the central vein (centrilobular) in Groups 3 (3/10) and 4 (5/10) that correlated with increased liver weights. In the 90-day study, 8 groups of rats were dosed with 0, 200, 400 and 800 mg test article/kg bw/day. Groups 5 to 8 had a 28-day recovery. There were no test article-linked changes in clinical observations, physical examinations, Functional Observation Battery, ophthalmology, Motor Activity Assessment, hematology, clinical chemistries and macropathology (all groups). With the exception of the liver and adrenal gland, no test article-linked pathology was observed. For all rats receiving the test article, histopathology showed hypertrophy of liver cells around the central vein. The increase of liver weight is most likely caused by hypertrophy due to up-regulation of the hepatic drug metabolizing enzymes. The hepatocellular hypertrophy was completely reversed in 28 days and was not considered to be an adverse effect. Vacuolization of the adrenal zona fasciculata was observed in the control and 800 mg test article/kg bw groups. The vacuolization of the zona fasciculata was of the same incidence and severity in treatment and control male rats and correlated with an increased in the weights of the adrenal glands. In addition, a statistically significant increase (p < 0.05) in adrenal-to-body weight ratios was observed for females receiving 800 mg test article/kg bw. This increase in adrenal-to-body weight ratio did not correlate with any of the pathology findings. The NOAEL for the test article is 800 mg/kg bw/day for female and 400 mg/kg bw/day for male Sprague Dawley rats. Abstract Case summary A 6-year-old female neutered domestic shorthair cat from Cyprus was presented with multiple ulcerated skin nodules. Cytology and histopathology of the lesions revealed granulomatous dermatitis with intracytoplasmic organisms, consistent with amastigotes of Leishmania species. Biochemistry identified a mild hyperproteinaemia. Blood extraction and PCR detected Leishmania species, Hepatozoon species and 'Candidatus Mycoplasma haemominutum' (CMhm) DNA. Subsequent sequencing identified Hepatozoon felis. Additionally, the rRNA internal transcribed spacer 1 locus of Leishmania infantum was partially sequenced and phylogeny showed it to cluster with species derived from dogs in Italy and Uzbekistan, and a human in France. Allopurinol treatment was administered for 6 months. Clinical signs resolved in the second month of treatment with no deterioration 8 months post-treatment cessation. Quantitative PCR and ELISA were used to monitor L infantum blood DNA and antibody levels. The cat had high L infantum DNA levels pretreatment that gradually declined during treatment but increased 8 months post-treatment cessation. Similarly, ELISA revealed high levels of antibodies pretreatment, which gradually declined during treatment and increased slightly 8 months post-treatment cessation. The cat remained PCR positive for CMhm and Hepatozoon species throughout the study. There was no clinical evidence of relapse 24 months post-treatment. Relevance and novel information To our knowledge, this is the first clinical report of a cat with leishmaniosis with H felis and CMhm coinfections. The high L infantum DNA levels post-treatment cessation might indicate that although the lesions had resolved, prolonged or an alternative treatment could have been considered. Abstract An epidemic of a novel coronavirus emerged from Wuhan, China, in late December 2019 and has since spread to several large Chinese cities. Should a scenario arise where this coronavirus spreads more broadly across China, we evaluate how patterns of international disease transmission could change. Abstract The localization of proteins to late-Golgi Abstract During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For liveattenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wildtype phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent TC-83. During serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy. Abstract Field epidemiology involves the implementation of quick and targeted public health interventions with the aid of epidemiological methods. In this article, we share our practical experiences in outbreak management and in safeguarding the population against novel diseases. Given that cities represent the financial nexuses of the global economy, global health security necessitates the safeguard of cities against epidemic diseases. Singapore's public health landscape has undergone a systemic and irreversible shift with global connectivity, rapid urbanization, ecological change, increased affluence, as well as shifting demographic patterns over the past two decades. Concomitantly, the threat of epidemics, ranging from severe acute respiratory syndrome and influenza A (H1N1) to the resurgence of vector-borne diseases as well as the rise of modern lifestyle-related outbreaks, have worsened difficulties in safeguarding public health amidst much elusiveness and unpredictability. One critical factor that has helped the country overcome these innate and man-made public health vulnerabilities is the development of a resilient field epidemiology service, which includes our enhancement of surveillance and response capacities for outbreak management, and investment in public health leadership. We offer herein the Singapore story as a case study in meeting the challenges of disease control in our modern built environment. Abstract Given the manifold ways that depression impairs Darwinian fitness, the persistence in the human genome of risk alleles for the disorder remains a much debated mystery. Evolutionary theories that view depressive symptoms as adaptive fail to provide parsimonious explanations for why even mild depressive symptoms impair fitness-relevant social functioning, whereas theories that suggest that depression is maladaptive fail to account for the high prevalence of depression risk alleles in human populations. These limitations warrant novel explanations for the origin and persistence of depression risk alleles. Accordingly, studies on risk alleles for depression were identified using PubMed and Ovid MEDLINE to examine data supporting the hypothesis that risk alleles for depression originated and have been retained in the human genome because these alleles promote pathogen host defense, which includes an integrated suite of immunological and behavioral responses to infection. Depression risk alleles identified by both candidate gene and genome-wide association study (GWAS) methodologies were found to be regularly associated with immune responses to infection that were likely to enhance survival in the ancestral environment. Moreover, data support the role of specific depressive symptoms in pathogen host defense including hyperthermia, reduced bodily iron stores, conservation/withdrawal behavior, hypervigilance and anorexia. By shifting the adaptive context of depression risk alleles from relations with conspecifics to relations with the microbial world, the Pathogen Host Defense (PATHOS-D) hypothesis provides a novel explanation for how depression can be nonadaptive in the social realm, whereas its risk alleles are nonetheless represented at prevalence rates that bespeak an adaptive function. Abstract Monomolecular arrays of protein or glycoprotein subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope components. S-layers are generally the most abundantly expressed proteins, have been observed in species of nearly every taxonomical group of walled bacteria, and represent an almost universal feature of archaeal envelopes. The isoporous lattices completely covering the cell surface provide organisms with various selection advantages including functioning as protective coats, molecular sieves and ion traps, as structures involved in surface recognition and cell adhesion, and as antifouling layers. S-layers are also identified to contribute to virulence when present as a structural component of pathogens. In Archaea, most of which possess S-layers as exclusive wall component, they are involved in determining cell shape and cell division. Studies on structure, chemistry, genetics, assembly, function, and evolutionary relationship of S-layers revealed considerable application potential in (nano)biotechnology, biomimetics, biomedicine, and synthetic biology. Abstract COPD is characterized by an ongoing inflammatory process of the airways that leads to obstruction or limitation of airflow. It is mainly associated with exposure to cigarette smoke. In addition, it is considered, at present, a serious public health problem, ranking fourth in mortality worldwide. Many cells participate in the pathophysiology of COPD, the most important are neutrophils, macrophages and CD4+ and CD8+ T cells. Neutrophil migration to the inflammation area could be mediated largely by cytokines related to CD4+ Th17 lymphocytes, because it has been shown that IL-17A, IL-17F and IL-22 act as inducers for CXCL8, CXCL1, CXCL5, G-CSF, and GM-CSF secretion by epithelial cells of the airways. The aims of these molecules are differentiation, proliferation and recruitment of neutrophils. Furthermore, it is believed that CD4+ lymphocytes Th17 may be involved in protection against pathogens for which Th1 and Th2 are not prepared to fight. In COPD exacerbations, there is an increased cellularity in the lung region and respiratory tract. Therefore, the increase in the number of neutrophils and macrophages in the airways and the increase in proinflammatory cytokines are directly related to the severity of exacerbations and that is the importance of the functions of Th17 profile in this entity. Abstract Numerous viral infections have arisen and affected global healthcare facilities. Millions of people are at severe risk of acquiring several evolving viral infections through several factors. In the present article we have described about risk factors, chance of infection, and prevention methods of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV), human coronaviruses (CoVs) frequently cause a normal cold which is mild and self-restricting. Zoonotic transmission of CoVs such as the newly discovered MERS-CoV and SARS-CoV, may be associated with severe lower respiratory tract infection. The present review provides the recent clinical and pathological information on MERS and SARS. The task is to transform these discoveries about MERS and SARS pathogenesis and to develop intervention methods that will eventually allow the effective control of these recently arising severe viral infections. Global health sector has learnt many lessons through the recent outbreak of MERS and SARS, but the need for identifying new antiviral treatment was not learned. In the present article we have reviewed the literature on the several facets like transmission, precautions and effectiveness of treatments used in patients with MERS-CoV and SARS infections. Abstract Secondary lymphoid organ chemokine (SLC) is expressed in high endothelial venules and in T cell zones of spleen and lymph nodes (LNs) and strongly attracts naive T cells. In mice homozygous for the paucity of lymph node T cell ( plt ) mutation, naive T cells fail to home to LNs or the lymphoid regions of spleen. Here we demonstrate that expression of SLC is undetectable in plt mice. In addition to the defect in T cell homing, we demonstrate that dendritic cells (DCs) fail to accumulate in spleen and LN T cell zones of plt mice. DC migration to LNs after contact sensitization is also substantially reduced. The physiologic significance of these abnormalities in plt mice is indicated by a markedly increased sensitivity to infection with murine hepatitis virus. The plt mutation maps to the SLC locus; however, the sequence of SLC introns and exons in plt mice is normal. These findings suggest that the abnormalities in plt mice are due to a genetic defect in the expression of SLC and that SLC mediates the entry of naive T cells and antigen-stimulated DCs into the T cell zones of secondary lymphoid organs. Abstract Emergence of drug resistance among the causative organisms for respiratory tract infections represents a critical challenge to the global health care community. Further, although vaccination can prevent disease, vaccine development is impeded by several factors. Therefore, novel approaches to treat and manage respiratory infections are urgently needed. Passive immunization represents a possible alternative to meet this need. Immunoglobulin Y antibodies (IgYs) from the yolk of chicken eggs have previously been used against bacterial and viral infections in human and animals. Their advantages include lack of reaction with mammalian Fc receptors, low production cost, and ease of extraction. Compared to mammalian IgGs, they have higher target specificity and greater binding avidity. They also possess remarkable pathogen-neutralizing activity in the respiratory tract and lungs. In this review, we provide an overview of avian IgYs and describe their potential therapeutic applications for the prevention and treatment of respiratory infections.ARTICLE HISTORY Abstract Virus-induced apoptosis is thought to be the primary mechanism of cell death following reovirus infection. Induction of cell death following reovirus infection is initiated by the incoming viral capsid proteins during cell entry and occurs via NF-B-dependent activation of classical apoptotic pathways. Prototype reovirus strain T3D displays a higher cell-killing potential than strain T1L. To investigate how signaling pathways initiated by T3D and T1L differ, we methodically analyzed cell death pathways activated by these two viruses in L929 cells. We found that T3D activates NF-B, initiator caspases, and effector caspases to a significantly greater extent than T1L. Surprisingly, blockade of NF-B or caspases did not affect T3D-induced cell death. Cell death following T3D infection resulted in a reduction in cellular ATP levels and was sensitive to inhibition of the kinase activity of receptor interacting protein 1 (RIP1). Furthermore, membranes of T3D-infected cells were compromised. Based on the dispensability of caspases, a requirement for RIP1 kinase function, and the physiological status of infected cells, we conclude that reovirus can also induce an alternate, necrotic form of cell death described as necroptosis. We also found that induction of necroptosis requires synthesis of viral RNA or proteins, a step distinct from that necessary for the induction of apoptosis.Thus, our studies reveal that two different events in the reovirus replication cycle can injure host cells by distinct mechanisms.Citation Berger AK, Danthi P. 2013. Reovirus activates a caspase-independent cell death pathway. mBio 4(3):e00178-13. Abstract Background. Acute upper respiratory tract infections are a common cause of emergency department (ED) visits and often result in unnecessary antibiotic treatment.Methods. We conducted a randomized clinical trial to evaluate the impact of a rapid, multipathogen respiratory panel (RP) test vs usual care (control). Patients were eligible if they were ≥12 months old, had symptoms of upper respiratory infection or influenzalike illness, and were not on antibiotics. The primary outcome was antibiotic prescription; secondary outcomes included antiviral prescription, disposition, and length of stay (ClinicalTrials.gov# NCT02957136).Results. Of 191 patients enrolled, 93 (49%) received RP testing; 98 (51%) received usual care. Fifty-three (57%) RP and 7 (7%) control patients had a virus detected and reported during the ED visit (P = .0001). Twenty (22%) RP patients and 33 (34%) usual care patients received antibiotics during the ED visit (-12%; 95% confidence interval, -25% to 0.4%; P = .06/0.08); 9 RP patients received antibiotics despite having a virus detected. The magnitude of antibiotic reduction was greater in children (-19%) vs adults (-9%, post hoc analysis). There was no difference in antiviral use, length of stay, or disposition.Conclusions. Rapid RP testing was associated with a trend toward decreased antibiotic use, suggesting a potential benefit from more rapid viral tests in the ED. Future studies should determine if specific groups are more likely to benefit from testing and evaluate the relative cost and effectiveness of broad testing, focused testing, and a combined diagnostic and antimicrobial stewardship approach. Abstract Cannabis sativa cannabinoids cannabidiol CBD toxicology olive oil hemp extract CYP liver mutagenicity CW hemp Charlotte's Web, Inc.Cannabinoids are extracted from Cannabis sativa L. and are used for a variety of medicinal purposes. Recently, there has been a focus on the cannabinoid Cannabidiol (CBD) and its potential benefits. This study investigated the safety of a proprietary extract of C. sativa, consisting of 9% hemp extract (of which 6.27% is CBD) and 91% olive oil. The mutagenic potential of the hemp extract was evaluated with the AMES assay inclusive of a hepatic drug metabolizing mix (S9) rich in CYP enzymes. The test article did not elicit evidence of bacterial mutagenicity. GLP compliant 14-day and a 90-day toxicity study were conducted. Olive oil was used as a control. The 90-day study had a 28-day recovery period. Treatments for the 14-day non-recovery range-finding study were 0, 1000, 2000 and 4000 mg test article/kg body weight (bw)/day for 14 days. There was a non-statistically significant (p > 0.05) decrease in body weights for the male and female rats receiving the test article. Hypoactivity, hyperactivity, reduced food consumption and piloerection were observed in the rats receiving 4000 mg test article/kg bw. Histopathology showed an increase in the size of liver cells (hypertrophy) around the central vein (centrilobular) in Groups 3 (3/10) and 4 (5/10) that correlated with increased liver weights. In the 90-day study, 8 groups of rats were dosed with 0, 200, 400 and 800 mg test article/kg bw/day. Groups 5 to 8 had a 28-day recovery. There were no test article-linked changes in clinical observations, physical examinations, Functional Observation Battery, ophthalmology, Motor Activity Assessment, hematology, clinical chemistries and macropathology (all groups). With the exception of the liver and adrenal gland, no test article-linked pathology was observed. For all rats receiving the test article, histopathology showed hypertrophy of liver cells around the central vein. The increase of liver weight is most likely caused by hypertrophy due to up-regulation of the hepatic drug metabolizing enzymes. The hepatocellular hypertrophy was completely reversed in 28 days and was not considered to be an adverse effect. Vacuolization of the adrenal zona fasciculata was observed in the control and 800 mg test article/kg bw groups. The vacuolization of the zona fasciculata was of the same incidence and severity in treatment and control male rats and correlated with an increased in the weights of the adrenal glands. In addition, a statistically significant increase (p < 0.05) in adrenal-to-body weight ratios was observed for females receiving 800 mg test article/kg bw. This increase in adrenal-to-body weight ratio did not correlate with any of the pathology findings. The NOAEL for the test article is 800 mg/kg bw/day for female and 400 mg/kg bw/day for male Sprague Dawley rats. Abstract The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a right hand with a flexible, positively charged b-hairpin and a hydrophobic palm. This domain was shown to interact with the genomic RNA for coronavirus infectious bronchitis virus (IBV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Based on its 3D structure, we used site-directed mutagenesis to identify residues essential for the RNA-binding activity of the IBV N protein and viral infectivity. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells. In contrast, mutation of amino acid Gln-74 to an alanine, which does not affect the binding activity of the N-terminal domain, showed minimal, if any, detrimental effect on the infectivity of IBV. This study thus identifies residues critical for RNA binding on the nucleocapsid surface, and presents biochemical and genetic evidence that directly links the RNA binding capacity of the coronavirus N protein to the viral infectivity in cultured cells. This information would be useful in development of preventive and treatment approaches against coronavirus infection. Abstract Hepatitis E virus (HEV) infection uncommonly causes chronic hepatitis and neurologic disease. We describe a case of genotype 3a HEV meningoencephalitis diagnosed by metagenomic next-generation sequencing, illustrating the power of an unbiased molecular approach to microbial testing and the first reported case of HEV infection presumably acquired through lung transplantation. Abstract A major focus of systems biology is to characterize interactions between cellular components, in order to develop an accurate picture of the intricate networks within biological systems. Over the past decade, protein microarrays have greatly contributed to advances in proteomics and are becoming an important platform for systems biology. Protein microarrays are highly flexible, ranging from large-scale proteome microarrays to smaller customizable microarrays, making the technology amenable for detection of a broad spectrum of biochemical properties of proteins. In this article, we will focus on the numerous studies that have utilized protein microarrays to reconstruct biological networks including protein-DNA interactions, posttranslational protein modifications (PTMs), lectin-glycan recognition, pathogen-host interactions and hierarchical signaling cascades. The diversity in applications allows for integration of interaction data from numerous molecular classes and cellular states, providing insight into the structure of complex biological systems. We will also discuss emerging applications and future directions of protein microarray technology in the global frontier. Abstract Introduction: Bovine parainfluenza virus-3 (BPIV3) and bovine respiratory syncytial virus (BRSV) are the cause of respiratory disease in cattle worldwide. With other pathogens, they cause bovine respiratory disease complex (BRDC) in ruminants. The aim of the study was the detection and molecular characterisation of BPIV3 and BRSV from nasal swabs and lung samples of cows in and around the Erzurum region of eastern Turkey. Material and Methods: In total, 155 samples were collected. Of animals used in the study 92 were males and 63 females. The age of the animals was between 9 months and 5 years, mean 1.4 years. Most males were in the fattening period and being raised in open sheds; females were in the lactating period and kept in free stall barns. All samples were tested for the presence of viral genes using RT-PCR. Genespecific primers in a molecular method (RT-PCR) identified BRSV (fusion gene) and BPIV3 (matrix gene) strains at the genus level. Results: RNA from BRSV and BPIV3 was detected in two (1.29%) and three (1.93%) samples, respectively, one of each of which was sequenced and the sequences were aligned with reference virus strains. Phylogenetic analyses clustered the strains in genotype C/BPIV3 and subgroup III/BRSV. Conclusion: The results indicate that BRSV and BPIV3 contribute to bovine respiratory disease cases in Turkey. This is the first report on their detection and molecular characterisation in ruminants in Turkey. Abstract To describe the trends in the incidence rates of 5 most common cancers, communicable diseases, and non-communicable diseases in Saudi Arabia over the last decade.The incidence rates of cancers (2001-2014), communicable diseases (2003-2016), and noncommunicable diseases were retrieved, classified, and analyzed retrospectively during November 2017, based on data available with the Ministry of Health and were analyzed at the Imam Abdulrahman Bin Faisal University in Dammam, Kingdom of Saudi Arabia.Results: Age-standardized incidence rate (ASR) (per 100,000 population) of breast cancer among women increased dramatically from 11.8 in 2001 to 22.7 in 2014, indicating a 92.4% increase over the decade. Colorectal cancer incidence was the highest among men, and its ASR per 100,000 population increased from 5.0 to 10.6 in men and from 5.0 to 8.2 in women. Among communicable diseases, incidences of hepatitis B, measles, chickenpox, and brucellosis decreased while dengue fever increased. An alarming increase was observed in the incidence rate of non-communicable diseases namely, obesity, diabetes, and hypertension.The incidence rate of non-communicable diseases increased over the decade and was associated with increased mortality and disability, reduced quality of life, and increased health-care costs, indicating an urgent need to establish prevention and control programs. The rising trend in the incidence of cancers may also become a health care issue in Saudi Arabia in the coming years. Abstract Objective: Spontaneous intracerebral hemorrhage (ICH) is caused by the rupture of small blood vessels and other health problems. In ICH patients, hematoma enlargement is the most critical risk factor for poor outcomes. Tranexamic acid, an anti-fibrinolytic agent, has been used to reduce hematoma expansion. We analyzed the risk factors for hematoma expansion in ICH patients and compared the predictability of hematoma expansion in ICH patients with the use of tranexamic acid. Methods: We performed retrospective analysis of ICH patients who underwent follow-up computed tomography scans from October 2008 to October 2018. Of the 329 included patients, 67 who received tranexamic acid and 262 who did not receive tranexamic acid were compared. We also analyzed the risk factors of 45 and 284 patients who did and did not experience hematoma expansion, respectively. Results: Hematoma expansion was observed in 7 (10.4%) of 67 patients in the tranexamic acid group and 38 (14.5%) of the 262 patients who did not receive tranexamic acid. There was no statistically significant difference between patients who did and did not received tranexamic acid (p=0.389). In the multivariate logistic regression analysis of risk factors for hematoma expansion, spot sign and a maximal diameter of 40 mm were identified as risk factors. Conclusion: We could not confirm the effect of tranexamic acid on hematoma expansion in ICH patients. Spot sign and the maximal diameter of hematomas were confirmed as risk factors of hematoma expansion. If the maximal diameter is greater than 40 mm, the hematoma should be closely monitored. Abstract Abbreviations: CCL2, CC-chemokine ligand 2; CM, conditioned medium; ER, estrogen receptor; MCP-1, monocyte chemoattractant protein 1; MR, tumor-associated macrophage from a tamoxifen-resistant tumor microenvironment; MS, tumor-associated macrophage from a tamoxifen-sensitive tumor microenvironment; PFS, progression-free survival; TAM, tumor-associated macrophage; TME, tumor microenvironment.Breast cancer is the most prevalent malignancy among women. Although endocrine therapy is effective, the development of endocrine resistance is a major clinical challenge. The tumor microenvironment (TME) promotes tumor malignancy, and tumor-associated macrophages (TAM) within the TME play a crucial role in endocrine resistance. Herein, we aimed to elucidate the relationship between TAM and the endocrine-resistant phenotype of breast cancer. Macrophages were cultured with conditioned medium (CM) from tamoxifen-sensitive (MCF7-S) or -resistant (MCF7-R) MCF7 breast cancer cells. M2 polarization was detected by CD163 immunofluorescence. To determine the effect on endocrine resistance, MCF7 cells were cultured in the supernatant of different TAM, and then treated with tamoxifen. CC-chemokine ligand 2 (CCL2) immunohistochemistry was carried out on pathological sections from 100 patients with invasive estrogen receptor-positive breast cancer. We found that macrophages cultured in the CM of MCF7-S and MCF7-R cells were induced into TAM, with a more obvious M2 polarization in the latter. Tamoxifen resistance was increased by culture in TAM medium. TAM secreted CCL2, which increased endocrine resistance in breast cancer cells through activation of the PI3K/Akt/mTOR signaling pathway. High expression of CCL2 was correlated with infiltration of CD163+macrophages (r = 0.548, P < .001), and patients with high CCL2 expression presented shorter progression-free survival than those with low CCL2 expression (P < .05). We conclude that CCL2 secreted by TAM activates PI3K/Akt/mTOR signaling and promotes an endocrine resistance feedback loop in the TME, suggesting that CCL2 and TAM may be novel therapeutic targets for patients with endocrineresistant breast cancer. Abstract Middle East Respiratory Syndrome (MERS) is an acute viral respiratory illness with high mortality caused by a new strain of betacoronavirus (MERS-CoV). Since the report of the first patient in Saudi Arabia in 2012, large-scale outbreaks through hospital-acquired infection and inter-hospital transmission have been reported. Most of the patients reported in South Korea were also infected in hospital settings. Therefore, to eliminate the spread of MERS-CoV, infection prevention and control measures should be implemented with rigor. The present guideline has been drafted on the basis of the experiences of infection control in the South Korean hospitals involved in the recent MERS outbreak and on domestic and international infection prevention and control guidelines. To ensure efficient MERS-CoV infection prevention and control, care should be taken to provide comprehensive infection control measures including contact control, hand hygiene, personal protective equipment, disinfection, and environmental cleaning. Abstract Infectious diseases caused by viral agents kill millions of people every year. The improvement of prevention and treatment of viral infections and their associated diseases remains one of the main public health challenges. Towards this goal, deciphering virus-host molecular interactions opens new perspectives to understand the biology of infection and for the design of new antiviral strategies. Indeed, modelling of an infection network between viral and cellular proteins will provide a conceptual and analytic framework to efficiently formulate new biological hypothesis at the proteome scale and to rationalize drug discovery. Therefore, we present the first release of VirHostNet (Virus-Host Network), a public knowledge base specialized in the management and analysis of integrated virus-virus, virushost and host-host interaction networks coupled to their functional annotations. VirHostNet integrates an extensive and original literature-curated dataset of virus-virus and virus-host interactions (2671 nonredundant interactions) representing more than 180 distinct viral species and one of the largest human interactome (10 672 proteins and 68 252 nonredundant interactions) reconstructed from publicly available data. The VirHostNet Web interface provides appropriate tools that allow efficient query and visualization of this infected cellular network. Public access to the VirHostNet knowledge-based system is available at Abstract Uniquely among RNA viruses, replication of the $30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this 'improved' recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, $20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity. Abstract Influenza virus infections are a major public health concern worldwide. Conventional treatments against the disease are designed to target viral proteins. However, the emergence of viral variants carrying drug-resistant mutations can outpace the development of pathogen-targeting antivirals. Diphyllin and bafilomycin are potent vacuolar ATPase (V-ATPase) inhibitors previously shown to have broad-spectrum antiviral activity. However, their poor water solubility and potential off-target effect limit their clinical application. Methods: In this study, we report that nanoparticle encapsulation of diphyllin and bafilomycin improves the drugs' anti-influenza applicability. Results: Using PEG-PLGA diblock copolymers, sub-200 nm diphyllin and bafilomycin nanoparticles were prepared, with encapsulation efficiency of 42% and 100%, respectively. The drug-loaded nanoparticles have sustained drug release kinetics beyond 72 hours and facilitate intracellular drug delivery to two different influenza virus-permissive cell lines. As compared to free drugs, the nanoparticulate V-ATPase inhibitors exhibited lower cytotoxicity and greater in vitro antiviral activity, improving the therapeutic index of diphyllin and bafilomycin by approximately 3 and 5-fold, respectively. In a mouse model of sublethal influenza challenge, treatment with diphyllin nanoparticles resulted in reduced body weight loss and viral titer in the lungs. In addition, following a lethal influenza viral challenge, diphyllin nanoparticle treatment conferred a survival advantage of 33%. Conclusions: These results demonstrate the potential of the nanoparticulate V-ATPase inhibitors for host-targeted treatment against influenza. Abstract Graphical Abstract Highlights d PI4KB activity expedites the formation of coxsackievirus replication organelles (ROs) d PI4KB inhibition impairs polyprotein processing, which is rescued by a 3A mutation d Upon PI4KB inhibition, this mutant replicates at the Golgi in the absence of ROs d Innate immune responses are not enhanced when RO biogenesis is delayed Correspondence m.barcena@lumc.nl (M.B.), f.j.m.vankuppeveld@uu.nl (F.Enteroviruses reorganize cellular endomembranes into replication organelles (ROs) for genome replication. Although enterovirus replication depends on phosphatidylinositol 4-kinase type IIIb (PI4KB), its role, and that of its product, phosphatidylinositol 4-phosphate (PI4P), is only partially understood. Exploiting a mutant coxsackievirus resistant to PI4KB inhibition, we show that PI4KB activity has distinct functions both in proteolytic processing of the viral polyprotein and in RO biogenesis. The escape mutation rectifies a proteolytic processing defect imposed by PI4KB inhibition, pointing to a possible escape mechanism. Remarkably, under PI4KB inhibition, the mutant virus could replicate its genome in the absence of ROs, using instead the Golgi apparatus. This impaired RO biogenesis provided an opportunity to investigate the proposed role of ROs in shielding enteroviral RNA from cellular sensors. Neither accelerated sensing of viral RNA nor enhanced innate immune responses was observed. Together, our findings challenge the notion that ROs are indispensable for enterovirus genome replication and immune evasion. Abstract To evaluate the uptake of a mandatory meningococcal, a highly recommended influenza, and an optional pneumococcal vaccine, and to explore the key factors affecting vaccination rate among health care workers (HCWs) during the Hajj.An anonymous cross-sectional online survey was distributed among HCWs and trainees who worked or volunteered at the Hajj 2015-2017 through their line managers, or by visiting their hospitals and healthcare centres in Makkah and Mina. Overseas HCWs who accompanied the pilgrims or those who work in foreign Hajj medical missions were excluded. Pearson's χ 2 test was used to compare categorical variables and odds ratio (OR) was calculated by "risk estimate" statistics along with 95% confidence interval (95%CI).A total of 138 respondents aged 20 to 59 (median 25.6) years with a male to female ratio of 2.5:1 participated in the survey. Only 11.6% (16/138) participants reported receiving all three vaccines, 15.2% (21/138) did not receive any vaccine, 76.1% (105/138) received meningococcal, 68.1% (94/138) influenza and 13.8% (19/138) pneumococcal vaccine. Females were more likely to receive a vaccine than males (OR 3.6, 95%CI: 1.0-12.7, P < 0.05). Willingness to follow health authority's recommendation was the main reason for receipt of vaccine (78.8%) while believing that they were up-to-date with vaccination (39.8%) was the prime reason for non-receipt.Some HCWs at Hajj miss out the compulsory and highly recommended vaccines; lack of awareness is a key barrier and authority's advice is an important motivator. Health education followed by stringent measures may be required to improve their vaccination rate. Abstract Introduction: The aetiology of acute exacerbations of chronic obstructive pulmonary disease (COPD) remains incompletely understood and strategies for treatment and prevention have not altered significantly for many years. Improved understanding of the role of respiratory pathogens in acute exacerbations of COPD (AECOPD) is required and the use of molecular microbiological techniques may lead to insights into host-pathogen interactions and the development of more targeted therapeutic approaches.Trial registration number: ClinicalTrials.gov NCT01360398.Bourne S, Cohet C, Kim V, et al. Abstract We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration Abstract Regulated secretory cells have two pathways that transport secreted proteins from the Golgi complex to the cell surface . To identify carrier vesicles involved in regulated and constitutive secretion, PC12 pheochromocytoma cells were labeled with [33S]sulfate to identify markers for the two secretory pathways, then mechanically permeabilized and incubated in vitro. Small constitutive secretory vesicles, containing mostly sulfated proteoglycans, accumulated during an in vitro incubation with ATP In the presence of M EMBRANE traffic between compartments involves carrier vesicles that bud from a donor compartment and fuse with the acceptor compartment . To explain how membrane proteins are sorted to different organelles, we need to know how the carrier vesicle selects a subset of the proteins present in the donor membrane compartment and how it fuses exclusively with the acceptor. With these questions in mind, considerable effort has been expended in identifying carrier vesicles involved in ER to Golgi transport, intra-Golgi transport and post-trans-Golgi Abstract We describe the aetiology of community-acquired pneumonia in children before and after the introduction of the pneumococcal conjugate vaccination (PCV) programme in 2006.Prospective studies were conducted in 2001-2002 (pre-vaccine) and 2009-2011 (post-vaccine) of children aged 0-16 years with radiologically confirmed pneumonia seen in hospital. Investigations included culture, serology, immunofluorescence antibody and urine antigen testing, with an increased use of PCR assays and expanded panels of pathogens in the post-vaccine study. 241 and 160 children were enrolled in the pre-and post-vaccine studies, respectively (73% aged ,5 years). Identification of a causative pathogen was higher post-vaccination (61%) than pre-vaccination (48.5%) (p50.019). Rates of bacterial infections were not different between post-and pre-vaccine studies (17.5% versus 24%, p50.258). Viral (31%) and mixed (12.5%) infections were found more often postvaccination (19.5%, p50.021) than pre-vaccination (5%, p50.015). Rates of identified pneumococcal infections were comparable between pre-and post-vaccine studies (14.7% versus 17.4%, p50.557). Diagnosis of pneumococcal infection post-vaccination improved when PCR was used compared to culture (21.6% versus 6%, p50.0004). Serotypes included in PCV13 but not PCV7 were identified in 75% (18 out of 24) post-vaccination.Infection with nonvaccine pneumococcal serotypes continues to be a significant cause of pneumonia in children in the UK.@ERSpublications Aetiology of community-acquired pneumonia in children following a pneumococcal conjugate vaccination programme Abstract The rodent arenavirus glycoprotein complex encodes a stable signal peptide (SSP) that is an essential structural component of mature virions. The SSP, GP1, and GP2 subunits of the trimeric glycoprotein complex noncovalently interact to stud the surface of virions and initiate arenavirus infectivity. Nascent glycoprotein production undergoes two proteolytic cleavage events: first within the endoplasmic reticulum (ER) to cleave SSP from the remaining precursor GP1/2 (glycoprotein complex [GPC]) glycoprotein and second within the Golgi stacks by the cellular SKI-1/S1P for GP1/2 processing to yield GP1 and GP2 subunits. Cleaved SSP is not degraded but retained as an essential glycoprotein subunit. Here, we defined functions of the 58amino-acid lymphocytic choriomeningitis virus (LCMV) SSP in regard to glycoprotein complex processing and maturation. Using molecular biology techniques, confocal microscopy, and flow cytometry, we detected SSP at the plasma membrane of transfected cells. Further, we identified a sorting signal (FLLL) near the carboxyl terminus of SSP that is required for glycoprotein maturation and trafficking. In the absence of SSP, the glycoprotein accumulated within the ER and was unable to undergo processing by SKI-1/S1P. Mutation of this highly conserved FLLL motif showed impaired glycoprotein processing and secretory pathway trafficking, as well as defective surface expression and pH-dependent membrane fusion. Immunoprecipitation of SSP confirmed an interaction between the signal peptide and the GP2 subunit; however, mutations within this FLLL motif disrupted the association of the GP1 subunit with the remaining glycoprotein complex.a neglected yet essential role in glycoprotein biosynthesis. Without this 6-kDa polypeptide, the glycoprotein precursor is retained within the endoplasmic reticulum, and trafficking to the plasma membrane where SSP, GP1, and GP2 localize for glycoprotein assembly into infectious virions is inhibited. To investigate SSP contributions to glycoprotein maturation and function, we created an SSP-tagged glycoprotein to directly detect and manipulate this subunit. This resource will aid future studies to identify host factors that mediate glycoprotein maturation.Citation Bederka LH, Bonhomme CJ, Ling EL, Buchmeier MJ. 2014. Arenavirus stable signal peptide is the keystone subunit for glycoprotein complex organization. mBio 5(6): Abstract Diarrhea outbreaks in pig farms have raised major concerns in Europe and USA, as they can lead to dramatic pig losses. During a suspected outbreak in Belgium of porcine epidemic diarrhea virus (PEDV), we performed viral metagenomics to assess other potential viral pathogens. Although PEDV was detected, its low abundance indicated that other viruses were involved in the outbreak. Interestingly, a porcine bocavirus and several enteroviruses were most abundant in the sample. We also observed the presence of a porcine enterovirus genome with a gene insertion, resembling a C28 peptidase gene found in toroviruses, which was confirmed using re-sequencing, bioinformatics, and proteomics approaches. Moreover, the predicted cleavage sites for the insertion suggest that this gene was being expressed as a single protein, rather than a fused protein. Recombination in enteroviruses has been reported as a major mechanism to generate genetic diversity, but gene insertions across viral families are rather uncommon. Although such inter-family recombinations are rare, our finding suggests that these events may significantly contribute to viral evolution. Abstract Porcine epidemic diarrhea (PED) is an intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV); manifestations of the disease are diarrhea, vomiting and dehydration. Starting from the end of 2010, a PED outbreak occurred in several pig-producing provinces in southern China. Subsequently, the disease spread throughout the country and caused enormous economic losses to the pork industry. Accumulating studies demonstrated that new PEDV variants that appeared in China were responsible for the PED outbreak. In the current mini-review, we summarize PEDV epidemiology and vaccination in China. Abstract This investigation enrolled 570 healthy young males gathered from all over the country for military service at the Republic of Korea Air Force boot camp. It confirmed RSV IgG seroprevalence by utilizing the enzyme immunoassay method just prior to undergoing basic training. The mean age of this study was 20.25 ± 1.34 yr old. The results of their immunoassay seroprofiles showed that 561 men (98.4%) were positive, 2 (0.4%) were negative and 7 (1.2%) were equivocal belonging to the grey zone. It was confirmed that RSV is a common respiratory virus and RSV infection was encountered by almost all people before reaching adulthood in Korea. Nine basic trainees belonging to the RSV IgG negative and equivocal grey zone categories were prospectively observed for any particular vulnerability to respiratory infection during the training period of two months. However, these nine men completed their basic training without developing any specific respiratory illness. Abstract In recent years, growth of international travel and trade, as well as climate change, has resulted in the frequent emergence and reemergence of infectious diseases such as Ebola, Zika, and MERS. In 2016, Taiwan used the Joint External Evaluation ( JEE) tool to evaluate its public health emergency response capacities and understand important areas for improvement. This article presents Taiwan's disaster and public health emergency response organizational structure, realtime integrated information, response processes, and command center structure. After reviewing the results of the JEE tool and drawing lessons from emergency response efforts in the United States, we provide 3 recommendations that may enhance Taiwan's public health emergency response capacities: establish common principles for disaster response regardless of which agency is in charge, standardize operation procedures, and perform regular training that includes nongovernmental organizations and a range of government departments. Abstract An emerging picture of the nature of immune systems across animal phyla reveals both conservatism of some features and the appearance among and within phyla of novel, lineage-specific defense solutions. The latter collectively represent a major and underappreciated form of animal diversity. Factors influencing this macroevolutionary (above the species level) pattern of novelty are considered and include adoption of different life styles, life histories, and body plans; a general advantage of being distinctive with respect to immune defenses; and the responses required to cope with parasites, many of which afflict hosts in a lineage-specific manner. This large-scale pattern of novelty implies that immunological phenomena can affect microevolutionary processes (at the population level within species) that can eventually lead to macroevolutionary events such as speciation, radiations, or extinctions. Immunologically based phenomena play a role in favoring intraspecific diversification, specialization and host specificity of parasites, and mechanisms are discussed whereby this could lead to parasite speciation. Host switching -the acquisition of new host species by parasites -is a major mechanism that drives parasite diversity and is frequently involved in disease emergence. It is also one that can be favored by reductions in immune competence of new hosts. Mechanisms involving immune phenomena favoring intraspecific diversification and speciation of host species are also discussed. A macroevolutionary perspective on immunology is invaluable in today's world, including the need to study a broader range of species with distinctive immune systems. Many of these species are faced with extinction, another macroevolutionary process influenced by immune phenomena. Abstract A case-control study was performed to identify clinical predictors for Middle East respiratory syndrome coronavirus (MERS-CoV) infection among patients with acute febrile illness during the nosocomial outbreak. Patients with MERS-CoV were more likely to have monocytosis with normal white blood cell (WBC) count and lower C-reactive protein (CRP) level. Simple laboratory data such as complete blood counts (CBC) with differential count could be a useful marker for the prediction of MERS and triage at the initial presentation of acute febrile patients in outbreak setting. Abstract Purpose: The aims of this study were to compare serum procalcitonin (PCT) levels between febrile children with Kawasaki disease (KD) and those with bacterial or viral infections, and assess the clinical usefulness of PCT level in predicting KD. Methods: Serum PCT levels were examined in febrile pediatric patients admitted between August 2013 and August 2014. The patients were divided into 3 groups as follows: 49 with KD, 111 with viral infec tions, and 24 with bacterial infections. Results: The mean PCT level in the KD group was significantly lower than that in the bacterial infection group (0.82±1.73 ng/mL vs. 3.11±6.10 ng/mL, P=0.002) and insignificantly different from that in the viral infection group (0.23±0.34 ng/mL,P=0.457). The mean erythrocyte sedimentation rate (ESR) and Creactive protein (CRP) level in the KD group were significantly higher than those in the viral and bacterial infection groups (P<0.001 and P<0.001 for ESR, P<0.001 and P=0.005 for CRP, respectively). The proportion of patients in the KD group with PCT levels of >1.0 ng/mL was significantly higher in the nonresponders to the initial intravenous immunoglobulin treatment than in the responders (36% vs. 8%, P=0.01). Conclusion: PCT levels may help to differentiate KD from bacterial infections. A combination of disease markers, including ESR, CRP, and PCT, may be useful for differentiating between KD and viral/bacterial infections. Abstract Hemophagocytic lymphohistiocytosis (HLH) covers a wide array of related life-threatening conditions featuring ineffective immunity characterized by an uncontrolled hyperinflammatory response. HLH is often triggered by infection. Familial forms result from genetic defects in natural killer cells and cytotoxic T-cells, typically affecting perforin and intracellular vesicles. HLH is likely under-recognized, which contributes to its high morbidity and mortality. Early recognition is crucial for any reasonable attempt at curative therapy to be made. Current treatment regimens include immunosuppression, immune modulation, chemotherapy, and biological response modification, followed by hematopoietic stem-cell transplant (bone marrow transplant). A number of recent studies have contributed to the understanding of HLH pathophysiology, leading to alternate treatment options; however, much work remains to raise awareness and improve the high morbidity and mortality of these complex conditions. Abstract Background: Human bocavirus (HBoV) is a newly identified viral pathogen, and its clinical epidemiology and significance in respiratory infections have not yet been completely elucidated. We investigated the prevalence of HBoV infection and the association between viral (HBoV) load and clinical features of the infection in patients of all age-groups. Methods: Nasopharyngeal aspirates from patients with symptoms of respiratory infection were tested for presence of HBoV by using real-time polymerase chain reaction. HBoV-positive patients were categorized into low-and high-viral-load groups using 1.0× 10 6 copies/mL as the threshold value of viral load. Results: Detection rate of HBoV was 4.8% (N=93) in a total of 1,926 samples with peak incidence of infection being observed in patients aged 6-12 months. HBoV infection was more frequently observed in young children, especially, in children aged less than 5 yr, and the HBoV load decreased with increase in age. HBoV was codetected with other respiratory viruses in 17 (18.3%) of the 93 HBoV-positive patients and 15 patients (88.2%) belonged to the low-viral-load group. Patients infected with HBoV alone showed a higher viral load than those patients in whom HBoV was codetected with other respiratory viruses (median load, 3.78× 10 5 copies/ mL vs. 1.94×10 4 copies/mL, P =0.014). Higher pulse rate (P =0.007) and respiratory rate (P =0.021) were observed in patients with a high-viral-load. Conclusions: Our results suggest that HBoV may be the causative agent of respiratory infection in the high-viral-load group. Abstract Recombination is a process that unlinks neighboring loci allowing for independent evolutionary trajectories within genomes of many organisms. If not properly accounted for, recombination can compromise many evolutionary analyses. In addition, when dealing with organisms that are not obligately sexually reproducing, recombination gives insight into the rate at which distinct genetic lineages come into contact. Since June 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has caused 1,106 laboratory-confirmed infections, with 421 MERS-CoV-associated deaths as of 16 April 2015. Although bats are considered as the likely ultimate source of zoonotic betacoronaviruses, dromedary camels have been consistently implicated as the source of current human infections in the Middle East. In this article, we use phylogenetic methods and simulations to show that MERS-CoV genome has likely undergone numerous recombinations recently. Recombination in MERS-CoV implies frequent co-infection with distinct lineages of MERS-CoV, probably in camels given the current understanding of MERS-CoV epidemiology. Abstract The role of rhinovirus asymptomatic infections in the transmission among close contacts subjects is unknown. We tested health care workers, a pair of one child and a family member and immunocompromised patients (n =191). HRV were detected on 22.9% symptomatic and 3.6% asymptomatic cases suggesting lower transmission among contacts. Rhinovirus infections are among the most frequent causes of the common colds (18). It is the most common etiology of viral respiratory infections among diverse populations, including adults, children and more recent studies have linked HRVs to more severe lower respiratory illnesses in otherwise healthy young children (11-12), immunocompromised (4,6), and elderly patients. (5,13, 23). In Brazil, there are few HRV studies, Arruda et.al. (1991), realized the first Brazilian study detecting a 45.5% rate on symptomatic children in Fortaleza -CE. Another study realized in Salvador -BA also showed a high prevalence (48.5%) (Souza ,2003). The only study Abstract Despite its poorly adapted codon usage, Abstract The purpose of this study was to detect porcine epidemic diarrhea virus (PEDV) subclinically infected pigs shipped from non-case farms to slaughterhouses. Systematic sampling was conducted at two slaughterhouses. A total of 1,556 blood samples were collected from 80 case and non-case farms from pigs over 6 months old. Blood samples were centrifuged to obtain sera. Serial serum dilutions were subjected to serological examination for PEDV presence using Neutralization test (NT). The cut-off titer was set at titer of 1:2 dilution and farms with at least one positive sample in duplicate were classified as PED-positive farms. Several non-case farms (9.4%, 6/64) and 100% (16/16) of the case farms were indeed positive for PEDV. The proportion of seropositive animals from case farms was 63.7%, significantly different from that of non-case farms (4.3%, P<0.05). In both case and non-case farms, the proportion of seropositive animals in farrow-to-finish farms was significantly higher than in wean-to-finish farms (P<0.05). Seropositive animals in non-case farms were detected by NT in a sero-survey by sampling at slaughterhouses. Therefore, subclinically infected pigs should be considered prior to shipment.KEY WORDS: passive surveillance, porcine epidemic diarrhea, subclinical infection, swine Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, causes acute diarrhea and dehydration in piglets, among which mortality rate can reach up to 100% [5, 16] . PEDV results in significant economic losses due to the high morbidity and mortality in neonatal piglets [1, 2, 10, 11, 17, 21] . There is an age-dependent resistance to pathogenic PEDV infection in pigs [15] , where 2-and 7-day-old pigs inoculated with PEDV developed severe diarrhea and died. On the other hand, 2and 4-week-old pigs presented with mild diarrhea and survived after inoculation with PEDV. The 8-and 12-week-old pigs did not show any clinical signs, but antibody against PEDV were detected by Neutralization test (NT) and immunofluorescence assay [15] .The virus was first identified in the United Kingdom in 1971 [20] and is now widespread throughout the United States, Canada, and many Asian countries including Japan. In Japan, PED cases were first observed in 1982 [18] and only sporadic cases were reported between 1997 and 2006, with no cases were reported from 2007 to 2012 [9] . However, large-scale PED outbreaks occurred during 2013-2015. Over 800 infected farms were identified in 38 out of 47 prefectures, resulting in at least 40,000 mortal cases, predominantly in piglets [8, 9] . Out of these cases, over 40% were confirmed in Kyushu island which possesses the largest Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel betacoronavirus that has been circulating in the Arabian Peninsula since 2012 and causing severe respiratory infections in humans. While bats were suggested to be involved in human MERS-CoV infections, a direct link between bats and MERS-CoV is uncertain. On the other hand, serological and virological data suggest dromedary camels as the potential animal reservoirs of MERS-CoV. Recently, we isolated MERS-CoV from a camel and its infected owner and provided evidence for the direct transmission of MERS-CoV from the infected camel to the patient. Here, we extend this work and show that identical MERS-CoV RNA fragments were detected in an air sample collected from the same barn that sheltered the infected camel in our previous study. These data indicate that the virus was circulating in this farm concurrently with its detection in the camel and in the patient, which warrants further investigations for the possible airborne transmission of MERS-CoV.IMPORTANCE This work clearly highlights the importance of continuous surveillance and infection control measures to control the global public threat of MERS-CoV. While current MERS-CoV transmission appears to be limited, we advise minimal contact with camels, especially for immunocompromised individuals, and the use of appropriate health, safety, and infection prevention and control measures when dealing with infected patients. Also, detailed clinical histories of any MERS-CoV cases with epidemiological and laboratory investigations carried out for any animal exposure must be considered to identify any animal source. Abstract The present studies have evaluated the effect of age on the induction of syngeneic graft-versushost disease (SGVHD) after syngeneic bone marrow transplantation (BMT) and cyclosporine (CsA) therapy. The results clearly document an inverse correlation of age with the incidence of SGVHD. Virtually a 100% incidence of SGVHD occurs in Lewis rats when syngeneic BMT and CsA therapy are started when the animals are 4 wk of age . Thereafter, there is a dramatic decline in the incidence of SGVHD with the increasing age of the animals. Although the age of the recipient was important, the most significant effect was the age of the marrow donor . Marrow from animals 6 mo of age was virtually incapable of eliciting SGVHD after BMT and CsA therapy. Furthermore, mixing the marrow from mature and immature animals resulted in a decreased incidence of SGVHD, implicating a regulatory effect present in the marrow from older rats. This regulatory effect was due to the presence of mature T cells in the marrow from animals 6 mo of age. Despite the fact that marrow from young animals possesses mature T lymphocytes, this regulatory activity was absent, suggesting that the host resistance mediated by T lymphocytes develops as the animal ages. These data further implicate the importance of a host resistance mechanism in preventing the induction of SGVHD with CsA, which appears to be mediated by the clonal inactivation of autoreactive cells . Abstract Objective: The six core competencies designated by Accreditation Council for Graduate Medical Education (ACGME) are essential for establishing a patient centre holistic medical system. The authors developed a faculty programme to promote the postgraduate year 1 (PGY 1 ) resident, ACGME six core competencies. The study aims to assess the clinical instructors' perception, attitudes and subjective impression towards the various sessions of the 'faculty development programme for teaching ACGME competencies.'instructors participated in the programme to establish their ability to teach and assess PGY 1 residents about ACGME competencies.Results: The participants in the faculty development programme reported that the skills most often used while teaching were learnt during circuit and itinerant bedside, physical examination teaching, mini-clinical evaluation exercise (mini-CEX) evaluation demonstration, training workshop and videotapes of 'how to teach ACGME competencies.' Participants reported that circuit bedside teaching and mini-CEX evaluation demonstrations helped them in the interpersonal and communication skills domain, and that the itinerant teaching demonstrations helped them in the professionalism domain, while physical examination teaching and mini-CEX evaluation demonstrations helped them in the patients' care domain. Both the training workshop and videotape session increase familiarity with teaching and assessing skills. Participants who applied the skills learnt from the faculty development programme the most in their teaching and assessment came from internal medicine departments, were young attending physician and had experience as PGY 1 clinical instructors.Conclusions: According to the clinical instructors' response, our faculty development programme effectively increased their familiarity with various teaching and assessment skills needed to teach PGY 1 residents and ACGME competencies, and these clinical instructors also then subsequently apply these skills. Abstract The World Health Organization (WHO) published 2 alcohol-based formulations to be used in healthcare settings and for outbreak-associated infections, but inactivation efficacies of these products have not been determined against (re-)emerging viruses. In this study, we evaluated the virucidal activity of these WHO products in a comparative analysis. Zika virus (ZIKV), Ebola virus (EBOV), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) as (re-)emerging viral pathogens and other enveloped viruses could be efficiently inactivated by both WHO formulations, implicating their use in healthcare systems and viral outbreak situations. Abstract Background: In this study, we evaluated the analytical performance and clinical potential of a one-step multiplex real-time PCR assay for the simultaneous detection of 14 types of respiratory viruses using the AdvanSure RV real-time PCR Kit (LG Life Sciences, Korea).Methods: Three hundred and twenty clinical specimens were tested with the AdvanSure RV real-time PCR Kit and conventional multiplex reverse transcription (RT)-PCR assay. The assay results were analyzed and the one-step AdvanSure RV real-time PCR Kit was compared with the conventional multiplex RT-PCR assay with respect to the sensitivity and specificity of the detection of respiratory viruses.Results: The limit of detection (LOD) was 1.31 plaque-forming units (PFU)/mL for human rhinoviruses (hRVs), 4.93 PFU/mL for human coronavirus HCoV-229E/NL63, 2.67 PFU/mL for human coronavirus HCoV-OC43, 18.20 PFU/mL for parainfluenza virus 1 (PIV)-1, 24.57 PFU/mL for PIV-2, 1.73 PFU/mL for PIV-3, 1.79 PFU/mL for influenza virus group (Flu) A, 59.51 PFU/mL for FluB, 5.46 PFU/mL for human respiratory syncytial virus (hRSV)-A, 17.23 PFU/mL for hRSV-B, 9.99 PFU/mL for human adenovirus (ADVs). The cross-reactivity test for this assay against 23 types of non-respiratory viruses showed negative results for all viruses tested. The agreement between the one-step AdvanSure multiplex real-time PCR assay and the conventional multiplex RT-PCR assay was 98%.Conclusions: The one-step AdvanSure RV multiplex real-time PCR assay is a simple assay with high potential for specific, rapid and sensitive laboratory diagnosis of respiratory viruses compared to conventional multiplex RT-PCR. Abstract Among the various nano/biomaterials used in cancer treatment, the beauty and benefits of DNA nanocomposites are outstanding. The specificity and programmability of the base pairing of DNA strands, together with their ability to conjugate with different types of functionalities have realized unsurpassed potential for the production of two-and threedimensional nano-sized structures in any shape, size, surface chemistry and functionality. This review aims to provide an insight into the diversity of static DNA nanodevices, including DNA origami, DNA polyhedra, DNA origami arrays and bioreactors, DNA nanoswitch, DNA nanoflower, hydrogel and dendrimer as young but promising platforms for cancer theranostics. The utility and potential of the individual formats in biomedical science and especially in cancer therapy will be discussed. Abstract Effective management of seasonal and pandemic influenza is a high priority internationally.Guidelines in many countries recommend antiviral treatment for older people and individuals with co-morbidity at increased risk of complications. However, antivirals are not often prescribed in primary care in Europe, because its clinical benefit has been insufficiently demonstrated by non-industry funded and pragmatic studies.ALIC 4 E is a European multi-national, multi-centre, phase IV, open-labelled, non-industry funded, pragmatic, adaptive-platform, randomised controlled trial (RCT). Initial trial arms will be best usual primary care, and best usual primary care plus treatment with oseltamivir for five days. We aim to recruit at least 2500 participants ≥1 year old presenting with influenza-like illness (ILI), with symptom duration ≤72 hours in primary care over three consecutive periods of confirmed high influenza incidence. Participant outcomes will be followed-up to 28 days by diary and telephone. The primary objective is to determine whether adding antiviral treatment to best usual primary care is effective in reducing time to return to usual daily activity with fever, head-and muscle-ache reduced to minor severity or less. Secondary objectives include determining cost-effectiveness, benefits in subgroups of participants according to age (<12, 12-64, >64 years), severity of symptoms (low, medium, high), comorbidity (yes/no), and duration of symptoms (≤48hours/>48-72 hours), decrease inFor peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Abstract Background. The microbial etiology of community-acquired pneumonia (CAP) is often unclear in clinical practice, and previous studies have produced variable results. Population-based studies examining etiology and incidence are lacking. This study examined the incidence and etiology of CAP requiring hospitalization in a population-based cohort as well as risk factors and outcomes for specific etiologies.Methods. Consecutive admissions due to CAP in Reykjavik, Iceland were studied. Etiologic testing was performed with cultures, urine-antigen detection, and polymerase chain reaction analysis of airway samples. Outcomes were length of stay, intensive care unit admission, assisted ventilation, and mortality.Results. The inclusion rate was 95%. The incidence of CAP requiring hospitalization was 20.6 cases per 10 000 adults/year. A potential pathogen was detected in 52% (164 of 310) of admissions and in 74% (43 of 58) with complete sample sets. Streptococcus pneumoniae was the most common pathogen (61 of 310, 20%; incidence: 4.1/10 000). Viruses were identified in 15% (47 of 310; incidence: 3.1/10 000), Mycoplasma pneumoniae were identified in 12% (36 of 310; incidence: 2.4/10 000), and multiple pathogens were identified in 10% (30 of 310; incidence: 2.0/10 000). Recent antimicrobial therapy was associated with increased detection of M pneumoniae (P < .001), whereas a lack of recent antimicrobial therapy was associated with increased detection of S pneumoniae (P = .02). Symptoms and outcomes were similar irrespective of microbial etiology.Conclusions. Pneumococci, M pneumoniae, and viruses are the most common pathogens associated with CAP requiring hospital admission, and they all have a similar incidence that increases with age. Symptoms do not correlate with specific agents, and outcomes are similar irrespective of pathogens identified. Abstract If accidentally released, mammalian-transmissible influenza A/H5N1 viruses could pose a greater threat to public health than possibly any other infectious agent currently under study in laboratories, because of such viruses' likely combination of transmissibility and virulence to humans. We advocate explicit risk-benefit assessments before work on such pathogens is permitted or funded, improvement of biosafety practices and enforcement, and harmonization of criteria for permitting such experiments across government agencies, as well as internationally. Such potential pandemic pathogens, as they have been called, jeopardize not only laboratory workers and their contacts, but also the wider population, who should be involved in assessments of when such risks are acceptable in the service of scientific knowledge that may itself bear major public health benefits. Abstract Background: In the past decade, the identification and characterization of antiviral genes with the ability to interfere with virus replication has established cell-intrinsic innate immunity as a third line of antiviral defense in addition to adaptive and classical innate immunity. Understanding how cellular factors have evolved to inhibit HIV-1 reveals particularly vulnerable points of the viral replication cycle. Many, but not all, antiviral proteins share type I interferon-upregulated expression and sensitivity to viral counteraction or evasion measures. Whereas well-established restriction factors interfere with early post-entry steps and release of HIV-1, recent research has revealed a diverse set of proteins that reduce the infectious quality of released particles using individual, to date poorly understood modes of action. These include induction of paucity of mature glycoproteins in nascent virions or self-incorporation into the virus particle, resulting in poor infectiousness of the virion and impaired spread of the infection.A better understanding of these newly discovered antiviral factors may open new avenues towards the design of drugs that repress the spread of viruses whose genomes have already integrated. Abstract The success of lentiviral vectors in curing fatal genetic and acquired diseases has opened a new era in human gene therapy. However, variability in the efficacy and safety of this therapeutic approach has been reported in human patients. Consequently, lentiviral-vector-based gene therapy is limited to incurable human diseases, with little understanding of the underlying causes of adverse effects and poor efficacy. To assess the role that host genetic variation has on efficacy of gene therapy, we characterized lentiviral-vector gene therapy within a set of 12 collaborative cross mouse strains. Lentiviral vectors carrying the firefly luciferase cDNA under the control of a liver-specific promoter were administered to female mice, with total-body and hepatic luciferase expression periodically monitored through 41 weeks post-vector administration. Vector copy number per diploid genome in mouse liver and spleen was determined at the end of this study. We identified major strain-specific contributions to overall success of transduction, vector biodistribution, maximum luciferase expression, and the kinetics of luciferase expression throughout the study. Our results highlight the importance of genetic variation on gene-therapeutic efficacy; provide new models with which to more rigorously assess gene therapy approaches; and suggest that redesigning preclinical studies of gene-therapy methodologies might be appropriate. Abstract Production of large quantities of viral vectors is crucial for the success of gene therapy in the clinic. There is a need for higher titers of herpes simplex virus-1 (HSV-1) vectors both for therapeutic use as well as in the manufacturing of clinical grade adenoassociated virus (AAV) vectors. HSV-1 yield increased when primary human fibroblasts were treated with anti-inflammatory drugs like dexamethasone or valproic acid. In our search for compounds that would increase HSV-1 yield, we investigated another anti-inflammatory compound, aurintricarboxylic acid (ATA). Although ATA has been previously shown to have antiviral effects, we find that low (micromolar) concentrations of ATA increased HSV-1 vector production yields. Our results showing the use of ATA to increase HSV-1 titers have important implications for the production of certain HSV-1 vectors as well as recombinant AAV vectors. Abstract Leishmania is associated with a broad spectrum of diseases, ranging from simple cutaneous to invasive visceral leishmaniasis. Here, the sequences of ten cysteine proteases of types A, B and C of Leishmania major were obtained from GeneDB database. Prediction of MHC class I epitopes of these cysteine proteases was performed by NetCTL program version 1.2. In addition, by using BcePred server, dif ferent structural properties of the proteins were predicted to f ind out their potential B cell epitopes. According to this computational analysis, nine regions were predicted as B cell epitopes. The results provide useful information for designing peptide-based vaccines. Abstract Metabolite-responsive RNA pseudoknots derived from prokaryotic riboswitches have been shown to stimulate −1 programmed ribosomal frameshifting (PRF), suggesting −1 PRF as a promising gene expression platform to extend riboswitch applications in higher eukaryotes. However, its general application has been hampered by difficulty in identifying a specific ligand-responsive pseudoknot that also functions as a ligand-dependent -1 PRF stimulator. We addressed this problem by using the −1 PRF stimulation pseudoknot of SARS-CoV (SARS-PK) to build a ligand-dependent −1 PRF stimulator. In particular, the extra stem of SARS-PK was replaced by an RNA aptamer of theophylline and designed to couple theophylline binding with the stimulation of −1 PRF. Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophyllinedependent −1 PRF stimulation activity in a stable human 293T cell-line. Thus, RNA-ligand interaction repertoire provided by in vitro selection becomes accessible to ligand-specific −1 PRF stimulator engineering using SARS-PK as the scaffold for synthetic biology application. Abstract Background Isolation of cases and contact tracing is used to control outbreaks of infectious diseases, and has been used for coronavirus disease 2019 (COVID-19). Whether this strategy will achieve control depends on characteristics of both the pathogen and the response. Here we use a mathematical model to assess if isolation and contact tracing are able to control onwards transmission from imported cases of COVID-19.We developed a stochastic transmission model, parameterised to the COVID-19 outbreak. We used the model to quantify the potential effectiveness of contact tracing and isolation of cases at controlling a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-like pathogen. We considered scenarios that varied in the number of initial cases, the basic reproduction number (R 0 ), the delay from symptom onset to isolation, the probability that contacts were traced, the proportion of transmission that occurred before symptom onset, and the proportion of subclinical infections. We assumed isolation prevented all further transmission in the model. Outbreaks were deemed controlled if transmission ended within 12 weeks or before 5000 cases in total. We measured the success of controlling outbreaks using isolation and contact tracing, and quantified the weekly maximum number of cases traced to measure feasibility of public health effort.Findings Simulated outbreaks starting with five initial cases, an R 0 of 1·5, and 0% transmission before symptom onset could be controlled even with low contact tracing probability; however, the probability of controlling an outbreak decreased with the number of initial cases, when R 0 was 2·5 or 3·5 and with more transmission before symptom onset. Across different initial numbers of cases, the majority of scenarios with an R 0 of 1·5 were controllable with less than 50% of contacts successfully traced. To control the majority of outbreaks, for R 0 of 2·5 more than 70% of contacts had to be traced, and for an R 0 of 3·5 more than 90% of contacts had to be traced. The delay between symptom onset and isolation had the largest role in determining whether an outbreak was controllable when R 0 was 1·5. For R 0 values of 2·5 or 3·5, if there were 40 initial cases, contact tracing and isolation were only potentially feasible when less than 1% of transmission occurred before symptom onset.Interpretation In most scenarios, highly effective contact tracing and case isolation is enough to control a new outbreak of COVID-19 within 3 months. The probability of control decreases with long delays from symptom onset to isolation, fewer cases ascertained by contact tracing, and increasing transmission before symptoms. This model can be modified to reflect updated transmission characteristics and more specific definitions of outbreak control to assess the potential success of local response efforts. Abstract Unexplained fever is one of the most common and difficult diagnostic problems faced daily by clinicians. This study evaluated the differences in health service utilization, health care expenditures, and quality of care provided to patients with unexplained fever before and after global budget (GB) implementation in Taiwan.The National Health Insurance Research Database was used for analyzing the health care expenditures and quality of care before and after implementation of the GB system. Patients diagnosed as having unexplained fever during 2000-2001 were recruited; their 2000-2001 and 2004-2005 data were considered baseline and postintervention data, respectively.Data of 259 patients with unexplained fever were analyzed. The mean lengths of stay (LOSs) before and after GB system implementation were 4.22 ± 0.35 days and 5.29 ± 0.70 days, respectively. .28 after implementation. The mean rates of revisiting the emergency department within 3 days and readmission within 14 days increased respectively from 10.5% ± 2.7% and 8.3% ± 2.4% before implementation to 6.3% ± 2.2% and 4.0% ± 1.7% after implementation.GB significantly increased LOS and incremental total costs for patients with unexplained fever; but improved the quality of care. Abbreviations: BNHI = Bureau of National Health Insurance, CMV = cytomegalovirus, CT = computed tomography, ED = emergency department, FDG-PET = fluorodeoxyglucose positron emission tomography, FFS = fee-for-service, FUO = fever of unknown origin, GB = global budget, HIV = human immunodeficiency virus, IRB = Institutional Review Board, IRR = incidence-rate ratio, LOS = length of stay, NHI = National Health Insurance, NHIRD = National Health Insurance Research Database, NHRI = National Health Research Institutes, SARS = severe acute respiratory syndrome, SPECT = single-photon emission computed tomography, TB = tuberculosis. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) still causes outbreaks despite public awareness and implementation of health care measures, such as rapid viral diagnosis and patient quarantine. Here we describe the current epidemiological picture of MERS-CoV, focusing on humans and animals affected by this virus and propose specific intervention strategies that would be appropriate to control MERS-CoV. One-third of MERS-CoV patients develop severe lower respiratory tract infection and succumb to a fatal outcome; these patients would require effective therapeutic antiviral therapy. Because of the lack of such intervention strategies, supportive care is the best that can be offered at the moment. Limiting viral spread from symptomatic human cases to health care workers and family members, on the other hand, could be achieved through prophylactic administration of MERS-CoV neutralizing antibodies and vaccines. To ultimately prevent spread of the virus into the human population, however, vaccination of dromedary camelscurrently the only confirmed animal host for MERS-CoVmay be the best option to achieve a sustained drop in human MERS cases in time. In the end, a One Health approach combining all these different efforts is needed to tackle this zoonotic outbreak. Abstract Hepatitis B virus (HBV) infection is considered a major public health problem worldwide, and a significant number of reports on nosocomial and occupational outbreaks have been reported. This systematic investigation of HBV stability and susceptibility to different antiseptics revealed that HBV infectivity was very stable, with a half-life of >22 days at 37°C. At 4°C, infectivity was barely reduced for up to 9 months. Different alcohols and commercially available hand antiseptics had a virucidal effect against HBV. We propose that very strict compliance with established hygienic guidelines should be mandatory to avoid and prevent HBV infections. Abstract Animal models that mimic cardiovascular diseases are indispensable tools for understanding the mechanisms underlying the diseases at the cellular and molecular level. This review focuses on various methods in preclinical research to create small animal models of cardiac diseases, such as myocardial infarction, dilated cardiomyopathy, heart failure, myocarditis and cardiac hypertrophy, and the related stem cell treatment for these diseases Abstract Export of cargo from the ER occurs through the formation of 60-70-nm COPII-coated vesicular carriers. We have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of ER export sites in vivo and in vitro. We find that ER buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. The basic organization of an export complex can be divided into an active COPIIcontaining budding zone on a single ER cisterna, which is adjacent to budding zones found on distantly connected ER cisternae. These budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain COPI coats involved in retrograde transport. Vesicles within these export complexes contain concentrated cargo molecules. The structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. We conclude that budding from the ER through recruitment of COPI! is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by COPI. Abstract Graphical Abstract Highlights d Influenza A virus PA-X targets the majority of host mRNAs for destruction d Downregulation by PA-X correlates with the number of splice sites in a transcript d Splicing renders RNAs susceptible to PA-X d The cellular CFIm complex interacts with PA-X and contributes to PA-X activity Correspondence craig.mccormick@dal.ca (C.M.), d.khaperskyy@dal.ca (D.A.K.), marta.gaglia@tufts.edu (M.M.G.) In Brief Gaucherand et al. uncover a unique relationship between RNA degradation by the influenza A virus ribonuclease PA-X and host RNA splicing, which allows PA-X to selectively target host RNAs for destruction. Abstract An efficient Z1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote Z1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several Z1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of Z1 PRF. Structurebased mutagenesis on hTPK-DU177 reveals that the Z1 PRF efficiency of this stimulator can be modulated by sequential removal of base-triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem-loop interactions and their relative positions to the helical junction play crucial roles for the Z1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate Z1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate Z1 PRF. Furthermore, it suggests that Z1 PRF, induced by an in-trans RNA via specific basetriple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs. Abstract Bacterial enteric infections resulting in diarrhea, dysentery, or enteric fever constitute a huge public health problem, with more than a billion episodes of disease annually in developing and developed countries. In this study, the deadly agent of hemorrhagic diarrhea and hemolytic uremic syndrome, Escherichia coli O157:H7 was investigated with extensive computational approaches aimed at identifying novel and broad-spectrum antibiotic targets. A systematic in silico workflow consisting of comparative genomics, metabolic pathways analysis, and additional drug prioritizing parameters was used to identify novel drug targets that were essential for the pathogen's survival but absent in its human host. Comparative genomic analysis of Kyoto Encyclopedia of Genes and Genomes annotated metabolic pathways identified 350 putative target proteins in E. coli O157:H7 which showed no similarity to human proteins. Further bioinformatic approaches including prediction of subcellular localization, calculation of molecular weight, and web-based investigation of 3D structural characteristics greatly aided in filtering the potential drug targets from 350 to 120. Ultimately, 44 non-homologous essential proteins of E. coli O157:H7 were prioritized and proved to have the eligibility to become novel broad-spectrum antibiotic targets and DNA polymerase III alpha (dnaE) was the top-ranked among these targets. Moreover, druggability of each of the identified drug targets was evaluated by the DrugBank database. In addition, 3D structure of the dnaE was modeled and explored further for in silico docking with ligands having potential druggability. Finally, we confirmed that the compounds N-coeleneterazine and N-(1,4-dihydro-5H-tetrazol-5-ylidene)-9-oxo-9H-xanthene-2-sulfonamide were the most suitable ligands of dnaE and hence proposed as the potential inhibitors of this target protein. The results of this study could facilitate the discovery and release of new and effective drugs against E. coli O157:H7 and other deadly human bacterial pathogens. Abstract Screening for thousands of viruses and other pathogenic microorganisms, including bacteria, fungi, and parasites, in human tumor tissues will provide a better understanding of the contributory role of the microbiome in the predisposition for, causes of, and therapeutic responses to the associated cancer. Metagenomic assays designed to perform these tasks will have to include rapid and economical processing of large numbers of samples, supported by straightforward data analysis pipeline and flexible sample preparation options for multiple input tissue types from individual patients, mammals, or environmental samples. To meet these requirements, the PathoChip platform was developed by targeting viral, prokaryotic, and eukaryotic genomes with multiple DNA probes in a microarray format that can be combined with a variety of upstream sample preparation protocols and downstream data analysis. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and deep sequencing methods. These studies demonstrate the use of the PathoChip technology combined with PCR and deep sequencing as a valuable strategy for detecting the presence of pathogens in human cancers and other diseases.Citation Baldwin DA, Feldman M, Alwine JC, Robertson ES. 2014. Metagenomic assay for identification of microbial pathogens in tumor tissues. mBio 5(5):e01714-14. Abstract Methamphetamine (METH) is a psychostimulant with high abuse potential and severe neurotoxicity. Recent studies in animal models have indicated that METH can impair the blood-brain barrier (BBB), suggesting that some of the neurotoxic effects resulting from METH abuse could be due to barrier disruption. We report here that while chronic exposure to METH disrupts barrier function of primary human brain microvascular endothelial cells (HBMECs) and human umbilical vein endothelial cells (HUVECs), an early pro-survival response is observed following acute exposure by induction of autophagic mechanisms. Acute METH exposure induces an early increase in Beclin1 and LC3 recruitment. This is mediated through inactivation of the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70S6K pathway, and upregulation of the ERK1/2. Blockade of Kappa opioid receptor (KOR), and treatment with autophagic inhibitors accelerated METH-induced apoptosis, suggesting that the early autophagic response is a survival mechanism for endothelial cells and is mediated through the kappa opioid receptor.Our studies indicate that kappa opioid receptor can be therapeutically exploited for attenuating METH-induced BBB dysfunction.Methamphetamine (METH) is a central nervous system (CNS) stimulant that is similar in structure to amphetamine. The escalating abuse of this drug in recent years and its longlasting neurotoxic effects have placed an increased burden upon health care costs. Previous studies have shown that METH-induced autophagy in neurons is associated with the formation of ubiquitin-positive aggregates and multilamellar bodies. 1,2 In addition to its direct effects on neurons, bloodbrain barrier (BBB) dysfunction could also contribute to METH-induced neurotoxicity. 3 The BBB is a specialized structure formed by brain endothelial cells that are tightly interconnected to form a boundary between the CNS and periphery . 4 Recent findings suggest that METH impairment of glucose transporter protein-1(GLUT1) at the brain endothelium may affect energy-associated disruption of tight junction assembly and loss of BBB integrity. 5 Furthermore, METH can act directly on cultured primary rat brain microvascular endothelial cells to compromise their barrier properties inducing eNOS activation and enhanced transcytosis. 3 The primary goal of the current study was to investigate whether METH treatment induces autophagy in endothelial cells. Macroautophagy (hereafter referred to as autophagy) is an intracellular lysosomal (vacuolar) degradation process that is characterized by the formation of a cytosolic doublemembrane vesicle, the autophagosome. During autophagy, cytoplasmic proteins, organelles or other materials are surrounded by phagophores, which expand and close to form double-membrane autophagosomes. These autophagosomes fuse with lysosomes (or vacuoles) to form autolysosomes, in which the cytoplasmic cargos are degraded by lysosomal enzymes. The primary role of autophagy is to ensure cell survival under stress conditions. 6 Most assays for autophagy modulators use the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3) as a readout for autophagic activity. LC3 is a mammalian orthologue of the yeast ATG 8 protein, a ubiquitin-like protein that becomes lipidated and tightly associated with the autophagosomal membranes. 7 Lysosomal-associated membrane protein1 (LAMP1) is among the most abundant lysosomal membrane protein. It is a heavily glycosylated protein creating a sugar coat or glycocalyx on the inner side of the lysosomal membrane which aids to the protection of the membrane from the hydrolytic enzymes and degradation. LAMP1 is expressed mainly in the endosome-lysosomal membrane of cells which is involved in maintaining lysosomal membrane integrity and phagolysosome formation. 8, 9 We show that METH exposure-induced autophagy early on and progressively changed into apoptosis in a time-dependent manner. The METH-induced autophagy was specific and initiated even in the presence of nutrient-rich medium. Inhibition of METH-induced autophagy accelerated apoptosis of endothelial cells. METH-induced autophagy was mediated 1 Abstract Socio-economic factors are widely believed to have been an important driver of the transmission of Ebola Virus Disease (EVD) during the West African outbreak of 2014-16, however, studies that have investigated the relationship between socio-economic status (SES) and EVD have found inconsistent results. Using nationally representative household survey data on whether respondents knew a close friend or family member with Ebola, we explore the SES determinants of EVD exposure along individual, household, and community lines in Liberia and Sierra Leone. While we find no overall association between household wealth and EVD exposure, we find that pooled data mask important differences observed within countries with higher wealth households more likely to have been exposed to EVD in Sierra Leone and the opposite relationship in Liberia. Finally, we also generally find a positive association between education and EVD exposure both at the individual and the community levels in the full sample. There is an urgent need to better understand these relationships to examine both why the outbreak spread and to help prepare for future outbreaks. Abstract Since the emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, there have been a number of clusters of human-to-human transmission. These cases of human-tohuman transmission involve close contact and have occurred primarily in healthcare settings, and they are suspected to result from repeated zoonotic introductions. In this study, we sequenced whole MERS-CoV genomes directly from respiratory samples collected from 23 confirmed MERS cases in the United Arab Emirates (UAE). These samples included cases from three nosocomial and three household clusters. The sequences were analysed for changes and relatedness with regard to the collected epidemiological data and other available MERS-CoV genomic data. Sequence analysis supports the epidemiological data within the clusters, and further, suggests that these clusters emerged independently. To understand how and when these clusters emerged, respiratory samples were taken from dromedary camels, a known host of MERS-CoV, in This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. the same geographic regions as the human clusters. Middle East respiratory syndrome coronavirus genomes from six virus-positive animals were sequenced, and these genomes were nearly identical to those found in human patients from corresponding regions. These data demonstrate a genetic link for each of these clusters to a camel and support the hypothesis that human MERS-CoV diversity results from multiple zoonotic introductions. Abstract To improve understanding of the relative burden of different causative respiratory pathogens on respiratory syndromic indicators monitored using syndromic surveillance systems in England. Abstract This study was performed to characterize respiratory viral infections in pediatric patients undergoing hematopoietic stem cell transplantation (HSCT). Study samples included 402 respiratory specimens obtained from 358 clinical episodes that occurred in the 116 children of the 175 consecutive HSCT cohort at Seoul National University Children's Hospital, Korea from 2007 to 2010. Multiplex reverse-transcription polymerase chain reactions were performed for rhinovirus, respiratory syncytial virus (RSV), parainfluenza viruses (PIVs), adenovirus, human coronavirus (hCoV), influenza viruses and human metapneumovirus. Viruses were identified in 89 clinical episodes that occurred in 58 patients. Among the 89 clinical episodes, frequently detected viruses were rhinovirus in 25 (28.1%), RSV in 23 (25.8%), PIV-3 in 16 (18.0%), adenovirus in 12 (13.5%), and hCoV in 10 (11.2%). Lower respiratory tract infections were diagnosed in 34 (38.2%). Neutropenia was present in 24 (27.0%) episodes and lymphopenia was in 31 (34.8%) episodes. Sixty-three percent of the clinical episodes were hospital-acquired. Three patients died of respiratory failure caused by respiratory viral infections. Respiratory viral infections in pediatric patients who have undergone HSCT are common and are frequently acquired during hospitalization. Continuous monitoring is required to determine the role of respiratory viruses in immunocompromised children and the importance of preventive strategies. Abstract Canine parvovirus type 2 (CPV-2) is the main etiological agent of viral enteritis in dogs. Actually in literature, CPV-2 has been reported with clinical signs that vary from the classical disease, and immunochromatography test and PCR technique have been introduced to veterinary hospitals to confirm CPV-2 diagnosis and other infections. However, the reliability of these techniques has been poorly analyzed. In this study, we evaluated the sensitivity and specificity of veterinary clinical diagnosis, immunochromatography test and PCR technique. Our data indicate that variations in the clinical signs of CPV-2 complicate the gathering of an appropriate diagnosis; and immunochromatography test and PCR technique do not have adequate sensitivity to diagnose positive cases. Abstract Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARS-CoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us. Abstract Background: Salmonellosis is a major cause of morbidity and mortality in neonatal calves, often occurring before preventative vaccines can be administered.Hypothesis/Objective: To evaluate the protective effect on calves of colostrum from cows vaccinated with a commercially available Salmonella Newport bacterin against a Salmonella Typhimurium challenge.Animals: Twenty Holstein bull calves from a university dairy farm.Methods: Nonrandomized placebo-controlled trial in which colostrum was harvested from 30 cows that received 2 doses of either Salmonella bacterin or saline before calving. Colostrum collected from each group was pooled and fed to 2 groups of 10 calves at birth. At approximately 2 weeks of age, calves were challenged with Salmonella Typhimurium. Clinical, hematologic, microbiological, and postmortem findings were compared between the 2 groups.: No differences in mortality, clinical findings, hematology results, blood and fecal cultures, or necropsy findings between the 2 groups were observed. Vaccinated cows had higher colostral titers, and calves fed this colostrum had higher serum titers (mean difference, 0.429; mean [SE], 0.852 [0.02] for vaccinated versus 0.423 [0.02] for control calves).Conclusions and Clinical Importance: Transfer of colostral immunoglobulins from Salmonella enterica serotype Newport bacterin to neonatal calves was not sufficient to decrease mortality, clinical signs, sepsis, intestinal damage, or fecal shedding when exposed to a highly pathogenic Salmonella isolate. A large-scale randomized controlled clinical trial is needed to evaluate the efficacy of this bacterin when administered in the dry period for prevention of salmonellosis in neonatal calves. Abstract Murine hepatitis virus (strain A59), (MHV-A59) is a coronavirus that buds into pre-Golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. The fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. MHV-A59 also infects, albeit inefficiently, AtT20 calls, murine pituitary tumor cells with a regulated as well as a constitutive exoeytic pathway. Here we examine AtT20 cells at early times after the infection, when the Golgi apparatus retains its morphological and biochemical integrity. We observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same Golgi stacks and accumulate in the trans-Golgi network. Their pathways diverge at this site, the condensed secretory proteins including the ACTH going to the secretory granules and the coronavirus to post-Golgi transport vesicles devoid of ACTH. On very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-Golgi vesicles. We conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein ACTH, diverge at the exit from the trans-Golgi network. Abstract Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential. Abstract Cetuximab improves the survival of patients with metastatic colorectal cancer. The main limitation is primary and secondary resistance, the underlying mechanism of which requires extensive investigation. We proved that PRSS expression levels are significantly negatively associated with the sensitivity of cancer cells to cetuximab. Detailed mechanistic analysis indicated that PRSS can cleave cetuximab, leading to resistance. Cetuximab or bevacizumab combined with SPINK1, a PRSS inhibitor, inhibited cell growth more efficiently than cetuximab or bevacizumab alone in xenograft models. PRSS levels in the serum of 156 patients with mCRC were analyzed, and poor efficacy of cetuximab therapy was observed in patients with aberrant PRSS expression. PRSS expression in monoclonal antibody (mAb)-treated patients with cancer from The Cancer Genome Atlas database was also evaluated to determine whether patients with higher PRSS expression have significantly reduced progression-free survival. Our work provides a strong scientific rationale for targeting PRSS in combination with cetuximab therapy. Abstract Canine infectious respiratory disease complex (CIRDC) viruses have been detected in dogs with respiratory illness. Canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAdV-2) and canine herpesvirus 1 (CaHV-1), are all associated with the CIRDC. To allow diagnosis, two conventional multiplex polymerase chain reactions (PCR) were developed to simultaneously identify four RNA and two DNA viruses associated with CIRDC. The two multiplex PCR assays were then validated on 102 respiratory samples collected from 51 dogs with respiratory illness by sensitivity and specificity determination in comparison to conventional simplex PCR and a rapid three-antigen test kit. All six viruses were detected in either individual or multiple infections. The developed multiplex PCR assays had a >87% sensitivity and 100% specificity compared to their simplex counterpart. Compared to the three-antigen test kit, the multiplex PCR assays yielded 100% sensitivity and more than 83% specificity for detection of CAdV-2 and CDV, but not for CIV. Therefore, the developed multiplex PCR modalities were able to simultaneously diagnose a panel of CIRDC viruses and facilitated specimen collection through being suitable for use of nasal or oral samples. KEY WORDS: canine infectious respiratory disease complex (CIRDC), diagnosis, multiplex PCRs, Thailand Abstract The infection dynamics of bovine respiratory syncytial virus (BRSV) were studied in randomly selected Norwegian dairy herds. A total of 134 herds were tested twice, six months apart. The herds were classified as positive for BRSV if at least one animal between 150 and 365 days old tested positive for antibodies against BRSV, thereby representing herds that had most likely had the virus present during the previous year. The prevalence of positive herds at the first and second sampling was 34 per cent and at 41 per cent, respectively, but varied greatly between regions. Negative herds were found in close proximity to positive herds. Some of these herds remained negative despite several new infections nearby. Of the herds initially being negative, 42 per cent changed status to positive during the six months. This occurred at the same rate during summer as winter, but a higher rate of animals in the herds was positive if it took place during winter. Of the herds initially being positive, 33 per cent changed to negative. This indicates that an effective strategy to lower the prevalence and the impact of BRSV could be to employ close surveillance and place a high biosecurity focus on the negative herds. Abstract Introduction Studies examining acute respiratory infections (ARIs) in emergency department (EDs), particularly in rural and remote areas, are rare. This study aimed to examine the burden of ARIs among Aboriginal and non-Aboriginal children presenting to Western Australian (WA) EDs from 2002 to 2012. Method Using a retrospective population-based cohort study linking ED records to birth and perinatal records, we examined presentation rates for metropolitan, rural and remote Aboriginal and non-Aboriginal children from 469 589 births. We used ED diagnosis information to categorise presentations into ARI groups and calculated age-specific rates. Negative binomial regression was used to investigate association between risk factors and frequency of ARI presentation. results Overall, 26% of presentations were for ARIs. For Aboriginal children, the highest rates were for those aged <12 months in the Great Southern (1233 per 1000 childyears) and Pilbara regions (1088 per 1000 child-years). Rates for non-Aboriginal children were highest in children <12 months in the Southwest and Kimberley (400 and 375 per 1000 child-years, respectively). Presentation rates for ARI in children from rural and remote WA significantly increased over time in all age groups <5 years. Risk factors for children presenting to ED with ARI were: male, prematurity, caesarean delivery and residence in the Kimberley region and lower socio-economic areas. Conclusion One in four ED presentations in WA children are for ARIs, representing a significant out-of-hospital burden with some evidence of geographical disparity. Planned linkages with hospital discharge and laboratory detection data will aid in assessing the sensitivity and specificity of ARI diagnoses in ED. Abstract Enteroviruses invade the host by crossing the intestinal mucosa, which is lined by polarized epithelium. A number of enteroviruses, including echoviruses (EV) and group B coxsackieviruses (CVB), initiate infection by attaching to decayaccelerating factor (DAF), a molecule that is highly expressed on the apical surface of polarized epithelial cells. We previously observed that entry of DAF-binding CVB3 into polarized intestinal epithelial cells occurs by an unusual endocytic mechanism that requires caveolin but does not involve clathrin or dynamin. Here we examined the entry of a DAF-binding echovirus, EV7. We found that drugs, small interfering RNAs (siRNAs), and dominant negative mutants that target factors required for clathrinmediated endocytosis, including clathrin and dynamin, inhibited both EV7 infection and internalization of virions from the cell surface. Once virus had entered the cell, it colocalized with markers of early endosomes (EEA1) and then late endosomes (LAMP-2). Inhibition of endosomal maturation-with siRNAs or dominant negative mutants targeting Rab5 and Rab7-inhibited infection and prevented release of viral RNA into the cell. These results indicate that EV7 is internalized by clathrin-mediated endocytosis and then moves to early and late endosomes before releasing its RNA. Trafficking through endosomes is known to be important for viruses that depend on low pH or endosomal cathepsin proteases to complete the entry process. However, we found that EV7 infection required neither low pH nor cathepsins.The cellular cues required for release of viral RNA from the enterovirus capsid ("uncoating") remain poorly defined. We found that EV7 moved to late endosomes and that release of RNA depended on endosomal maturation; nonetheless, EV7 did not depend on the endosomal factors implicated in uncoating and entry by other viruses. The results suggest either that an unidentified endosomal factor is essential for uncoating of EV7 or that trafficking through the endosome is an essential step in a pathway that leads to another intracellular organelle where uncoating is completed.Citation Kim C, Bergelson JM. 2012. Echovirus 7 entry into polarized intestinal epithelial cells requires clathrin and Rab7. mBio 3(2):e00304-11. Abstract Background: Medical tourism (MT) refers to circumstances in which people travel for medical treatments. The present study focuses on determining factors affecting MT in Iran.Methods: The study uses a mixed method approach. Initially, through a qualitative study, 12 experts were interviewed deeply; then, 22 participants in three equal focus groups expressed their ideas about growth and development of MT in Iran. Based on the expressed ideas, 120 factors were identified and accordingly a structured questionnaire was developed. Some members from the focus groups confirmed the questionnaire's face and content validity. The reliability of pertinent items was confirmed using Cronbach's alpha=0.8. Afterwards, 61 eligible subjects filled out this questionnaire.Results: The findings showed that "healthcare quality" and "high level of expertise" are two most attractive factors in MT. However, other factors such as "healthcare costs", and "visa facilities" are among key factors as well. Also, the role of "the healthcare providers" was found to be more prominent than the roles of "the government" and "the general tourist services".Conclusion: Although some attractive MT factors are present currently, MT expansion to a desirable level in Iran requires a comprehensive plan of which its factors were discussed in this paper. Abstract Interest in bat-borne diseases and parasites has grown in the past decade over concerns for human health. However, the drivers of parasite diversity among bat host species are understudied as are the links between parasite richness and emerging risks. Thus, we aimed at exploring factors that explain macro and microparasite species richness in bats from Southeast Asia, a hotspot of emerging infectious diseases. First, we identified bat species that need increased sampling effort for pathogen discovery. Our approach highlights pathogen investigation disparities among species within the same genus, such as Rhinolophus and Pteropus. Secondly, comparative analysis using independent contrasts method allowed the identification of likely factors explaining parasite and viral diversity of bats. Our results showed a key role of bat distribution shape, an index of the fragmentation of bat distribution, on parasite diversity, linked to a decrease for both viral and endoparasite species richness. We discuss how our study may contribute to a better understanding of the link between parasite species richness and emergence. Abstract The provincial government of Jeju, South Korea, was notified that a 42-year-old man infected with the Middle East Respiratory Syndrome (MERS) coronavirus had gone sightseeing in Jeju Island. Although the visiting period might be interpreted as the incubation period of MERS, the province decided to conduct active surveillance to prevent a worst-case scenario. Based on the channel of movement of the patient, healthy isolation and active monitoring were conducted for persons who came in contact with the patient. During the active surveillance, none of the 56 persons in self-isolation and 123 persons under active monitoring became infected. This fact supports that MERS is not contagious during the incubation period. Abstract Bioactive peptides cleaved from the egglaying hormone precursor in the bag cell neurons of Abstract Recently, the risk of viral infection has dramatically increased owing to changes in human ecology such as global warming and an increased geographical movement of people and goods. However, the efficacy of vaccines and remedies for infectious diseases is limited by the high mutation rates of viruses, especially, RNA viruses. Here, we comprehensively review the effectiveness of several probiotics and paraprobiotics (sterilized probiotics) for the prevention or treatment of virally-induced infectious diseases. We discuss the unique roles of these agents in modulating the cross-talk between commensal bacteria and the mucosal immune system. In addition, we provide an overview of the unique mechanism by which viruses are eliminated through the stimulation of type 1 interferon production by probiotics and paraprobiotics via the activation of dendritic cells. Although further detailed research is necessary in the future, probiotics and/or paraprobiotics are expected to be among the rational adjunctive options for the treatment of various viral diseases. Abstract Macroautophagy (autophagy) involves the formation of a double-membrane organelle called the autophagosome, which sequesters cytoplasmic components that are degraded upon its fusion with the lysosome (Stolz et al., 2014; Kaur and Debnath, 2015) . Studies in yeast have identified >30 autophagy-related (ATG) proteins, many of which have identified mammalian orthologues. The canonical process of autophagy begins with initiation at the phagophore assembly site, mediated by the UNC51-like kinase (ULK) complex, which is composed of the ATG proteins ULK1/2, ATG13, FIP200, and ATG101. The ULK complex supports the activation of a class III phosphatidylinositol 3-kinase complex consisting of Beclin 1 (ATG6), ATG14, and VPS34 to produce phosphatidylinositol triphosphate, which serves as the initial membrane mark recognized by early autophagic effector proteins. Phagophore membrane expansion is mediated by the two ubiquitin-like conjugation systems involving multiple ATG proteins that ultimately lead to the lipidation of microtubule-associated protein 1 light chain 3 (LC3), the mammalian orthologue of ATG8. LC3 is recognized by autophagy cargo receptors that promote the selective capture and engulfment of proteins, organelles, or microbes. The closed autophagosome is then trafficked to and fuses with the lysosome to degrade its cargo (Stolz et al., 2014; Kaur and Debnath, 2015) . ATG proteins traditionally have been studied for their roles in autophagosome formation and maturation, but we have recently begun to appreciate that they may have pleiotropic roles beyond autophagy (Bestebroer et al., 2013) . In this issue, through a comprehensive functional screen for ATG proteins in the control of viral replication, Mauthe et al. highlight the importance of the nontraditional functions of ATG proteins in the host viral response.In a tour-de-force study examining multiple models of viral infection, Mauthe et al. (2016) interrogated the individual roles of ATG proteins in the replication of herpes simplex virus type 1, mouse hepatitis virus (MHV), vaccinia virus (VaV), semliki forest virus, encephalomyocarditis virus, and influenza A virus. The authors generated a curated siRNA library to target all ATG proteins individually as well as functionally redundant groups concurrently to evaluate how they promote the replication of these six viruses in two model cell lines (HeLa and U2OS). Indeed, their screen reveals that unconventional functions of ATG proteins are widespread; overall, 16 out of the 44 tested ATG proteins (36%) appear to have unconventional functions. Rarely does knockdown of individual ATG genes result in concordant effects on viral replication, arguing against a general role for autophagy. Moreover, the authors fail to identify any single ATG protein that regulates viral replication in all of the tested viruses; rather, specific ATG genes influenced the replication of each virus. Remarkably, most of the ATG proteins show a beneficial role for VaV replication in U2OS cells yet negatively impact VaV replication in HeLa cells. Overall, the effect of ATG loss of function on viral replication is exquisitely ATG specific, virus specific, and cell line specific.To demonstrate functions of ATG proteins independent of autophagy, the authors scrutinized ATG13 and FIP200, two ULK complex proteins that initiate formation of the autophagosome, and found that these proteins exert effects on viral replication distinct from the effects of other ULK complex components. Mauthe et al. (2016) demonstrate that depletion of ATG13 and FIP200 reduces the replication of two picornaviruses, encephalomyocarditis virus and coxsackievirus (CV) B3, whereas the other ULK complex components promote their replication. Furthermore, a principal upstream regulator of ULK complex activity, mammalian target of rapamycin complex 1, showed no changes in activity during infection or knockdown of the ULK complex components. Altogether, this evidence indicates that ATG13 and FIP200 act independently of the ULK complex to modify picornaviral replication outside the context of autophagy.Although the precise mechanism of this unconventional function of ATG13 and FIP200 remains to be elucidated, Mauthe et al. (2016) uncovered that these proteins control viral replication as opposed to viral entry or viral translation. However, ATG13 and FIP200 do not interact with any CV proteins or affect the morphology of CV replication structures, suggesting Autophagy-related (ATG) proteins have increasingly demonstrated functions other than cellular self-eating. In this issue, Mauthe et al. (2016. J. Cell Biol. http ://dx .doi .org /10 .1083 /jcb .201602046) conduct an unbiased RNA interference screen of the ATG proteome to reveal numerous noncanonical roles for ATG proteins during viral infection. Abstract Drug combinations are synergistic when their combined efficacy exceeds the sum of the individual actions, but they rarely include ineffective drugs that become effective only in combination. We identified several ''enabling pairs'' of neutralizing and non-neutralizing anti-ebolavirus monoclonal antibodies, whose combination exhibited new functional profiles, including transforming a non-neutralizing antibody to a neutralizer. Sub-neutralizing concentrations of antibodies 2G4 or m8C4 enabled non-neutralizing antibody FVM09 (IC 50 >1 mM) to exhibit potent neutralization (IC 50 1-10 nM). While FVM09 or m8C4 alone failed to protect Ebola-virusinfected mice, a combination of the two antibodies provided 100% protection. Furthermore, nonneutralizers FVM09 and FVM02 exponentially enhanced the potency of two neu-tralizing antibodies against both Ebola and Sudan viruses. We identified a hotspot for the binding of these enabling antibody pairs near the interface of the glycan cap and GP2. Enabling cooperativity mayThis is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). be an underappreciated phenomenon for viruses, with implications for the design and development of immunotherapeutics and vaccines.In Brief: Howell et al. describe the phenomenon of "enabling, cooperative neutralization" where weakly neutralizing and/or non-neutralizing ebolavirus monoclonal antibodies exhibit potent neutralization when combined. The previously identified antibodies m8C4 and FVM09, which are non-protective alone, offer 100% protection against Ebola and Sudan virus challenge in mice when combined.Howell et al. Abstract Cadmium is an environmentally hazardous metal, which causes toxicity in humans. Inhalation of cigarette smoke and industrial fumes containing cadmium are sources of cadmium exposure. It is responsible for the malfunction of various organs, leading to disease particularly in the lungs, liver and kidneys. In the present study, the effect of cadmium chloride (CdCl 2 ) on cell viability, and the expression levels of interleukin (IL)-1α and IL-10 cytokines at various concentrations and incubation durations were assessed in MRC-9 human normal lung and A549 human lung cancer cells to elucidate the mechanism of cadmium toxicity. Cell viability was measured using a crystal violet dye binding assay. The expression levels of the cytokines were measured by cytokine specific enzyme-linked immunosorbent assay kits. The viability assay results revealed higher sensitivity of the A549 lung cancer cells to CdCl 2 compared with the normal MRC-9 lung cells. In the normal MRC-9 lung cells, higher expression levels of the cytokines were observed at the lowest CdCl 2 concentration at a shorter exposure time compared with the lung cancer cells. Higher levels of the cytokines were observed in the A549 lung cancer cells at all other times and concentrations compared with the MRC-9 cells, indicating higher levels of inflammation. The cytokine levels were reduced at higher CdCl 2 concentrations and longer exposure durations, demonstrating the toxic effect of cadmium. The results indicated that CdCl 2 affected the expression levels of the cytokines and led to cytotoxicity in human lung cells, and suggested that compounds which reduce inflammation may prevent cadmium toxicity. Abstract Perspective Since the Middle East respiratory syndrome (MERS) outbreak in the Republic of Korea (hereafter Korea) began on May 11, 2015, a total of 186 persons have been infected by the MERS coronavirus, 38 of whom have died. With this number, Korea becomes second only to the Kingdom of Saudi Arabia in the ranking of cumulative MERS cases. In this paper Korea's unique experience of an outbreak of MERS will be summarized and discussed briefly. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) infections continue to be a serious emerging disease problem internationally with well over 1000 cases and a major outbreak outside of the Middle East region. While the hypothesis that dromedary camels are the likely major source of MERS-CoV infection in humans is gaining acceptance, conjecture continues over the original natural reservoir host(s) and specifically the role of bats in the emergence of the virus. Dromedary camels were imported to Australia, principally between 1880 and 1907 and have since become a large feral population inhabiting extensive parts of the continent. Here we report that during a focussed surveillance study, no serological evidence was found for the presence of MERS-CoV in the camels in the Australian population. This finding presents various hypotheses about the timing of the emergence and spread of MERS-CoV throughout populations of camels in Africa and Asia, which can be partially resolved by testing sera from camels from the original source region, which we have inferred was mainly northwestern Pakistan. In addition, we identify bat species which overlap (or neighbour) the range of the Australian camel population with a higher likelihood of carrying CoVs of the same lineage as MERS-CoV. Both of these proposed follow-on studies are examples of "proactive surveillance", a concept that has particular relevance to a One Health approach to emerging zoonotic diseases with a complex epidemiology and aetiology. Abstract siRNA-aptamer chimeras have emerged as one of the most promising approaches for targeted delivery of siRNA due to the modularity of their diblock RNA structure, relatively lower cost over other targeted delivery approaches, and, most importantly, the outstanding potential for clinical translation. However, additional challenges must be addressed for efficient RNA interference (RNAi), in particular, endosomal escape. Currently, vast majority of siRNA delivery vehicles are based on cationic materials, which form complexes with negatively charged siRNA. Unfortunately, these approaches complicate the formulations again by forming large complexes with heterogeneous sizes, unfavorable surface charges, colloidal instability, and poor targeting ligand orientation. Here, we report the development of a small and simple protein tag that complements the therapeutic and targeting functionalities of chimera with two functional domains: a dsRNA binding domain (dsRBD) for siRNA docking and a pH-dependent polyhistidine to disrupt endosomal membrane. The protein selectively tags along the siRNA block of individual chimera, rendering the overall size of the complex small, desirable for deep tissue penetration, and the aptamer block accessible for target recognition. More interestingly, we found that extending the c-terminal polyhistidine segment in the protein tag to 18 amino acids completely abolishes the RNA binding function of dsRBD. s iRNA is of considerable current interest because it can elicit potent, target-specific knockdown of virtually any mRNA, creating new opportunities for personalized medicine and for addressing a broad range of traditionally undruggable disease targets using small molecules 1-3 . Similar to other antisense approaches, however, cell-specific delivery of siRNA technology in vivo still represents a major technical hurdle 4 . To guide siRNA to diseased cells, targeting ligands such as small molecules, lipids, peptides, and proteins have been identified and linked directly to siRNA or on the surface of siRNA nanocarriers 5-10 . Considering the complex physical and chemical structures of various formulations, the siRNA-targeting ligand-delivery vehicle complexes face difficulty in large-scale production and regulatory approval for clinical uses.Recently, siRNA-aptamer chimera, employing only RNA molecules, has emerged as a highly promising approach for cell type-specific RNAi, owing to its low immunogenicity, ease of chemical synthesis and modification, small size, and the targeting specificity of aptamers. RNA-based aptamers are identified through in vitro enrichment known as SELEX (systematic evolution of ligands by exponential enrichment) 11-14 . Similar to antibodies, they are capable of binding to various molecular targets including small molecules, proteins, and cells, while offering key advantages as they can be completely identified and produced with desired chemical modifications in vitro through automated processes. For aptamer-guided siRNA delivery, exciting works by McNamara, Dassie, and coworkers show that chimeras composed of aptamer targeting prostate specific membrane antigen (PSMA) and siRNA targeting anti-apoptotic genes (Plk1) have been made and optimized 15,16 . The aptamer block recognizes PSMA on prostate tumor cell surface and leads to chimera cell internalization, whereas the siRNA block enzymatically cleaved from the chimera promotes cell death. Significantly reduced tumor mass was observed in mouse xenograft models of prostate tumor after administration of the chimera, though the exact mechanism of chimera endosomal escape remains unclear 17 . This limitation helps explain why high concentrations of chimera were required in these pioneer studies to treat prostate tumors 15, 16 .An obvious solution to this problem is to combine chimeras with nanocarriers with endosome rupturing capabilities. Common delivery vehicles include lipids, polymers, and inorganic nanoparticles such as gold, silica, magnetic, and semiconductor nanoparticles [18] [19] [20] [21] [22] . For siRNA immobilization, condensation, stabilization against enzymatic degradation, and endosomal escape, virtually all these nanocarriers are positively charged, and so are their siRNA complexes. Unfortunately, the electrostatically induced nanocarrier-chimera condensation almost completely defies the purpose of simple formulation for siRNA clinical translation because the final nanoparticles become complex again, with mixed sizes, surface properties, aptamer conformations and orientations, and batchto-batch variations. For example, the size difference between the original intact chimeras (nanometers) with the OPEN SUBJECT AREAS: DRUG DELIVERY NANOPARTICLES Abstract We describe a fatal case of Japanese encephalitis virus infection following short-term travel to Thailand. Viral RNA was detected in urine and whole blood out to 26 and 28 days, respectively, after the onset of symptoms. Live virus was isolated from a urine specimen from day 14. Abstract A high percentage (up to 90%) of dromedary camels in the Middle East as well as eastern and central Africa have antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV). Here we report comparably high positivity of MERS-CoV antibodies in dromedary camels from northern Mali. This extends the range of MERS-CoV further west in Africa than reported to date and cautions that MERS-CoV should be considered in cases of severe respiratory disease in the region. Abstract Humans carry trillions of viruses that thrive because of their ability to exploit the host. In this exploitation, viruses promote their own replication by suppressing the host antiviral response and by inducing changes in host biosynthetic processes, often with extremely small genomes of their own. In the review, we discuss the phenomenon of histone mimicry by viral proteins and how this mimicry allows the virus to dial in to the cell's transcriptional processes and establish a cell state that promotes infection. We suggest that histone mimicry is part of a broader viral strategy to use intrinsic protein disorder as a means to overcome the size limitations of its own genome and to maximize its impact on host protein networks. In particular, we discuss how intrinsic protein disorder may enable viral proteins to interfere with phase-separated host protein condensates, including those that contribute to chromatin-mediated control of gene expression. Abstract Objectives The safety and efficacy of the 3C-like protease inhibitor GC376 was tested on a cohort of client-owned cats with various forms of feline infectious peritonitis (FIP). Methods Twenty cats from 3.3-82 months of age (mean 10.4 months) with various forms of FIP were accepted into a field trial. Fourteen cats presented with wet or dry-to-wet FIP and six cats presented with dry FIP. GC376 was administered subcutaneously every 12 h at a dose of 15 mg/kg. Cats with neurologic signs were excluded from the study. Results Nineteen of 20 cats treated with GC376 regained outward health within 2 weeks of initial treatment. However, disease signs recurred 1-7 weeks after primary treatment and relapses and new cases were ultimately treated for a minimum of 12 weeks. Relapses no longer responsive to treatment occurred in 13 of these 19 cats within 1-7 weeks of initial or repeat treatment(s). Severe neurologic disease occurred in 8/13 cats that failed treatment and five cats had recurrences of abdominal lesions. At the time of writing, seven cats were in disease remission. Five kittens aged 3.3-4.4 months with wet FIP were treated for 12 weeks and have been in disease remission after stopping treatment and at the time of writing for 5-14 months (mean 11.2 months). A sixth kitten was in remission for 10 weeks after 12 weeks of treatment, relapsed and is responding to a second round of GC376. The seventh was a 6.8-year-old cat with only mesenteric lymph node involvement that went into remission after three relapses that required progressively longer repeat treatments over a 10 month period. Side effects of treatment included transient stinging upon injection and occasional foci of subcutaneous fibrosis and hair loss. There was retarded development and abnormal eruption of permanent teeth in cats treated before 16-18 weeks of age. Conclusions and relevance GC376 showed promise in treating cats with certain presentations of FIP and has opened the door to targeted antiviral drug therapy. Abstract Owing to advancements in molecular diagnostics, recent years have seen an increasing number of laboratories adopting respiratory viral panels to detect respiratory pathogens. In December 2015, the NxTAG respiratory pathogen panel (NxTAG RPP) was approved by the United States Food and Drug Administration. We compared the clinical performance of this new assay with that of the xTAG respiratory viral panel (xTAG RVP) FAST v2 using 142 clinical samples and 12 external quality assessment samples. Discordant results were resolved by using a laboratory-developed respiratory viral panel. The NxTAG RPP achieved 100% concordant negative results and 86.6% concordant positive results. It detected one coronavirus 229E and eight influenza A/H3N2 viruses that were missed by the xTAG RVP FAST v2. On the other hand, the NxTAG RPP missed one enterovirus/rhinovirus and one metapneumovirus that were detected by FAST v2. Both panels correctly identified all the pathogens in the 12 external quality assessment samples. Overall, the NxTAG RPP demonstrated good diagnostic performance. Of note, it was better able to subtype the influenza A/H3N2 viruses compared with the xTAG RVP FAST v2. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract During canonical translation, the ribosome moves along an mRNA from the start to the stop codon in exact steps of one codon at a time. The collinearity of the mRNA and the protein sequence is essential for the quality of the cellular proteome. Spontaneous errors in decoding or translocation are rare and result in a deficient protein. However, dedicated recoding signals in the mRNA can reprogram the ribosome to read the message in alternative ways. This review summarizes the recent advances in understanding the mechanisms of three types of recoding events: stop-codon readthrough, -1 ribosome frameshifting and translational bypassing. Recoding events provide insights into alternative modes of ribosome dynamics that are potentially applicable to other non-canonical modes of prokaryotic and eukaryotic translation. Abstract Background: Micro-organisms transmitted from vertebrate animalsincluding livestockto humans account for an estimated 60% of human pathogens. Micro-organisms can be transmitted through inhalation, ingestion, via conjunctiva or physical contact. Close contact with animals is crucial for transmission. The role of intensity and type of contact patterns between livestock and humans for disease transmission is poorly understood. In this systematic review we aimed to summarise current knowledge regarding patterns of human-livestock contacts and their role in micro-organism transmission. Methods: We included peer-reviewed publications published between 1996 and 2014 in our systematic review if they reported on human-livestock contacts, human cases of livestock-related zoonotic diseases or serological epidemiology of zoonotic diseases in human samples. We extracted any information pertaining the type and intensity of human-livestock contacts and associated zoonoses. Results: 1522 papers were identified, 75 were included: 7 reported on incidental zoonoses after brief animalhuman contacts (e.g. farm visits), 10 on environmental exposures and 15 on zoonoses in developing countries where backyard livestock keeping is still customary. 43 studies reported zoonotic risks in different occupations. Occupations at risk included veterinarians, culling personnel, slaughterhouse workers and farmers. For culling personnel, more hours exposed to livestock resulted in more frequent occurrence of transmission. Slaughterhouse workers in contact with live animals were more often positive for zoonotic micro-organisms compared to co-workers only exposed to carcasses. Overall, little information was available about the actual mode of micro-organism transmission. Conclusions: Little is known about the intensity and type of contact patterns between livestock and humans that result in micro-organism transmission. Studies performed in occupational settings provide some, but limited evidence of exposure response-like relationships for livestock-human contact and micro-organism transmission. Better understanding of contact patterns driving micro-organism transmission from animals to humans is needed to provide options for prevention and thus deserves more attention. Abstract Introduction: N95 or higher filtering respirators have been recommended in healthcare settings, although there is still a risk of infection due to the improper selection and wearing of respirators. We aimed to assess the effects of training with N95 or higher filter respirators on the protection performance of respirators among healthcare providers in the emergency medical center (EMC).Methods: This randomized crossover study evaluated 23 healthcare providers. Quantitative fit tests (QNFTs) were performed before and after training using three types of N95 or higher filter respirators (cup-type, fold-type, valve-type). Training was performed by lecture, real-time feedback, and fit check. The primary outcome was the fit factor, and the secondary outcomes were overall fit factor, adequate protection rate, and respiratory preference.Results: Fit factors, overall fit factor, and adequate protection rate were higher after training than before training for the 3 types of respirators (all P < .05). For normal breathing, fit factors before and after training were 121 (10-185) vs 192 (161-200) for cup-type, 200 (39-200) vs 200 (200-200) for fold-type, and 85 (18-157) vs 173 (117-200) for valve-type. For normal breathing, the adequate protection rates before and after training were 62 (0-100) vs 100 (90-100) for cup-type, 100 (0-100) vs 100 (100-100) for fold-type, and 19 (0-100) vs 100 (44-100) for valve-type (all P < .05). The most preferred respirator type was the valve-type (10 persons, 45.5%).Conclusions: Training on wearing an N95 or higher respirator improved the protection performance of respirators among healthcare providers working in the EMC. The selection of proper respirators and training would be beneficial to the safety of healthcare providers. Abbreviations: CDC = American Centers for Disease Control and Prevention, EMC = emergency medical center, FFP = filtering facepiece respirator, MERS = Middle East respiratory syndrome, NIOSH = National Institute for Occupational Safety and Health, OSHA = Occupational Safety and Health Administration, PPE = personal protective equipment, QLFT = qualitative fit tests, QNFT = quantitative fit tests. Abstract Glioblastoma multiforme (GBM) is the most frequent and most devastating of the primary central nervous system tumors, with few patients living beyond 2 years postdiagnosis. The damage caused by the disease and our treatments for the patients often leave them physically and cognitively debilitated. Generally, GBMs appear after very short clinical histories and are discovered by imaging (using magnetic resonance imaging [MRI]), and the diagnosis is validated by pathology, following surgical resection. The treatment response and diagnosis of tumor recurrence are also tracked by MRI, but there are numerous problems encountered with these monitoring modalities, such as ambiguous interpretation and forms of pseudoprogression. Diagnostic, prognostic, and predictive biomarkers would be an immense boon in following treatment schemes and in determining recurrence, which often requires an invasive intracranial biopsy to verify imaging data. Extracellular vesicles (EVs) are stable, membrane-enclosed, virussized particles released from either the cell surface or from endosomal pathways that lead to the systemic release of EVs into accessible biofluids, such as serum/plasma, urine, cerebrospinal fluid, and saliva. EVs carry a wide variety of proteins, nucleic acids, lipids, and other metabolites, with many common features but with enough individuality to be able to identify the cell of origin of the vesicles. These components, if properly interrogated, could allow for the identification of tumor-derived EVs in biofluids, indicating tumor progression, relapse, or treatment failure. That knowledge would allow clinicians to continue with treatment regimens that were actually effective or to change course if the therapies were failing. Here, we review the features of GBM EVs, in terms of EV content and activities that may lead to the use of EVs as serially accessible biomarkers for diagnosis and treatment response in neuro-oncology. Abstract The basic reproduction number is one of the conceptual cornerstones of mathematical epidemiology. Its classical definition as the number of secondary cases generated by a typical infected individual in a fully susceptible population finds a clear analytical expression in homogeneous and stratified mixing models. Along with the generation time (the interval between primary and secondary cases), the reproduction number allows for the characterization of the dynamics of an epidemic. A clear-cut theoretical picture, however, is hardly found in real data. Here, we infer from highly detailed sociodemographic data two multiplex contact networks representative of a subset of the Italian and Dutch populations. We then simulate an infection transmission process on these networks accounting for the natural history of influenza and calibrated on empirical epidemiological data. We explicitly measure the reproduction number and generation time, recording all individual-level transmission events. We find that the classical concept of the basic reproduction number is untenable in realistic populations, and it does not provide any conceptual understanding of the epidemic evolution. This departure from the classical theoretical picture is not due to behavioral changes and other exogenous epidemiological determinants. Rather, it can be simply explained by the (clustered) contact structure of the population. Finally, we provide evidence that methodologies aimed at estimating the instantaneous reproduction number can operationally be used to characterize the correct epidemic dynamics from incidence data. computational modeling | infectious diseases | multiplex networks | reproduction number | generation timeThe analysis of real epidemiological data has raised issues of the adequacy of the classic homogeneous modeling framework and quantities, such as the basic reproduction number in real-world situations. Based on high-quality sociodemographic data, here we generate a multiplex network describing the contact pattern of the Italian and Dutch populations. By using a microsimulation approach, we show that, for epidemics spreading on realistic contact networks, it is not possible to define a steady exponential growth phase and a basic reproduction number. We show the operational use of the instantaneous reproduction rate as a good descriptor of the transmission dynamics. Abstract Purpose: Wheezing following viral lower respiratory tract infections (LRTIs) in children <2 years of age is an important risk factor for the development of asthma later in life; however, not all children with viral LRTIs develop wheezing. This study investigated risk factors for the development of wheezing during viral LRTIs requiring hospitalization. Methods: The study included 142 children <2 years of age hospitalized for LRTIs with at least one virus identified as the cause and classified them into children diagnosed with LRTIs with wheezing (n=70) and those diagnosed with LRTIs without wheezing (n=72). Results: There were no significant differences in the viruses detected between the two groups. Multivariate logistic regression analysis showed that, after adjusting for potentially confounding variables including sex and age, the development of wheezing was strongly associated with parental history of allergic diseases (adjusted odds ratio [aOR], 20.19; 95% confidence interval [CI], 3.22-126.48), past history of allergic diseases (aOR, 13.95; 95% CI, 1.34-145.06), past history of hospitalization for respiratory illnesses (aOR, 21.36; 95% CI, 3.77-120.88), exposure to secondhand smoke at home (aOR, 14.45; 95% CI, 4.74-44.07), and total eosinophil count (aOR, 1.01; 95% CI, 1.01-1.02). Conclusion: Past and parental history of allergic diseases, past history of hospitalization for respiratory illnesses, exposure to secondhand smoke at home, and total eosinophil count were closely associated with the development of wheezing in children <2 years of age who required hospitalization for viral LRTIs. Clinicians should take these factors into consideration when treating, counseling, and monitoring young children admitted for viral LRTIs. Abstract Background: Nonendoscopic bronchoalveolar lavage (BAL) is a practical alternative for a deep nasopharyngeal swab (DNS) to sample the airways of a large number of calves in a short period of time. The extent of commensal overgrowth and agreement of BAL with DNS culture results in preweaned calves are unknown.Objectives: To compare commensal overgrowth and bacterial culture results between DNS and BAL samples. Animals: A total of 183 preweaned calves (144 with bovine respiratory disease and 39 healthy animals).Methods: Cross-sectional study. Deep nasopharyngeal swab and BAL samples were taken from each calf and cultured to detect Pasteurellaceae and Mycoplasma bovis. Agreement and associations between culture results of DNS and BAL samples were determined by kappa statistics and logistic regression.Results: Bronchoalveolar lavage samples were less often polymicrobial, more frequently negative and yielded more pure cultures compared to DNS, leading to a clinically interpretable culture result in 79.2% of the cases compared to only in 31.2% of the DNS samples. Isolation rates were lower in healthy animals, but not different between DNS and BAL samples. Only Histophilus somni was more likely to be isolated from BAL samples. In clinical cases, a polymicrobial DNS culture result did not increase the probability of a polymicrobial BAL result by ≥30%, nor did it influence the probability of a negative culture. A significant herd effect was noted for all observed relationships.Conclusions and Clinical Relevance: Nonendoscopic BAL samples are far less overgrown by bacteria compared to DNS samples under the conditions of this study, facilitating clinical interpretation and resulting in a higher return on investment in bacteriologic culturing. Abstract The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored, gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOHterminal, 58-amino acid residue cytoplasmic tail (CT) ). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence micros-9 The Rockefeller University Press, Abstract The epidemiology and clinical features of the Saffold cardiovirus (SAFV) remain ambiguous. The present study was designed to systematically and intensively investigate the epidemiological features of SAFV in pediatric patients in China. Three cohorts of pediatric patients were recruited from 2009 to 2012. Cohort 1 comprised patients with acute respiratory tract infections. Cohort 2 comprised patients with diarrhea. Cohort 3 comprised hand, foot, and mouth disease (HFMD) patients. A total of 115 patients (1.6%) among 6052 (17/1647, 12/2013, and 86/2392 in cohorts 1, 2, and 3, respectively) were SAFV-positive. The samples from 82 SAFV-positive patients were successfully sequenced, and four genotypes were identified: 8 SAFV-1, 41 SAFV-2, 29 SAFV-3, and 4 SAFV-6. A significantly higher detection rate was found in the HFMD patients than in other two cohorts (both P ,0.001). A higher frequency of severe clinical outcome and nervous system manifestation were also observed in the SAFV-positive HFMD patients. Additionally, 6 (3.5%) cerebrospinal fluid and 7 (2.2%) serum samples from the HFMD-associated encephalitis patients were SAFV-positive. Based on the VP1 sequences, all four genotypes displayed distinct geographical clustering. SAFV infection might be associated with a wide clinical spectrum and contribute to HFMD. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) infections continue to be a serious emerging disease problem internationally with well over 1000 cases and a major outbreak outside of the Middle East region. While the hypothesis that dromedary camels are the likely major source of MERS-CoV infection in humans is gaining acceptance, conjecture continues over the original natural reservoir host(s) and specifically the role of bats in the emergence of the virus. Dromedary camels were imported to Australia, principally between 1880 and 1907 and have since become a large feral population inhabiting extensive parts of the continent. Here we report that during a focussed surveillance study, no serological evidence was found for the presence of MERS-CoV in the camels in the Australian population. This finding presents various hypotheses about the timing of the emergence and spread of MERS-CoV throughout populations of camels in Africa and Asia, which can be partially resolved by testing sera from camels from the original source region, which we have inferred was mainly northwestern Pakistan. In addition, we identify bat species which overlap (or neighbour) the range of the Australian camel population with a higher likelihood of carrying CoVs of the same lineage as MERS-CoV. Both of these proposed follow-on studies are examples of "proactive surveillance", a concept that has particular relevance to a One Health approach to emerging zoonotic diseases with a complex epidemiology and aetiology. Abstract Since late 2013, several outbreaks of porcine epidemic diarrhea virus (PEDV) infection have emerged in Taiwan. Suckling piglets under 2 weeks of age showed severe vomiting and watery yellowish diarrhea with morbidity and mortality ranging from 80 to 100% and 90 to 100%, respectively. A total of 68 samples from 25 pig farms were confirmed as positive for PEDV and negative for rotavirus and transmissible gastroenteritis virus by reverse transcription PCR, and the partial S gene of PEDV was analyzed. Phylogenetic analysis places all 18 Taiwanese PEDV isolates collected during this outbreak in the same clade as the US strains of PEDV. This novel PEDV is prevailing and currently causing severe outbreaks in Taiwan. Abstract Background. The epidemiology and burden of respiratory syncytial virus (RSV) illness are not well defined in older adults. Methods. Adults ≥60 years old seeking outpatient care for acute respiratory illness were recruited from 2004-2005 through 2015-2016 during the winter seasons. RSV was identified from respiratory swabs by multiplex polymerase chain reaction. Clinical characteristics and outcomes were ascertained by interview and medical record abstraction. The incidence of medically attended RSV was estimated for each seasonal cohort.Results. RSV was identified in 243 (11%) of 2257 enrollments (241 of 1832 individuals), including 121 RSV type A and 122 RSV type B. The RSV clinical outcome was serious in 47 (19%), moderate in 155 (64%), and mild in 41 (17%). Serious outcomes included hospital admission (n = 29), emergency department visit (n = 13), and pneumonia (n = 23) and were associated with lower respiratory tract symptoms during the enrollment visit. Moderate outcomes included receipt of a new antibiotic prescription (n = 144; 59%), bronchodilator/nebulizer (n = 45; 19%), or systemic corticosteroids (n = 28; 12%). The relative risk of a serious outcome was significantly increased in persons aged ≥75 years (vs 60-64 years) and in those with chronic obstructive pulmonary disease or congestive heart failure. The average seasonal incidence was 139 cases/10 000, and it was significantly higher in persons with cardiopulmonary disease compared with others (rate ratio, 1.89; 95% confidence interval, 1.44-2.48).Conclusions. RSV causes substantial outpatient illness with lower respiratory tract involvement. Serious outcomes are common in older patients and those with cardiopulmonary disease. Abstract Since there is no available serological methods to detect antibodies to ferret coronavirus (FRcoV), an enzyme-linked immunosorbent assay (ELISA) using recombinant partial nucleocapsid (N) proteins of the ferret coronavirus (FRcoV) Yamaguchi-1 strain was developed to establish a serological method for detection of FRcoV infection. Many serum samples collected from ferrets recognized both a.a. 1-179 and a.a. 180-374 of the N protein, but two serum samples did not a.a. 180-374 of the N protein. This different reactivity was also confirmed by immunoblot analysis using the serum from the ferret.Therefore, the a.a. 1-179 of the N protein was used as an ELISA antigen. Serological test was carried out using sera or plasma of ferrets in Japan. Surprisingly, 89% ferrets in Japan had been infected with FRcoV. These results indicated that our established ELISA using a.a. 1-179 of the N protein is useful for detection of antibody to FRcoV for diagnosis and seroepidemiology of FRcoV infection. KEY WORDS: enzyme-linked immunosorbent assay (ELISA), ferret coronavirus (FRcoV), nucleocapsid (N) Abstract Enteroviruses cause a wide spectrum of clinical disease. In this study, we describe the case of a young man with orchitis and aseptic meningitis who was diagnosed with enterovirus infection. Using unbiased "metagenomic" massively parallel sequencing, we assembled a near-complete viral genome, the first use of this method for full-genome viral sequencing from cerebrospinal fluid. We found that the genome belonged to the subgroup echovirus 30, which is a common cause of aseptic meningitis but has not been previously reported to cause orchitis.A previously healthy man in his 20s developed unilateral testicular pain and swelling. Four days later, he developed headache and fever. He presented to our institution, where he was febrile to 102.7°F with mild meningismus; testicular swelling and tenderness had resolved. The combination of symptoms raised concern for mumps in the setting of a local outbreak, although parotitis was absent. Cerebrospinal fluid (CSF) analysis showed 17 total nucleated cells/µL (79% neutrophils, 12% monocytes, 9% lymphocytes), glucose of 65 mg/dL (serum glucose of 109 mg/dL), and total protein of 31 mg/dL. He was treated empirically with vancomycin, ceftriaxone, and acyclovir. Cerebrospinal fluid enterovirus polymerase chain reaction (PCR) was positive, antimicrobials were discontinued, and the patient recovered fully. Mumps serology was positive for immunoglobulin (Ig)G and negative for IgM, consistent with his reported history of vaccination. A urine culture for mumps performed by the Massachusetts Department of Health State Laboratory was negative. Cerebrospinal fluid Gram stain and culture were negative, as were CSF herpes simplex virus 1 and 2 PCR, Lyme PCR, and testing for syphilis via the Venereal Disease Research Laboratory test. Given the relatively unusual presentation of orchitis and meningitis, we performed metagenomic sequencing to obtain additional genomic information about the particular strain of enterovirus and to identify any potential copathogens including mumps virus.We performed metagenomic sequencing and enterovirus genome assembly using methods developed and validated by our group [1, 2] . Full methods are described in the Supplementary Appendix.Unbiased metagenomic sequencing allows detection of any potential pathogens in a sample. We performed metagenomic analysis of all sequencing reads from this patient's CSF and identified enterovirus; no other pathogen, including mumps virus, was found. We assembled a near-full-length enterovirus genome (7212 base pair [bp], median depth of coverage 10×), which included the 5'-untranslated region (UTR) and near-complete coding region, but not the 3' end (21 bp) of the coding region or the 3'-UTR. By comparing this genome to published enterovirus genomes, we found that this virus belongs to echovirus subgroup 30 ( Figure 1A) , a member of the highly diverse enterovirus B species group, which includes most echoviruses, coxsackie B viruses, and coxsackie A9 virus [3] .Echovirus 30 has not previously been associated with orchitis, but other enteroviruses have, including echovirus 6 [4] and coxsackieviruses A9 and B [5, 6]. Therefore, we investigated whether the virus we identified was a recombinant between echovirus 30 and a subgroup that is more commonly associated with orchitis, because recombination between enteroviruses occurs frequently [3] . We first examined the VP1 gene, which encodes a capsid protein that interacts with host cell receptors and may therefore confer tropism for specific tissue such as the testes. In phylogenetic analysis of the VP1 gene, our virus again clustered with echovirus 30, rather than a subgroup more Open Forum Infectious Diseases ® Abstract Background: Cholesterol deficiency (CD), a newly identified autosomal recessive genetic defect in Holstein cattle, is associated with clinical signs of diarrhea, failure to thrive, and hypocholesterolemia.Hypothesis/Objectives: The objective is to describe the clinicopathological phenotype of affected Holstein cattle homozygous for the causative apolipoprotein B gene (APOB) mutation.Animals: Six Holstein cattle, 5 calves with a clinical history of chronic diarrhea, and 1 heifer with erosions in the buccal cavity and neurologic symptoms were admitted to the Clinic for Ruminants.Methods: This case review included a full clinical examination, a complete blood count, blood chemistry, and measurements of cholesterol and triglycerides. The animals were euthanized and necropsied. A PCR-based direct gene test was applied to determine the APOB genotype.Results: All 6 animals were inbred, could be traced back to the sire Maughlin Storm, and were confirmed homozygous for the APOB mutation. The clinical phenotype included poor development, underweight, and intermittent diarrhea in the calves, and neurologic signs in the heifer included hypermetria and pacing. Hypocholesterolemia and low triglycerides concentrations were present in all animals. The pathological phenotype of all animals was steatorrhea with enterocytes of the small intestine containing intracytoplasmic lipid vacuoles. The peripheral nervous system of the heifer displayed degenerative changes.Conclusions and clinical importance: Suspicion of CD in Holstein cattle is based on the presence of chronic diarrhea with no evidence of primary infections. Confirmation of the associated APOB gene mutation is needed. Additionally, the heifer demonstrated primarily signs of neurologic disease providing an unexpected phenotype of CD. Abstract Increased nasopharyngeal colonization density has been associated with pneumonia. We used experimental human pneumococcal carriage to investigate whether upper respiratory tract viral infection predisposes individuals to carriage. A total of 101 healthy subjects were screened for respiratory virus before pneumococcal intranasal challenge. Virus was associated with increased odds of colonization (75% virus positive became colonized vs. 46% virus-negative subjects; P ¼ 0.02). Nasal Factor H (FH) levels were increased in virus-positive subjects and were associated with increased colonization density. Using an in vitro epithelial model we explored the impact of increased mucosal FH in the context of coinfection. Epithelial inflammation and FH binding resulted in increased pneumococcal adherence to the epithelium. Binding was partially blocked by antibodies targeting the FH-binding protein Pneumococcal surface protein C (PspC). PspC epitope mapping revealed individuals lacked antibodies against the FH binding region. We propose that FH binding to PspC in vivo masks this binding site, enabling FH to facilitate pneumococcal/epithelial attachment during viral infection despite the presence of anti-PspC antibodies. We propose that a PspC-based vaccine lacking binding to FH could reduce pneumococcal colonization, and may have enhanced protection in those with underlying viral infection. Abstract Mouse hepatitis coronavirus (MHV) buds into pleomorphic membrane structures with features expected of the intermediate compartment between the ER and the Golgi complex. Here, we characterize the MHV budding compartment in more detail in mouse L cells using streptolysin O (SLO) permeabilization which allowed us to better visualize the membrane structures at the ER-Golgi boundary. The MHV budding compartment shares membrane continuities with the rough ER as well as with cisternal elements on one side of the Golgi stack. It also labeled with p58 and rab2, two markers of the intermediate compartment, and with PDI, usually considered to be a marker of the rough ER. The membranes of the budding compartment, as well as the budding virions themselves, but not the rough ER, labeled with the N-acetyl-galactosamine (GalNAc)-specific lectin Helix pomatia. When the SLO-permeabilized cells were treated with guanosine 5'-(3-O-thio)triphosphate (GTP',/S), the budding compartment accumulated a Abstract Introduction Immune reconstitution after haematopoietic stem cell transplantation (HSCT) is a complex and dynamic process, varying from a state of nearly complete immunosuppression to an expected full immune recovery. Specific vaccination guidelines recommend reimmunisation after HSCT but data regarding vaccine efficacy in this unique population are scarce. New immune functional assays could enable prediction of vaccine response in the setting of HSCT. Methods and analysis A prospective, longitudinal single-centre cohort study of autologous and allogeneic HSCT recipients was designed in order to determine the vaccine response to five vaccine targets (pneumococcus, hepatitis B virus, Haemophilus Influenzae type b, tetanus and diphtheria) and to correlate it to immune function parameters. A workflow was set up to study serological response to vaccines and to describe the functional immune status of 100 HSCT recipients (50 autologous and 50 allogeneic) before and 3, 12 and 24 months after primary immunisation. At each time point, 'basic' immune status recording (serology, immunophenotyping of lymphocyte subsets by flow cytometry) will be assessed. The immune response will furthermore be evaluated before and 3 months after primary vaccination by two ex vivo immune functional assays assessing: (1) tumour necrosis factor alpha, interferon gamma production and host messenger RNA expression on whole-blood stimulation by lipopolysaccharide or Staphylococcus aureus enterotoxin B and (2) T-lymphocyte proliferation in response to a standard mitogen (phytohaemagglutinin) or to selected recall antigens. Reference intervals will be determined from a cohort of 30 healthy volunteers. This translational study will provide data describing vaccine response, immune functionality of HSCT recipients over time and will allow mapping HSCT recipients with regard to their immune function. Abstract Human activated T lymphocytes expressing dass II molecules are able to present only complex antigens that bind to their own surface receptors, and thus can be captured, internalized, and processed through the class II major histocompatibility complex processing pathway. We have used the antigen-presenting T cell system to identify the viral receptor used by hepatitis B virus (HBV) to enter cells, as well as the sequence of HB envelope antigen (HBenvAg) involved in this interaction. Results show that both CD4 + and CD8 + T clones can process and present HBenvAg to class II-restricted cytotoxic T lymphocytes and that the CD71 transferrin receptor (TfR) is involved in efficient HBenvAg uptake by T cells. Moreover, we provide evidence that the HBenvAg sequence interacting with the T cell surface is contained within the pre-S2 region. Since TfR is also expressed on hepatocytes, it might represent a portal of cellular entry for HBV infection. This system of antigen presentation by T cells may serve as a model to study both lymphocyte receptors used by lymphocytotropic viruses and viral proteins critical to bind them. Abstract The People's Republic of China has nearly the highest incidence of both diabetes mellitus (DM) and tuberculosis (TB) worldwide. DM increases the risk of TB by two to three times and adversely affects TB treatment outcomes. The increasing epidemic of DM in the People's Republic of China is due to decreased physical activity, unhealthy diet, and obesity. Over the last 20 years, the excellent free China National Tuberculosis Program has been set up, and the "DOTS" (directly observed treatment + short-course chemotherapy) model for TB control has successfully reduced the burden of TB, but the disease is still a considerable problem. Given the high burden of TB and DM in the People's Republic of China and the relationship between the two diseases, it is sensible to screen DM patients for TB. A bidirectional screening of the two diseases was conducted in the People's Republic of China from 2011 to 2012, which identified a TB incidence in patients with DM of about 958 per 100,000. Here, we report the findings of our recent study on the incidence of TB among diabetic patients in the People's Republic of China. The data agree with those of previous reports. Abstract Chest X-ray is a "golden standard" for the diagnosis and severity assessment of community-acquired pneumonia (CAP). However, it cannot be used as routine examination of CAP in children. The present study aims to investigate the roles of prealbumin (PA) in CAP in children and further determine the usefulness of PA in diagnosis and severity assessment of CAP in children.This was a retrospective analysis of 174 cases of hospitalized children with CAP. The following indicators were recorded: vital sign, inflammatory indexes, PA, and respiratory pathogens immunoglobulin M antibody test results. A total of 33 healthy children were selected as the control group. The results of laboratory tests between CAP and control groups were compared. CAP group was further divided into mild CAP and severe CAP groups, and vital signs and laboratory examination results of 2 groups were compared.The total positive rate of Mycoplasma pneumoniae in this study was 27.4%, and there was no significant difference in different seasons (P = 0.356). Compared with controls, there was no significant difference between procalcitonin and C-reactive protein in CAP group (P = 0.355, 0.061). The white blood cell count, percentage of neutrophils, neutrophil count, and erythrocyte sedimentation rate in the CAP group were significantly higher than those in control group, and PA was significantly lower than that in the control group (all P < 0.05). In the traditional cutoff value (<170 mg/L), the sensitivity of PA for the diagnosis of CAP was 0.847, which was significant higher than traditional inflammatory indicators. Moreover, it was found that PA was an independent protective factor for CAP in children based on multivariate analysis (odds ratio: 0.974; 95% confidence interval: 0.956-0.993; P = 0.008). PA level in severe CAP group was significantly lower than in mild CAP group (P = 0.001). With a cutoff value of 125 mg/L, the sensitivity and specificity of PA for the severity assessment of CAP were 0.703 and 0.714, respectively.Combined with traditional inflammatory markers, PA may improve the diagnostic efficacy of CAP in children. PA can be used as a reference marker to complement the chest X-rays for severity assessment of children CAP.Abbreviations: CAP = community-acquired pneumonia, CRP = C-reactive protein, ESR = erythrocyte sedimentation rate, LRI = lower respiratory infections, MP = Mycoplasma pneumoniae, PA = prealbumin, PCT = procalcitonin, RR = respiratory rate, WBC = white blood cell count. Abstract Brefeldin A has dramatic, well-documented, effects on the structural and functional organization of the Golgi complex . We have examined the effects of brefeldin A (BFA) on the Golgi-localized synthesis and addition of chondroitin sulfate glycosaminoglycan carbohydrate side chains . BFA caused a dose-dependent inhibition of chondroitin sulfate glycosaminoglycan elongation and sulfation onto the core proteins of the melanoma-associated proteoglycan and the major histocompatibility complex class II-associated invariant chain . In the presence of BFA, the melanoma proteoglycan core protein was retained in the ER but still acquired complex, sialylated, N-linked oligosaccharides, as measured by digestion with endoglycosidase H and neuraminidase. The initiation of glycosaminoglycan syn- Abstract Five Japanese Black embryo transfer calves from a single embryo flush, 30 to 45-daysold, including 4 live animals for clinical examination and 1 dead for necropsy, were presented with a history of decreased milk intake and hypoproteinemia. Consistent clinicopathological abnormalities in the 4 calves presented for clinical evaluation included hyperkalemia, hyperphosphatemia, hypoproteinemia, hypoalbuminemia, hyperbilirubinemia, increased creatine phosphokinase activity, and proteinuria. Four calves ultimately were necropsied and all had histologic evidence of immune complex glomerulonephritis. Glomerulonephritis in these calves was hypothesized to have resulted from the interaction of passively acquired antibodies at birth and active immunization at 7 and 28 days of age with a Salmonella Typhimurium core antigen vaccine. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) still causes outbreaks despite public awareness and implementation of health care measures, such as rapid viral diagnosis and patient quarantine. Here we describe the current epidemiological picture of MERS-CoV, focusing on humans and animals affected by this virus and propose specific intervention strategies that would be appropriate to control MERS-CoV. One-third of MERS-CoV patients develop severe lower respiratory tract infection and succumb to a fatal outcome; these patients would require effective therapeutic antiviral therapy. Because of the lack of such intervention strategies, supportive care is the best that can be offered at the moment. Limiting viral spread from symptomatic human cases to health care workers and family members, on the other hand, could be achieved through prophylactic administration of MERS-CoV neutralizing antibodies and vaccines. To ultimately prevent spread of the virus into the human population, however, vaccination of dromedary camelscurrently the only confirmed animal host for MERS-CoVmay be the best option to achieve a sustained drop in human MERS cases in time. In the end, a One Health approach combining all these different efforts is needed to tackle this zoonotic outbreak. Abstract Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5 0 -segment that initiates stable priming, and a short 3 0 -segment that determines targetspecific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR. Abstract T his letter addresses some concerns about two recent articles published by the same authors in mBio (1, 2), specifically many uncertainties regarding the potential applicability of their epidemiological data, which were obtained from dromedary camels (DCs) infected with Middle East respiratory syndrome coronavirus (MERS-CoV), to human public health.Aiming to investigate the possible role of DCs in the transmission of MERS-CoV to humans, the authors in their first article determined the seroprevalence of MERS-CoV infection in DCs throughout Saudi Arabia and arrived at three main conclusions (1). Their first conclusion was that DCs can be infected with MERS-CoV, as evident by the determination of (i) high loads of viral nucleic acids (RNA) and antibodies to MERS-CoV in archived and freshly collected samples and (ii) a high resemblance (Ͼ99%) in the collected samples in three regions of phylogenetically analyzed MERS-CoV genomic sequence. Their second conclusion is that there are seroprevalence differences in MERS-CoV infection depending on the camel's age (95% of the adults compared to 35% of the juveniles) and the region of the country (ranging from 90% in the East to 5% in the Southwest). The third conclusion is that airborne transmission is the main mode of MERS-CoV transmission, as evidenced by the more frequent detection of viral nucleic acids in nasal swabs than in rectal specimens. Furthermore, the authors in their second article described complete genomic sequencing of MERS-CoV isolated from both DCs and humans and arrived at two more conclusions (2). First, they demonstrated that DCs can be simultaneously infected with three genetic variants (genotypes) of MERS-CoV; second, they showed that the alignment of the complete genomic sequence of one MERS-CoV genotype (claimed to be a quasispecies) obtained from culturing the virus from nasal swab samples of DCs was indistinguishable from the genomic sequence of MERS-CoV recovered from humans. Based on all these findings, the authors speculated that DCs may have a role or serve as a potential reservoir or vector of MERS-CoV in human infection and they clearly argued that we ought to orient future investigations on MERS disease among humans toward direct or indirect exposure to DCs.Although these findings are remarkable and obviously advance our knowledge in pursuing the evolutionary emergence of MERS disease, we argue that they cannot be taken as conclusive evidence in implicating DCs as harboring the infectious form of MERS-CoV and as serving as the source of infection for humans; much less, these findings are still only speculations and their utmost scientific importance is the assumption of an emerging interspecies transmission of MERS-CoV. In fact, our concerns can be better understood when discussed in the context of emergence (and reemergence) of infectious diseases, factors which were largely ignored by the authors over the mechanism of MERS-CoV emer-gence between humans and DCs and the most relevant to the debate.To elucidate this issue, four possible routes of MERS-CoV transmission should be epidemiologically assessed: human to human, camel to camel, camel to human, and human to camel. Regarding human-to-human MERS-CoV transmission, clusters of infection cases have indicated that MERS-CoV actually can be spread horizontally from human to human through close contact (3-5). Effective and successful emergence of MERS-CoV requires that the value of the basic reproduction number (R 0 ) should exceed 1 in the new host (presumably human) (6). In the case of MERS, the R 0 is currently less than 1 (most likely 0.5), indicating that MERS-CoV infection will inevitably die out; however, it is recommended, in such cases, to take into account the demographic stochasticity of MERS-CoV transmission (7). Despite the limited human-to-human transmission, the recent increases in the number of MERS-CoV infections among humans and the exceptionally high fatality rate associated with it as reported by WHO (8) represent a marked increase in MERS emergence (i.e., an increase in R 0 ) and subsequently an epidemic waiting to happen, which obviously is the key driver in the current debate.Regarding camel-to-camel transmission, although the identification of antibodies and viral nucleic acids of MERS-CoV in DCs is remarkable, these findings suggest only that DCs can be naturally infected with MERS-CoV and provide us with no clues as to how this transmission occurs. We recognize that these findings, together with the absence of viremia, as reported by the authors, and the lack of even a single case fatality in DCs, indicate that DCs may not only be a useful animal model for evaluating candidate vaccines and drugs against MERS but also a good reservoir of MERS-CoV. However, this form of transmission cannot yet be ascertained to implicate DCs as a significant reservoir species in the epidemiology of MERS-CoV, as noted by Nishiura et al. (9). These authors stated that two conditions should be objectively examined to confirm that an animal species constitutes a reservoir: (i) the reservoir is sufficient to maintain the disease by frequently transmitting the virus to another host, and (ii) the presence of the reservoir is essential for the continuous transmission of infection. The results presented by Lipkin and colleagues in mBio Abstract The number of patients receiving hematopoietic stem cell transplantation (HSCT) is rapidly rising worldwide. Despite substantial improvements in peri-transplant care, pulmonary complications resulting in respiratory failure remain a major contributor to morbidity and mortality in the post-transplant period, and represent a major barrier to the overall success of HSCT. Infectious complications include pneumonia due to bacteria, viruses, and fungi, and most commonly occur during neutropenia in the early post-transplant period. Non-infectious complications include idiopathic pneumonia syndrome, periengraftment respiratory distress syndrome, diffuse alveolar hemorrhage, pulmonary veno-occlusive disease, delayed pulmonary toxicity syndrome, cryptogenic organizing pneumonia, bronchiolitis obliterans syndrome, and post-transplant lymphoproliferative disorder. These complications have distinct clinical features and risk factors, occur at differing times following transplant, and contribute to morbidity and mortality. Abstract Dendritic cells (DCs) are critical to initiate the immune response and maintain tolerance, depending on different status and subsets. The expression profiles of microRNAs (miRNAs) in various DC subsets and haematopoietic stem cells (HSCs), which generate DCs, remain to be fully identified. Here we examine miRNomes of mouse bone marrow HSCs, immature DCs, mature DCs and IL-10/NO-producing regulatory DCs by deep sequencing. We identify numerous stage-specific miRNAs and histone modification in HSCs and DCs at different differentiation stages. miR-30b, significantly upregulated via a TGF-beta/Smad3-mediated epigenetic pathway in regulatory DCs, can target Notch1 to promote IL-10 and NO production, suggesting that miR-30b is a negative regulator of immune response. We also identify miRNomes of in vivo counterparts of mature DCs and regulatory DCs and systematically compare them with DCs cultured in vitro. These results provide a resource for studying roles of miRNAs in stem cell biology, development and functional regulation of DC subsets. Abstract Infection with hepatitis C virus (HCV), a major viral cause of chronic liver disease, frequently progresses to steatosis and cirrhosis, which can lead to hepatocellular carcinoma. HCV infection strongly induces host responses, such as the activation of the unfolded protein response, autophagy and the innate immune response. Upon HCV infection, the host induces the interferon (IFN)-mediated frontline defense to limit virus replication. Conversely, HCV employs diverse strategies to escape host innate immune surveillance. Type I IFN elicits its antiviral actions by inducing a wide array of IFN-stimulated genes (ISGs). Nevertheless, the mechanisms by which these ISGs participate in IFN-mediated anti-HCV actions remain largely unknown. In this review, we first outline the signaling pathways known to be involved in the production of type I IFN and ISGs and the tactics that HCV uses to subvert innate immunity. Then, we summarize the effector mechanisms of scaffold ISGs known to modulate IFN function in HCV replication. We also highlight the potential functions of emerging ISGs, which were identified from genome-wide siRNA screens, in HCV replication. Finally, we discuss the functions of several cellular determinants critical for regulating host immunity in HCV replication. This review will provide a basis for understanding the complexity and functionality of the pleiotropic IFN system in HCV infection. Elucidation of the specificity and the mode of action of these emerging ISGs will also help to identify novel cellular targets against which effective HCV therapeutics can be developed. Abstract Adoptive transfer of regulatory T cells (FOXP3 + Tregs) has been developed as a potential curative immune therapy to prevent and treat autoimmune and graft-versus-host diseases (GVHD). A major limitation that has hindered the use of Treg immunotherapy in humans is the difficulty of consistently isolating and obtaining highly purified Tregs after ex vivo expansion. Methods: We isolated bona fide Tregs from expansion cultures based on their selective surface expression of latency-associated peptide (LAP). The TCR Vβ diversity and intracellular cytokine production of Tregs were determined by flow cytometer. The TSDR methylation was determined by epigenetic human FOXP3 qPCR Assay. Their in vitro and in vivo potency was confirmed with suppression assay and humanized xenogeneic GVHD (xGVHD) murine model, respectively. Results: LAP + repurification results in >90% LAP + FOXP3 + Tregs, leaving behind FOXP3 − and FOXP3 + nonTregs within the LAP − population. After 4-week expansion, the LAP + Tregs were >1 billion cells, highly suppressive and anergic in vitro, >90% demethylated in the TSDR and able to maintain TCR Vβ diversity. In the xGVHD model, exogenous CD25 − PBMC administered alone results in a median survival of 32 days. The co-transfer of LAP + Tregs increased median survival to 47 days, while the LAP parent (CD25 + ) and LAP − nonTregs had median survival of 39 and 31 days, respectively. Conclusions: These preclinical data together provide evidence that LAP + Tregs are highly purified with fully suppressive function for cell therapy. This population results in a more effective and safer product for immunotherapy to treat GVHD and provides the necessary preclinical data for transition into a clinical trial with LAP + Tregs to prevent or treat GVHD and other autoimmune diseases. Abstract b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 575-583 h t t p : / / w w w . b j m i c r o b i o l . c o m . b r / Associate Editor: Mauricio Nogueira Keywords: Mamastrovirus 5 Canine astrovirus Dog Gastroenteritis MAstV5 a b s t r a c t Mamastrovirus 5 (MAstV5), belonging to the Astroviridae (AstV) family, previously known as canine astrovirus or astrovirus-like particles, has been reported in several countries to be associated with viral enteric disease in dogs since the 1980s. Astroviruses have been detected in fecal samples from a wide variety of mammals and birds that are associated with gastroenteritis and extra enteric manifestations. In the present study, RT-PCR was used to investigate the presence of MAstV5 in 269 dog fecal samples. MAstV5 was detected in 26% (71/269) of the samples. Interestingly, all MAstV5-positive samples derived from dogs displaying clinical signs suggestive of gastroenteritis, other enteric viruses were simultaneously detected (canine parvovirus, canine distemper virus, canine coronavirus, canine adenovirus and canine rotavirus). Based on genomic sequence analysis of MAstV5 a novel classification of the species into four genotypes, MAstV5a-MAstV5d, is proposed.Phylogenetic analyses based on the ORF2 amino acid sequences, samples described herein grouped into the putative genotype 'a' closed related with Chinese samples. Other studies are required to attempt the clinical and antigenic implications of these astrovirus genotypes in dogs. Abstract Newly emerging human viruses such as Ebola virus, severe acute respiratory syndrome (SARS) virus, and HIV likely originate within an extant population of viruses in nonhuman hosts and acquire the ability to infect and cause disease in humans. Although several mechanisms preventing viral infection of particular hosts have been described, the mechanisms and constraints on viral host expansion are ill defined. We describe here mycobacteriophage Patience, a newly isolated phage recovered using Mycobacterium smegmatis mc 2 155 as a host. Patience has genomic features distinct from its M. smegmatis host, including a much lower GC content (50.3% versus 67.4%) and an abundance of codons that are rarely used in M. smegmatis. Nonetheless, it propagates well in M. smegmatis, and we demonstrate the use of mass spectrometry to show expression of over 75% of the predicted proteins, to identify new genes, to refine the genome annotation, and to estimate protein abundance. We propose that Patience evolved primarily among lower-GC hosts and that the disparities between its genomic profile and that of M. smegmatis presented only a minimal barrier to host expansion. Rapid adaptions to its new host include recent acquisition of higher-GC genes, expression of out-of-frame proteins within predicted genes, and codon selection among highly expressed genes toward the translational apparatus of its new host. IMPORTANCE The mycobacteriophage Patience genome has a notably lower GC content (50.3%) than its Mycobacterium smegmatis host (67.4%) and has markedly different codon usage biases. The viral genome has an abundance of codons that are rare in the host and are decoded by wobble tRNA pairing, although the phage grows well and expression of most of the genes is detected by mass spectrometry. Patience thus has the genomic profile of a virus that evolved primarily in one type of host genetic landscape (moderate-GC bacteria) but has found its way into a distinctly different high-GC environment. Although Patience genes are ill matched to the host expression apparatus, this is of little functional consequence and has not evidently imposed a barrier to migration across the microbial landscape. Interestingly, comparison of expression levels and codon usage profiles reveals evidence of codon selection as the genome evolves and adapts to its new environment. Abstract In this article, we propose a 3-dimensional graphical representation of protein sequences based on 10 physicochemical properties of 20 amino acids and the BLOSUM62 matrix. It contains evolutionary information and provides intuitive visualization. To further analyze the similarity of proteins, we extract a specific vector from the graphical representation curve. The vector is used to calculate the similarity distance between 2 protein sequences. To prove the effectiveness of our approach, we apply it to 3 real data sets. The results are consistent with the known evolution fact and show that our method is effective in phylogenetic analysis. Abstract The Middle East respiratory syndrome (MERS) is a new human disease caused by a novel coronavirus (CoV). The disease is reported mainly in adults. Data in children are scarce. The disease caused by MERS-CoV in children presents with a wide range of clinical manifestations, and it is associated with a lower mortality rate compared with adults. Poor outcome is observed mainly in admitted patients with medical comorbidities. We report a new case of MERS-CoV infection in a 9-month-old child complicated by severe respiratory symptoms, multi-organ dysfunction, and death. We reviewed the literature in an attempt to characterize the mode of presentation, the risk factors, and outcome of MERS-CoV infection in the pediatric population. Abstract The study of cell-population heterogeneity in a range of biological systems, from viruses to bacterial isolates to tumor samples, has been transformed by recent advances in sequencing throughput. While the high-coverage afforded can be used, in principle, to identify very rare variants in a population, existing ad hoc approaches frequently fail to distinguish true variants from sequencing errors. We report a method (LoFreq) that models sequencing run-specific error rates to accurately call variants occurring in <0.05% of a population. Using simulated and real datasets (viral, bacterial and human), we show that LoFreq has near-perfect specificity, with significantly improved sensitivity compared with existing methods and can efficiently analyze deep Illumina sequencing datasets without resorting to approximations or heuristics. We also present experimental validation for LoFreq on two different platforms (Fluidigm and Sequenom) and its application to call rare somatic variants from exome sequencing datasets for gastric cancer. Source code and executables for LoFreq are freely available at http://sourceforge.net/projects/lofreq/. Abstract Objectives: The outbreak of Middle Eastern respiratory syndrome coronavirus (MERS-CoV) was one of the major events in South Korea in 2015. In particular, this study pays attention to formulating a mathematical model for MERS transmission dynamics and estimating transmission rates. Methods: Incidence data of MERS-CoV from the government authority was analyzed for the first aim and a mathematical model was built and analyzed for the second aim of the study. A mathematical model for MERS-CoV transmission dynamics is used to estimate the transmission rates in two periods due to the implementation of intensive interventions. Results: Using the estimates of the transmission rates, the basic reproduction number was estimated in two periods. Due to the superspreader, the basic reproduction number was very large in the first period; however, the basic reproduction number of the second period has reduced significantly after intensive interventions. Conclusion: It turned out to be the intensive isolation and quarantine interventions that were the most critical factors that prevented the spread of the MERS outbreak. The results are expected to be useful to devise more efficient intervention strategies in the future. Abstract Background. Seeking a noninvasive method to conduct surveillance for respiratory pathogens, we sought to examine the usefulness of 2 types of off-the-shelf aerosol samplers to detect respiratory viruses in Singapore.Methods. In this pilot study, we ran the aerosol samplers several times each week with patients present in the patient waiting areas at 3 primary health clinics during the months of April and May 2016. We used a SKC BioSampler with a BioLite Air Sampling Pump (run for 60 min at 8 L/min) and SKC AirChek TOUCH personal air samplers with polytetrafluoroethylene Teflon filter cassettes (run for 180 min at 5 L/min). The aerosol specimens and controls were studied with molecular assays for influenza A virus, influenza B virus, adenoviruses, and coronaviruses.Results. Overall, 16 (33.3%) of the 48 specimens indicated evidence of at least 1 respiratory pathogen, with 1 (2%) positive for influenza A virus, 3 (6%) positive for influenza B virus, and 12 (25%) positive for adenovirus.Conclusions. Although we were not able to correlate molecular detection with individual patient illness, patients with common acute respiratory illnesses were present during the samplings. Combined with molecular assays, it would suggest that aerosol sampling has potential as a noninvasive method for novel respiratory virus detection in clinical settings. Abstract Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.Bovine respiratory disease complex (BRDC) is one of the most prevalent cattle diseases, causing large economic losses in the cattle industry worldwide. For example, BRDC is estimated to account for 70-80% of all feedlot cattle morbidity and 40-50% of all cattle mortality, resulting in a loss of greater than US $500 million per year in the United States [16, 22] . In Japan, number of illness in the statistics mutual aid for livestock owners in 2014 reported by the Ministry of Agriculture, Forestry and Fisheries indicate that respiratory diseases accounted for 23% of illness in cattle (http://www.maff.go.jp/j/tokei/kouhyou/katiku_kyosai/) (in Japanese). Despite considerable research and attempts to manage hygiene to prevent BRDC, these measures have had a limited effect on reducing the impacts of this disease [29] .One of the difficulties in investigation and prevention of BRDC is its multi-factorial etiology, based on host interactions with multiple pathogens, such as virus and bacteria, including mycoplasma. The primary viral infection sometimes results in clinical symptoms, and it was exacerbated by secondary infections with bacterial pathogens [10] . Stressors, such as transportation, and host factors, such as nutritional status, raise the risk and severity of BRDC [17, 28] . Viruses, which are commonly associated with Abstract Purpose: Activation of Toll-like receptor 2 (TLR2) present on circulating monocytes in patients with Kawasaki disease (KD) can lead to the production of proinflammatory cytokines and interleukin-10 (IL-10). We aimed to determine the association of the frequency of circulating TLR2+/ CD14+ monocytes (FTLR2%) with the outcomes of KD, as well as to compare FTLR2% to the usefulness of sIL-10. Methods: The FTLR2% in patients with KD was measured by flow cytometry. Serum levels of IL-10 (sIL-10) were determined in 31 patients with KD before the initial treatment with intravenous immunoglobulin (IVIG) and in 21 febrile controls by using enzyme-linked immunosorbent assay. Patients were classified as having coronary artery lesions (CALs) based on the maximal internal diameters of the proximal right coronary artery and proximal left anterior descending coronary artery one month after the initial diagnosis. Results: We found that FTLR2% greater than 92.62% predicted CALs with 80% sensitivity and 68.4% specificity, whereas FTLR2% more than 94.61% predicted IVIG resistance with 66.7% sensitivity and 71.4% specificity. Moreover, sIL-10 more than 15.52 pg/mL predicted CALs and IVIG resistance with 40% and 66.7% sensitivity, respectively, and 73.7% and 76.2% specificity, respectively. Conclusion: We showed that measuring FTLR2% before the initial treatment could be useful in predicting CAL development with better sensitivity than sIL-10 and with results comparable to sIL-10 results for the prediction of IVIG resistance in patients with KD. However, further studies are necessary to validate FTLR2% as a marker of prognosis and severity of KD. Abstract Human rhinoviruses are the primary etiological agent of the common cold. This infection can be mild and self-limiting in immunocompetent hosts, but can be associated with bronchiolitis in infants, pneumonia in the immunosuppressed and exacerbations of pre-existing pulmonary conditions such as asthma or chronic obstructive pulmonary disease. Many of these conditions can place significant economic costs upon healthcare infrastructure. There is currently no licensed vaccine for rhinovirus, as the large variety of rhinovirus serotypes has posed significant challenges for research. In this review, we discuss current knowledge around antiviral drugs and small molecule inhibitors of rhinovirus infection, as well as antiviral host defense peptides as exciting prospects to approach the development of novel therapeutics which target human rhinovirus. Abstract Small-animal models have been developed for several Filoviridae species; however, serial adaptation was required to produce lethal infection. These adapted viruses have sequence changes in several genes, including those that modulate the host immune response. Nonhuman primate models do not require adaptation of filoviruses. Here, we describe lethal models of disease for Bundibugyo, Sudan, and Zaire species of Ebolavirus in the domestic ferret, using wild-type nonadapted viruses. Pathologic features were consistent with disease in primates. Of particular importance, this is the only known small-animal model developed for Bundibugyo and the only uniformly lethal animal model for Bundibugyo. Abstract Highlights d ENDU-2 nuclease regulates nucleotide metabolism and germ cell proliferation in worms d ENDU-2 expression is induced by nucleotide imbalance and other genotoxic stresses d ENDU-2 inhibits CTP synthase phosphorylation by repressing PKA and HDA-1 in the gut d ENDU-2 function may be conserved in mammalian cells Abstract Background: Acute respiratory distress syndrome (ARDS) is one of the most lethal diseases encountered in the pediatric intensive care unit (PICU). The etiological pathogens and prognostic factors of severe ARDS of pulmonary origin in children with respiratory virus infections were prospectively investigated. Methods: Enrolled children fulfilled the following criteria: (1) PICU admission; (2) age of 1 month to 16 years; (3) diagnosis of infectious pneumonia and respiratory virus infection; and (4) development of severe ARDS within 72 h after PICU admission. Pathogens were detected in the blood and tracheal lavage fluid using molecular techniques and a conventional culture system. The serum levels of inflammatory mediators on the day of PICU admission were examined. Results: Fifty-seven patients (32 boys; median age, 9 months) were enrolled. Multiple virus infections, co-infection with bacteria/fungus, and bacteremia/fungemia were observed in 60%, 49%, and 32% of children, respectively. Adenovirus-B, measles virus, and cytomegalovirus were detected predominantly in tracheal lavage fluid. There were no statistically significant differences between non-survivors and survivors regarding the types of pathogen, incidence of multiple virus infection, gender, age, clinical features, and treatment. The serum levels of interferon (IFN)-γ and the IFN-γ/interleukin (IL)-10 ratio were higher in non-survivors. Conclusions: IFN-γ upregulation as detected on the day of PICU admission was found to be one of the possible prognostic factors affecting a fatal outcome. These results suggest that modulation of inflammatory responses is critical for the clinical management of children with ARDS. Abstract Canine coronavirus (CCoV) is an important pathogen that causes enteritis in dogs, but there is no information on CCoV infection in Vietnam. To examine the prevalence of CCoV infection among Vietnamese dogs, 201 serum samples were analyzed by virus-neutralization (VN) test. The results showed that antibody against CCoV-II was present in 87 dogs (43.3%). To detect genes of CCoV, fecal samples collected from 30 diarrheic and 50 healthy dogs were examinated by RT-PCR, confirming that 2 diarrheic dogs and 5 healthy dogs were positive for CCoV. Nucleotide sequences of N-terminal region of spike (S) gene indicated that CCoV strains were divided into two subgenotypes, CCoV-IIa and -IIb, respectively. Furthemore, we succeeded in isolating CCoV/ dog/HCM47/2015, the isolate was plaque-purified three times, and 3'-terminal one-third of the genome was analyzed. Interestingly, the plaque-purified virus had a large deletion in ORF3abc and E genes (1,165 nt), and a short deletion in ORF7b gene (60 nt), suggesting that these regions are not necessary for in vitro replication of CCoV. Next, the antigenicity between the isolated CCoV-IIb and the other CCoV-IIa was compared by VN test, revealing that antigenicty of the isolated CCoV is equal or higher than that of the other CCoV. In summary, two subgenotypes of CCoV-II are spreading among Vietnamese dogs. The isolated virus with a large deletion after in vitro passage may be useful for the development of vaccine, owing to its antigenicity and efficient viral growth in vitro. Abstract This study investigated the role and mechanism of alprostadil in acute respiratory distress syndrome (ARDS) induced by oleic acid (OA) in rats.Sprague-Dawley rats were randomly divided into control, OA model, and OA + Alprostadil (2.5, 5, and 10 μg/kg, respectively) groups. The ARDS model was induced by femoral vein injection of OA, and alprostadil was administrated immediately. Lung injury was evaluated by lung wet-dry weight ratio (W/D) and histological analyses. Expressions of ACE, inflammatory mediators, apoptotic-related proteins, and proteins in the MAPKs and NF-kB signaling pathways were determined by Western blot or immunohistochemical staining.Compared with the control group, the OA model group had significantly increased W/D, lung injury score, and collagen deposition at 3 h after OA injection. However, alprostadil (10 μg/kg) treatment significantly reduced OA-induced elevation of these indicators. Additionally, OA-induced expression of TNF-a and IL-1b were suppressed by alprostadil. The OA-induced activation of nuclear factor (NF) kB p65 was also reduced by alprostadil. Furthermore, we found that Alprostadil had an inhibitory effect on the phosphorylation of JNK, ERK1/2, and p38 MAPKs. Alprostadil inhibited Bax but increased Bcl-2, indicating a suppressive role in apoptosis. Remarkably increased expression of ACE in the OA model group was observed, which was decreased by alprostadil.Alprostadil has a protective effect on ARDS induced by OA in rats, possibly through inhibiting apoptosis, suppressing the activation of MAPKs and NF-kB signaling pathways, and decreasing ACE protein expression. Therefore, the use of alprostadil in clinical ARDS treatment is promising. Abstract Viruses are the major contributors to the morbidity and mortality of upper and lower acute respiratory infections (ARIs) for all age groups. The aim of this study was to determine the frequencies for a large range of respiratory viruses using a sensitive molecular detection technique in specimens from outpatients of all ages with ARIs. Nasopharyngeal aspirates were obtained from 162 individuals between August 2007-August 2009. Twenty-three pathogenic respiratory agents, 18 respiratory viruses and five bacteria were investigated using multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay (IIF). Through IIF, 33 (20.4%) specimens with respiratory virus were recognised, with influenza virus representing over half of the positive samples. Through a multiplex real-time RT-PCR assay, 88 (54.3%) positive samples were detected; the most prevalent respiratory viral pathogens were influenza, human rhinovirus and respiratory syncytial virus (RSV). Six cases of viral co-detection were observed, mainly involving RSV. The use of multiplex real-time RT-PCR increased the viral detection by 33.9% and revealed a larger number of respiratory viruses implicated in ARI cases, including the most recently described respiratory viruses [human bocavirus, human metapneumovirus, influenza A (H1N1) pdm09 virus, human coronavirus (HCoV) NL63 and HCoV HKU1]. Abstract The past few months have yielded disconcerting news about viruses carried in mammalian reservoirs. What is the relevance of virus discoveries mushrooming in the literature? Will bats yield the next pandemic virus? Animal ecologists and virologists need to join forces. Abstract Cytotoxic T lymphocytes (CTL) kill ceils by perturbing the target's plasma membrane and by inducing the disintegration of the target cell's DNA into oligonudeosomal fragments, a process characteristic of apoptosis. We show that the DNA fragmentation event is distinct from the membrane lysis event and is dependent on the state of target cell activation or commitment into the mitotic cycle. Quiescent cells were refractory to DNA fragmentation, but not to membrane lysis. Log phase growth, transformation with c-myc, or infection of quiescent Go targets with herpes simplex virus-l, which induces a competent state for DNA synthesis, all enhanced target cell susceptibility to CTL-induced DNA fragmentation without altering the membrane lysis. These results suggest that Go cells are resistant to CTL-induced apoptosis, but that entry into G1 or a Gl-like state by growth factors, cellular transformation, or DNA virus infection renders them competent to enter the apoptotic pathway(s). W. K. Nishioka would like to thank Drs. J. Campisi, D. Green, E. Martz, and S. Silverstein for insightful discussions; A. Mahboubi for critical reading of the manuscript; M. Cole for the c-myc containing retrovirus; J. Stein for the BALB/c 3T3, done A31 cells; and D. G. Nishioka for support and motivation. Abstract The use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. Using sensitive sequence profile analysis methods, we characterize a novel superfamily (the SUKH superfamily) that unites a diverse group of proteins including Smi1/Knr4, PGs2, FBXO3, SKIP16, Syd, herpesviral US22, IRS1 and TRS1, and their bacterial homologs. Using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (CDI) systems of proteobacteria. By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. These include at least eight distinct types of DNases belonging to HNH/ EndoVII-and restriction endonuclease-fold, and RNases of the EndoU-like and colicin E3-like cytotoxic RNases-folds. The N-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the CDI systems) in proteobacteria, ESAT-6/WXG-like ATP-dependent secretory systems in Gram-positive bacteria and the conventional Sec-dependent system in several bacterial lineages. The hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. Unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the SUKH superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different C-terminal nuclease domains to N-terminal secretion-related domains. Across the bacterial superkingdom these systems might participate in discriminating 'self' or kin from 'non-self' or non-kin strains. Using structural analysis we demonstrate that the SUKH domain possesses a versatile scaffold that can be used to bind a wide range of protein partners. In eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. Similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (SuFu) superfamily has been recruited for comparable roles in eukaryotes. In animal DNA viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the SUKH domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins. Abstract BACKGROUND Abstract Background: Herpes B virus (BV) is a zoonotic disease caused by double-stranded enveloped DNA virus with cercopithecidae as its natural host. The mortality rate of infected people could be up to 70% with fatal encephalitis and encephalomyelitis. Up to now, there are no effective treatments for BV infection. Among the various proteins encoded by monkey B virus, gD, a conserved structural protein, harbors important application value for serological diagnosis of frequent variations of the monkey B virus.Objectives: This study aimed to expressed the gD protein of BV in Escherichia coli by a recombinant vector, and prepare specific monoclonal antibodies against gD of BV to pave the way for effective and quick diagnosis reagent research.The gD gene of BV was optimized by OptimWiz to improve codon usage bias and synthesis, and the recombinant plasmid, pET32a/gD, was constructed and expressed in E. coli Rosetta (DE3). The expressed fusion protein, His-gD, was purified and the BALB/c mice were immunized by this protein. Spleen cells from the immunized mice and SP2/0 myeloma cells were fused together, and the monoclonal cell strains were obtained by indirect enzyme-linked immunosorbent assay (ELISA) screening, followed by preparation of monoclonal antibody ascetic fluid.The optimized gD protein was highly expressed in E. coli and successfully purified. Five monoclonal antibodies (mAbs) against BV were obtained and named as 4E3, 3F8, 3E7, 1H3 and 4B6, and with ascetic fluid titers of 2 × 10 6 , 2 × 10 5 , 2 × 10 5 , 2 × 10 3 and 2 × 10 2 , respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass.The cell lines obtained in this work secreted potent, stable and specific anti-BV mAbs, which were suitable for the development of herpes B virus diagnosis reagents. Abstract ABBREVIATIONS: ARDS = acute respiratory distress syndrome; BLA = biologics license application; EMR = electronic medical record; IT = information technology; PR = pathogen reduction; SDP(s) = single-donor platelet(s); TA-GVHD = transfusionassociated graft-versus-host disease. Abstract RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant nonstructural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRpassociated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies. Abstract Ebola virus (EBOV) enters host cells by macropinocytosis, a poorly understood process. Recent studies have suggested that cell factors involved in autophagy, an evolutionally conserved pathway leading to the lysosomal degradation of protein aggregates and organelles during cellular stress, also have roles in macropinocytosis. Here, we demonstrate that autophagy-associated proteins are required for trafficking of EBOV into the cell body. Depleting cells of beclin 1, autophagy-related protein 7, or microtubule-associated protein 1A/B light chain 3B (LC3B) abolished EBOV uptake, owing to a block in vesicle formation at the cell surface. Both LC3B-I and LC3B-II interacted with macropinocytic structures. Our work indicates that, although various forms of LC3B possess an inherent ability to associate with forming macropinosomes, LC3B-II is critical for internalization of macropinocytic vesicles and, therefore, EBOV from the cell surface. Abstract The Taiwan Centers for Disease Control (Taiwan CDC) has established a 3-tier personal protective equipment (PPE) stockpiling framework that could maintain a minimum stockpile for the surge demand of PPE in the early stage of a pandemic.However, PPE stockpiling efforts must contend with increasing storage fees and expiration problems. In 2011, the Taiwan CDC initiated a stockpile replacement model in order to optimize the PPE stockpiling efficiency, ensure a minimum stockpile, use the government's limited funds more effectively, and achieve the goal of sustainable management. This stockpile replacement model employs a first-in-first-out principle in which the oldest stock in the central government stockpile is regularly replaced and replenished with the same amount of new and qualified products, ensuring the availability and maintenance of the minimum stockpiles. In addition, a joint electronic procurement platform has been established for merchandising the replaced PPE to local health authorities and medical and other institutions for their routine or epidemic use. In this article, we describe the PPE stockpile model in Taiwan, including the 3-tier stockpiling framework, the operational model, the components of the replacement system, implementation outcomes, epidemic supports, and the challenges and prospects of this model. Abstract Backgrounds: An ongoing outbreak of a novel coronavirus (2019-nCoV) pneumonia hit a major city in China, Wuhan, December 2019 and subsequently reached other provinces/regions of China and other countries. We present estimates of the basic reproduction number, R 0 , of 2019-nCoV in the early phase of the outbreak. Methods: Accounting for the impact of the variations in disease reporting rate, we modelled the epidemic curve of 2019-nCoV cases time series, in mainland China from January 10 to January 24, 2020, through the exponential growth. With the estimated intrinsic growth rate (g), we estimated R 0 by using the serial intervals (SI) of two other well-known coronavirus diseases, MERS and SARS, as approximations for the true unknown SI. Findings: The early outbreak data largely follows the exponential growth. We estimated that the mean R 0 ranges from 2.24 (95%CI: 1.96-2.55) to 3.58 (95%CI: 2.89-4.39) associated with 8-fold to 2-fold increase in the reporting rate. We demonstrated that changes in reporting rate substantially affect estimates of R 0 . Conclusion: The mean estimate of R 0 for the 2019-nCoV ranges from 2.24 to 3.58, and is significantly larger than 1. Our findings indicate the potential of 2019-nCoV to cause outbreaks. Abstract The Middle East respiratory syndrome coronavirus (MERS-CoV) presents an ideal example for developing One Health concepts. Dromedary camels are the principal reservoir for the virus. Infected camels shed the virus in body secretions, particularly nasal discharges. MERS-CoV has the potential to remain active in the environment for some time under optimum conditions of temperature and humidity. This shedding sustains the virus in endemic communities and thus contact with camels is considered a major risk factor for human infection. Reducing virus shedding from camels will have a great positive impact on reducing the human risk of infection. Our main objective is to highlight the potential aspects of reducing virus shedding from camels to the environment, thereby reducing the possibility of human infection. We will focus on the potential roles of camel markets, camel shows, importation, transportation and grazing in the amplification and shedding of the virus, providing some novel concepts for the control approaches for the MERS-CoV. Abstract Purpose: To investigate the association between necrotizing enterocolitis (NEC) and red blood cell transfusions in very low birth weight (VLBW) preterm infants. Methods: We studied were 180 VLBW preterm infants who were admitted to the neonatal intensive care unit of CHA Gangnam Hospital from January of 2006 to December of 2009. The subjects were divided into 2 groups: an NEC group (greater than stage II on the modified Bell's criteria) and a control group (less than stage II on the modified Bell's critieria). We defined red blood cell transfusion before NEC diagnosis as the frequency of transfusion until NEC diagnosis (mean day at NEC diagnosis, day 18) in the NEC group and the frequency of transfusion until 18 days after birth in the control group. Results: Of the 180 subjects, 18 (10%) belonged to the NEC group, and 14 (78%) of these 18 patients had a history of transfusion before NEC diagnosis. The NEC group received 3.1±2.9 transfusions, and the control group received 1.0±1.1 transfusions before the NEC diagnosis (P=0.005). In a multi variate logistic regression corrected for gestational age, Apgar score at 1 minute, the presence of respiratory distress syndrome, patent ductus arteriosus, premature rupture of membrane, disseminated intravascular coagulopathy and death were confounding factors. The risk of NEC increased 1.63 times (95% confidence interval, 1.145 to 2.305; P=0.007) with transfusion before the NEC diagnosis. Conclusion: The risk for NEC increased significantly with increased transfusion frequency before the NEC diagnosis. Abstract Telephone: +61 7 405 974 555 Facsimile: +61 7 3636 5578 Word count Abstract 249 words Main text 2394 words Key words respiratory viruses gastrointestinal infection children real-time polymerase chain reaction cohort studies Page 2 of 33 For peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open ARTICLE SUMMARY Article focus Infectious diseases are a common cause of morbidity in early childhood, even in developed economies.A diagnostic gap exists for common respiratory and gastrointestinal syndromes, with the likelihood that as yet undiscovered pathogens are involved.Existing knowledge about these common illnesses relies on research conducted before the rapid developments in molecular diagnostics of recent decades or focusses on disease at the severe end of the spectrum -hospitalisations -affecting a limited number of children and discounting the burden of more common, but less severe, community-managed illness.This protocol outlines a dynamic birth cohort study that will allow for a detailed description of the epidemiology of respiratory and gastrointestinal viruses during the first two years of life.The large biobank of specimens to be collated will act as a rich source of material to answer targeted research questions, including the role of virus acquisition and shedding on clinical illness and the discovery of new infectious agents.As study procedures, including specimen collection and return, are conducted by parents, findings will be free from Hawthorne effects from frequent interactions with study staff.Systematic weekly sampling will provide a control set of specimens for the individual and the cohort, allowing quantification of virus-specific attributable risk to illness.Non-random recruitment and enrolment requires awareness and assessment of potential bias and confounding prior to broad-based generalisation.Similar studies in the past have oversampled from higher socioeconomic households, and we attempt to avoid this by using a recruitment strategy that targets pregnant women in both public and private hospital settings. . van der Zalm MM, Uiterwaal CS, de Jong BM, et al. Viral specimen collection by parents increases response rate in population-based virus studies. J Allergy Clin Immunol 2006;117:955-6; author reply 56-7. Abstract Respiratory virus infections in children are a leading cause of morbidity and mortality worldwide. Methods: A total of 897 clinical specimens were collected from February 2007 to January 2008 and transported to the National Influenza Center. Two hundred and two samples belonged to children under the age of six from 897 specimens, described above, were selected. Then they were tested for influenza virus types and subtypes by real time PCR assay subsequently, the specimens were tested for RSV and hMPV by hemi-nested multiplex PCR and parainfluenza viruses type 1-4 by hemi-nested multiplex PCR and adenovirus by hemi-nested PCR. Results: The throat swab was taken from the Kawasaki case with the history of chicken`s contact. The specimen was tested for all influenza subtypes especially H5N1 and the results were negative. Meanwhile PCR was done for screening of other respiratory viruses that results came out positive for RSV and hMPV. Conclusion: In the present study, we demonstrated the possibility to detect dual infection caused by RSV and hMPV, but because of the extravagant pattern of this case, more investigation is suggested specially on Kawasaki patients. Abstract A novel human coronavirus (HCoV-EMC) was recently identified in the Middle East as the causative agent of a severe acute respiratory syndrome (SARS) resembling the illness caused by SARS coronavirus (SARS-CoV). Although derived from the CoV family, the two viruses are genetically distinct and do not use the same receptor. Here, we investigated whether HCoV-EMC and SARS-CoV induce similar or distinct host responses after infection of a human lung epithelial cell line. HCoV-EMC was able to replicate as efficiently as SARS-CoV in Calu-3 cells and similarly induced minimal transcriptomic changes before 12 h postinfection.Later in infection, HCoV-EMC induced a massive dysregulation of the host transcriptome, to a much greater extent than SARS-CoV. Both viruses induced a similar activation of pattern recognition receptors and the interleukin 17 (IL-17) pathway, but HCoV-EMC specifically down-regulated the expression of several genes within the antigen presentation pathway, including both type I and II major histocompatibility complex (MHC) genes. This could have an important impact on the ability of the host to mount an adaptive host response. A unique set of 207 genes was dysregulated early and permanently throughout infection with HCoV-EMC, and was used in a computational screen to predict potential antiviral compounds, including kinase inhibitors and glucocorticoids. Overall, HCoV-EMC and SARS-CoV elicit distinct host gene expression responses, which might impact in vivo pathogenesis and could orient therapeutic strategies against that emergent virus.HCoV-EMC and SARS-CoV. We used this information to predict potential effective drugs against HCoV-EMC, a method that could be more generally used to identify candidate therapeutics in future disease outbreaks. These data will help to generate hypotheses and make rapid advancements in characterizing this new virus. RS, Katze MG. 2013 . Cell host response to infection with novel human coronavirus EMC predicts potential antivirals and important differences with SARS coronavirus. mBio 4(3):e00165-13. Abstract See the Editorial Commentaries by Musher on pages 824-5 and Jain and Pavia on pages 826-8.)Background. The frequent lack of a microbiological diagnosis in community-acquired pneumonia (CAP) impairs pathogendirected antimicrobial therapy. This study assessed the use of comprehensive multibacterial, multiviral molecular testing, including quantification, in adults hospitalized with CAP.Methods. Clinical and laboratory data were collected for 323 adults with radiologically-confirmed CAP admitted to 2 UK tertiary care hospitals. Sputum (96%) or endotracheal aspirate (4%) specimens were cultured as per routine practice and also tested with fast multiplex real-time polymerase-chain reaction (PCR) assays for 26 respiratory bacteria and viruses. Bacterial loads were also calculated for 8 bacterial pathogens. Appropriate pathogen-directed therapy was retrospectively assessed using national guidelines adapted for local antimicrobial susceptibility patterns.Results. Comprehensive molecular testing of single lower respiratory tract (LRT) specimens achieved pathogen detection in 87% of CAP patients compared with 39% with culture-based methods. Haemophilus influenzae and Streptococcus pneumoniae were the main agents detected, along with a wide variety of typical and atypical pathogens. Viruses were present in 30% of cases; 82% of these were codetections with bacteria. Most (85%) patients had received antimicrobials in the 72 hours before admission. Of these, 78% had a bacterial pathogen detected by PCR but only 32% were culture-positive (P < .0001). Molecular testing had the potential to enable de-escalation in number and/or spectrum of antimicrobials in 77% of patients.Conclusions. Comprehensive molecular testing significantly improves pathogen detection in CAP, particularly in antimicrobialexposed patients, and requires only a single LRT specimen. It also has the potential to enable early de-escalation from broadspectrum empirical antimicrobials to pathogen-directed therapy. Abstract The purpose of this study was to obtain a natural antibiotic from Phenol-rich compounds; for the dressing and the treatment of chronic wounds.The Phenol-rich compound sweet gel was prepared by blending four natural herbal extracts, Acacia catechu (L.F.), Momia (Shilajit), Castanea sativa, and Ephedra sinica stapf, with combination of a sweet gel medium, including honey, maple saps, Phoenix dactylifera L. (date), pomegranate extract and Azadirachta indica gum as a stabilizer. The combinations were screened by using a well-diffusion assay with cloxacillin as a control. Pseudomonas spp. was tested with our novel antimicrobial compound. The zones of inhibition in agar culture were measured for each individual component and for the compound, and the results were compared with those of the control group which had been treated with cloxacillin. Data were ex-pressed as means ± standard deviations. Quantitative analyses were performed using the paired t-test.The antibiotic effect of the Phenol-rich compound sweet gel was statistically shown to be more significant than that of cloxacillin against Pseudomonas aeruginosa (P < 0.05).Our novel approach to fighting the antibiotic resistance of Pseudomonas proved to be successful. The Phenol-rich compound sweet gel was found to be suitable for use as an alternative medicine and bioactive dressing material, for the treatment of patients with various types of wounds, including burns, venous leg ulcers, ulcers of various etiologies, leg ulcers on the feet of diabetic, unhealed graft sampling sites, abscesses, boils, surgical wounds, necrotic process, post-operative and neonatal wound infection, and should be considered as an alternative to the usual methods of cure. Abstract The leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus Leishmania. LEISHDNAVAX is a multi-antigen, T-cell epitope-enriched DNA vaccine candidate against human leishmaniasis. The vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. Here, we describe the safety testing of LEISHDNAVAX in naive mice and rats, complemented by the demonstration of tolerability in Leishmania-infected mice. Biodistribution and persistence were examined following single and repeated intradermal (i.d.) administration to rats. DNA vectors were distributed systemically but did not accumulate upon repeated injections. Although vector DNA was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. Evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i.d. administration to naive, non-infected mice did not reveal any safety concerns. LEISHDNAVAX was also well tolerated in Leishmania-infected mice. Taken together, our results substantiate a favorable safety profile of LEISHDNAVAX in both naive and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine. Abstract In December 2019, a viral pneumonia outbreak caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV), began in Wuhan, China. We report the epidemiological and clinical features of the first patient with 2019-nCoV pneumonia imported into Korea from Wuhan. This report suggests that in the early phase of 2019-nCoV pneumonia, chest radiography would miss patients with pneumonia and highlights taking travel history is of paramount importance for early detection and isolation of 2019-nCoV cases. Abstract Hemophagocytic lymphohistiocytosis (HLH) is an aggressive inflammatory syndrome that results from inappropriate activation of the immune system. HLH has a high mortality if not treated. We describe a case of a fulminant HLH, associated with a reactivation of an EBV infection. The patient responded well to steroid treatment. Abstract The amyloid precursor protein plus presenilin-1 (APP/PS1) mice are a frequently-used model for Alzheimer's disease studies (AD). However, the data relevant to which proteins are involved in inflammatory mechanism are not sufficiently well-studied using the AD mouse model. Using behavioral studies, quantitative RT-PCR and Western-blot techniques, significant findings were determined by the expression of proteins involved in inflammation comparing APP/PS1 and Wild type mice. Increased GFAP expression could be associated with the elevation in number of reactive astrocytes. IL-3 is involved in inflammation and ABDF1 intervenes normally in the transport across cell membranes and both were found up-regulated in APP/PS1 mice compared to Wild type mice. Furthermore, CCR5 expression was decreased and both CCL3 and CCL4 chemokines were highly expressed indicating a possible gliosis and probably an increase in chemotaxis from lymphocytes and T cell generation. We also noted for the first time, a CCR8 increase expression with diminution of its CCL1 chemokine, both normally involved in protection from bacterial infection and demyelination. Control of inflammatory proteins will be the next step in understanding the progression of AD and also in determining the mechanisms that can develop in this disease. Abstract As discussed in the main text, the ProtoArrays' signal analysis in both Stage I and Stage II of the investigation involved multi-step processing. In both steps, aggregate raw signals ( Supplementary Fig. S1 ) were normalized within each array, using an implementation of the ProCAT algorithm 1 . Briefly, the algorithm was utilized with parameters implementing a sliding window of length 15, to take into account local background subtraction as well as local intensity normalization across each array. The normalized signal for each position i,j on the protein array is calculated using the sliding window signals and backgrounds as,M ij = Mean({window foreground signals}) , (foreground refers to either 635 nm wavelength for GST tag signal, or 532 nm wavelength for IgG reactivity signal), S ij ∈ {window signals} , MAbsDev ij = Mean({| S ij − M ij |}) , is the mean absolute deviation of the foreground window signals from the mean of the local sliding window, and b S,ij ∈ {local smoothed background} is the smoothed background of the local window. The smoothed background averages the local immediate background surrounding the spot of interest. Negative low signals (due to signal being smaller than background) are replaced by the minimum detectable signal value. The subsequent quantile normalization of the ProCAT normed arrays puts all distributions on the same scale based on rank, with ties replaced by the average of the values 2 . After Mann Abstract Routine molecular diagnostic testing by our laboratory, based on using a primer pair with conservative binding sites on the spike glycoprotein coding sequence, has indicated the recurring of a unique phylogenetic cluster of chicken infectious bronchitis viruses (IBV) in the Middle East since 2010. The nearly full-length S1 subunit of the spike gene phylogeny of selected strains, however, split up this grouping, suggesting potential recombination in the S1 gene. In order to clarify this, various bioinformatic analyses of the strains were carried out, which confirmed this supposition. Two patterns of recombination were found among the strains, one of which could also be identified in GenBank-deposited IBV sequences from the region. These findings demonstrate that IBV strains of different recombinant patterns occur simultaneously in the same geographic region and could circulate for an extended period of time, thus contributing to the knowledge on IBV evolution. Abstract We collected rectal swabs from dogs in Japan during 2011 to 2014, and canine coronavirus (CCoV) nucleocapsid gene was detected by RT-PCR. The relationship between CCoV infection and the manifestation of diarrhea symptoms was investigated, and a correlation was noted (df=1, χ 2 =8.90, P<0.005). The types of CCoV detected in samples from CCoV-infected dogs were CCoV-I in 88.9% and CCoV-II in 7.4%, respectively. We retrospectively investigated the seroprevalence of CCoV-I in dogs in Japan during 1998 to 2006. The sera were tested with a neutralizing antibody test. In the absence of CCoV-I laboratory strain, we used feline coronavirus (FCoV)-I that shares high sequence homology in the S protein with CCoV-I. 77.7% of the sera were positive for neutralizing anti-FCoV-I antibodies. Abstract Hendra virus was first described in 1994 in Australia, causally associated with a cluster of fatal equine and human cases at a thoroughbred racing stable in the Brisbane suburb of Hendra. This year marks the twentieth anniversary of the identification of pteropid bats (flying-foxes) as the natural host of the virus, and it is timely to reflect on a pivotal meeting of an eclectic group of scientists in that process. They included animal and public health experts, environmental scientists, veterinary and horse industry representatives, and wildlife experts. The task was to review and prioritise wildlife surveillance seeking the origin of the previously unknown virus. The group determined that the likely reservoir must occur in disparate locations, and be capable of moving between locations, or exist in continuous, overlapping populations spanning multiple locations. Flying-foxes were considered to be a more probable source of the novel virus than birds. Within weeks, antibodies were detected in several species of flying-fox, and the virus was subsequently isolated. While the identification of the natural host of Hendra virus within 18 months of its description was remarkable in itself, a broader legacy followed. In the subsequent years, a suite of zoonotic viruses including Australian bat lyssavirus, Nipah virus, SARS coronavirus, and Ebola and Marburg viruses have been detected in bats. Bats are now the "go to" taxa for novel viruses. History has repeatedly demonstrated that knowledge begets knowledge. This simple notion of bringing a diverse group of people together in an environment of mutual respect reinforced this principle and proves that the sum is often so much more powerful than the parts. Abstract One hypothesis for the etiology of central nervous system (CNS) autoimmune disease is that infection by a virus sharing antigenic epitopes with CNS antigens (molecular mimicry) elicits a virus-specific immune response that also recognizes self-epitopes. To address this hypothesis, transgenic mice were generated that express the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus (LCMV) as self in oligodendrocytes. Intraperitoneal infection with LCMV strain Armstrong led to infection of tissues in the periphery but not the CNS, and the virus was cleared within 7-14 d. After clearance, a chronic inflammation of the CNS resulted, accompanied by upregulation of CNS expression of MHC class I and II molecules. A second LCMV infection led to enhanced CNS pathology, characterized by loss of myelin and clinical motor dysfunction. Disease enhancement also occurred after a second infection with unrelated viruses that cross-activated LCMV-specific memory T cells. These findings indicate that chronic CNS autoimmune disease may be induced by infection with a virus sharing epitopes with a protein expressed in oligodendrocytes and this disease may be enhanced by a second infection with the same or an unrelated virus. These results may explain the association of several different viruses with some human autoimmune diseases. Abstract such as complement dependent cytotoxicity CDC) and antibody dependent cell-mediated cytotoxicity (ADCC), making human IgG1 a natural and desirable choice for therapeutic antibody development in oncology. Considerable effort has focused on improving the stability and Fc function of this IgG1 subclass through protein and glycan engineering. 2-8 Twenty-two of 27 FDA approved therapeutic antibodies belong to the human IgG1 Abstract Bats are unique mammals that are reservoirs of high levels of virus diversity. Although several of these viruses are zoonotic, the majority are not. Astroviruses, transmitted fecal-orally, are commonly detected in a wide diversity of bat species, are prevalent at high rates and are not thought to directly infect humans. These features make astroviruses useful in examining virus evolutionary history, epidemiology in the host, and temporal shedding trends. Our study screened for the presence of astroviruses in bats in Singapore, reconstructed the phylogenetic relations of the polymerase genes and tested for population characteristics associated with infection. Of the seven species screened, astroviruses were detected in Rhinolophus lepidus and Eonycteris spelaea. The R. lepidus sequences grouped with other Rhinolophus astrovirus sequences from China and Laos, while the Eoncyteris sequences formed a distinct clade with astroviruses from Rousettus spp. in Laos and Pteropus giganteus in Bangladesh, but not with other E. spelaea sequences. Longitudinal collections of Eonycteris feces demonstrated variable shedding. Juvenile status of bats was a risk factor for astroviruses. This study highlights the diversity of astroviruses in nectivorous and insectivorous bats in Singapore and provides a predictive framework for understanding astrovirus infection in these bats. It also suggests that in addition to host phylogenetic relatedness, host ecology, such as roosting behavior, may drive co-infections, virus maintenance and spillover. Abstract This study aims to analyze the different binational/multinational activities, programs, and structures taking place on the borders of Brazil and the U.S. between 2013 and 2015. A descriptive exploratory study of two border epidemiological surveillance (BES) systems has been performed. Two approaches were used to collect data: i) technical visits to the facilities involved with border surveillance and application of a questionnaire survey; ii) application of an online questionnaire survey. It was identified that, for both surveillance systems, more than 55% of the technicians had realized that the BES and its activities have high priority. Eighty percent of North American and 71% of Brazilian border jurisdictions reported an exchange of information between countries. Less than half of the jurisdictions reported that the necessary tools to carry out information exchange were available. Operational attributes of completeness, feedback, reciprocity, and quality of information were identified as weak or of low quality in both systems. Statements, guidelines, and protocols to develop surveillance activities are available at the U.S.-Mexico border area. The continuous systematic development of surveillance systems at these borders will create more effective actions and responses. Abstract We evaluated the mechanisms of thrombocytopenia and procoagulant changes in relation with clinical variables in a cohort of patients with acute hantavirus disease.Blood samples of 33 prospectively recruited, consecutive, hospitalized patients with acute Puumala virus-induced hemorrhagic fever with renal syndrome (HFRS) were collected acutely and at the recovery visit (control). Serum thrombopoietin (TPO) and activity of plasma microparticles (MPs) from various cell sources were measured with enzyme-linked immunosorbent assay-based methods. The results were related to data on platelet indices and functions, coagulation variables, and clinical disease.Serum TPO was nearly 4-fold higher acutely compared with the control (median 207 pg/mL, range 56-1258 pg/mL vs. median 58 pg/mL, range 11-241 pg/mL, P < 0.001) and coincided with high mean platelet volume (MPV) and immature platelet fraction (IPF%). Prothrombin fragments and D-dimer were high acutely compared with the control (F1 + 2 median 704 pmol/L, range 284-1875 pmol/ L vs. median 249 pmol/L, range 118-556 pmol/L, P < 0.001; D-dimer median 2.8 mg/L, range 0.6-34.0 mg/L vs. median 0.4 mg/L, range 0.2-1.1 mg/L, P < 0.001), and associated with low platelet count and severe acute kidney injury (AKI). MPs' procoagulant activity was high acutely only among patients with mild AKI (plasma creatinine below the median at the time of the measurement).Upregulated TPO together with high MPV and IPF% confirm active thrombopoiesis, but do not predict severity of HFRS. Simultaneously, elevated prothrombin fragments and D-dimer suggest increased consumption of platelets in patients with severe AKI. Activity of platelet-derived MPs in HFRS should be studied with flow cytometry in a larger cohort of patients.Abbreviations: AKI = acute kidney injury, ALT = alanine aminotransferase, CRP = C-reactive protein, HCPS = hantavirus cardiopulmonary syndrome, HFRS = hemorrhagic fever with renal syndrome, HIV = human immunodeficiency virus, IL-6 = interleukin 6, IPF% = immature platelet fraction %, MP = microparticle, MPV = mean platelet volume, PFA-100 = platelet factor analyzer 100, PUUV = Puumala virus, STAT = signal transducers and activators of transcription, TF = tissue factor, TGFb = transforming growth factor b, TPO = thrombopoietin. Abstract Neutrophils, immune cells crucial for protecting against invading pathogens, are important in sepsis. Neutrophil migration is regulated by chemokine receptors and their cognate ligands. Our previous study investigated the effect of n-butanol extract from Folium isatidis on lipopolysaccharide (LPS)-induced septic shock. The present study stimulated neutrophils with LPS to explore the influence of LPS on cell. Neutrophils were then pretreated with n-butanol extract from Folium isatidis followed by LPS to examine the effect of this extract on neutrophil chemotaxis. The results showed that LPS decreased the expression levels of CXC-chemokine receptor (CXCR)1, CXCR2 and L-selectin (CD62L), and increased the expression of interleukin-8 (IL-8) by neutrophils. The addition of n-butanol extract from Folium isatidis inhibited this LPS-induced downregulation of CXCR1, CXCR2 and CD62L, and decreased the expression of IL-8 on neutrophils. In addition, n-butanol extract promoted myeloperoxidase activity in neutrophils. Taken together, LPS downregulated the expression of chemokine receptors, leading to the failure of neutrophils to migrate to sites of infection. The addition of n-butanol extract, which promoted the ability of neutrophils to migrate, is a natural product and potential therapeutic agent with which to target neutrophil chemotaxis during LPS stimulation. Abstract Diversity and plasticity are two hallmarks of macrophages. M1 macrophages (classically activated macrophages) are pro-inflammatory and have a central role in host defense against infection, while M2 macrophages (alternatively activated macrophages) are associated with responses to anti-inflammatory reactions and tissue remodeling, and they represent two terminals of the full spectrum of macrophage activation. Transformation of different phenotypes of macrophages regulates the initiation, development, and cessation of inflammatory diseases. Here we reviewed the characters and functions of macrophage polarization in infection, atherosclerosis, obesity, tumor, asthma, and sepsis, and proposed that targeting macrophage polarization and skewing their phenotype to adapt to the microenvironment might hold great promise for the treatment of inflammatory diseases. Abstract Objectives and design: Developing an effective HIV-1 vaccine that elicits broadly neutralizing HIV-1 human antibodies (bnAbs) remains a challenging goal. Extensive studies on HIV-1 have revealed various strategies employed by the virus to escape host immune surveillance. Here, we investigated the human antibody gene repertoires of uninfected and HIV-1-infected individuals at genomic DNA (gDNA) and cDNA levels by deep sequencing followed by high-throughput sequence analysis to determine the frequencies of putative germline antibody genes of known HIV-1 monoclonal bnAbs (bnmAbs).Methods: Combinatorial gDNA and cDNA antibody libraries were constructed using the gDNAs and mRNAs isolated from uninfected and HIV-1-infected human peripheral blood mononuclear cells (PBMCs). All libraries were deep sequenced and sequences analysed using IMGT/HighV-QUEST software (http://imgt.org/HighV-QUEST/index. action). The frequencies of putative germline antibodies of known bnmAbs in the gDNA and cDNA libraries were determined.The human gDNA antibody libraries were more diverse in heavy and light chain V-gene lineage usage than the cDNA libraries, indicating that the human gDNA antibody gene repertoires may have more potential than the cDNA repertoires to develop HIV-1 bnAbs. The frequencies of the heavy and kappa and lambda light chain variable regions with identical V(D)J recombinations to known HIV-1 bnmAbs were extremely low in human antibody gene repertoires. However, we found relatively high frequencies of the heavy and kappa and lambda light chain variable regions that used the same V-genes and had the same CDR3 lengths as known HIV-1 bnmAbs regardless of (D)J-gene usage. B-cells bearing B-cell receptors of such heavy and kappa and lambda light chain variable regions may be stimulated to induce HIV-1 bnAbs. Abstract Dipeptidyl peptidase IV (DPP-4), an incretin glucagon-like peptide-1 (GLP-1) degrading enzyme, contains two forms and it can exert various physiological functions particular in controlling blood glucose through the action of GLP-1. In diabetic use, the DPP-4 inhibitor can block the DDP-4 to attenuate GLP-1 degradation and prolong GLP-1 its action and sensitize insulin activity for the purpose of lowering blood glucose. Nonetheless the adverse effects of DPP-4 inhibitors severely hinder their clinical applications, and notably there is a clinical demand for novel DPP-4 inhibitors from various sources including chemical synthesis, herbs, and plants with fewer side effects. In this review, we highlight various strategies, namely computational biology (in silico), in vitro enzymatic and cell assays, and in vivo animal tests, for seeking natural DPP-4 inhibitors from botanic sources including herbs and plants. The pros and cons of all approaches for new inhibitor candidates or hits will be under discussion. Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.Developing metrics for emergency care research in low-and middle-income countries De´velopper des outils de mesure pour la recherche en matie`re de soins d'urgence dans les pays a`faible et moyen revenus Introduction: There is little research on emergency care delivery in low-and middle-income countries (LMICs). To facilitate future research, we aimed to assess the set of key metrics currently used by researchers in these settings and to propose a set of standard metrics to facilitate future research. Methods: Systematic literature review of 43,109 published reports on general emergency care from 139 LMICs. Studies describing care for subsets of emergency conditions, subsets of populations, and data aggregated across multiple facilities were excluded. All facility-and patient-level statistics reported in these studies were recorded and the most commonly used metrics were identified. Results: We identified 195 studies on emergency care delivery in LMICs. There was little uniformity in either patient-or facility-level metrics reported. Patient demographics were inconsistently reported: only 33% noted average age and 63% the gender breakdown. The upper age boundary used for paediatric data varied widely, from 5 to 20 years of age. Emergency centre capacity was reported using a variety of metrics including annual patient volume (n = 175, 90%); bed count (n = 60, 31%), number of rooms (n = 48, 25%); frequently none of these metrics were reported (n = 16, 8%). Many characteristics essential to describe capabilities and performance of emergency care were not reported, including use and type of triage; level of provider training; admission rate; time to evaluation; and length of EC stay. Conclusion: We found considerable heterogeneity in reporting practices for studies of emergency care in LMICs. Standardised metrics could facilitate future analysis and interpretation of such studies, and expand the ability to generalise and compare findings across emergency care settings. Abstract Deciphering the way gene expression regulatory aspects are encoded in viral genomes is a challenging mission with ramifications related to all biomedical disciplines. Here, we aimed to understand how the evolution shapes the bacteriophage lambda genes by performing a high resolution analysis of ribosomal profiling data and gene expression related synonymous/silent information encoded in bacteriophage coding regions.We demonstrated evidence of selection for distinct compositions of synonymous codons in early and late viral genes related to the adaptation of translation efficiency to different bacteriophage developmental stages. Specifically, we showed that evolution of viral coding regions is driven, among others, by selection for codons with higher decoding rates; during the initial/progressive stages of infection the decoding rates in early/late genes were found to be superior to those in late/early genes, respectively. Moreover, we argued that selection for translation efficiency could be partially explained by adaptation to Escherichia coli tRNA pool and the fact that it can change during the bacteriophage life cycle.An analysis of additional aspects related to the expression of viral genes, such as mRNA folding and more complex/longer regulatory signals in the coding regions, is also reported. The reported conclusions are likely to be relevant also to additional viruses. Abstract Methods: This is a randomized, double-blind, placebo-controlled trial of adults aged 18-65 years with influenza-like illness and positive influenza rapid antigen test. Treatments were 1 g paracetamol four times a day, or matching placebo, for 5 days. Pernasal swabs were taken for influenza quantitative RT-PCR at Baseline and Days 1, 2 and 5. Temperature and symptom scores were recorded for 5-14 days or time of resolution respectively. The primary outcome variable was area under the curve (AUC) for quantitative PCR log10 viral load from Baseline to Day 5.Results: A total of 80 participants were randomized: no one was lost to follow up, and one withdrew after 4 days.There were 22 and 24 participants who were influenza PCR-positive in placebo and in paracetamol groups respectively. Mean (SD) AUC PCR log10 viral load was 4.40 (0.91) in placebo and 4.64 (0.88) in paracetamol; difference was −0.24, 95% CI: −0.78 to 0.29, P = 0.36. In all participants there were no differences in symptom scores, temperature, time to resolution of illness and health status, with no interaction between randomized treatment and whether influenza was detected by PCR. Conclusion: Regular paracetamol had no effect on viral shedding, temperature or clinical symptoms in patients with PCR-confirmed influenza. There remains an insufficient evidence base for paracetamol use in influenza infection. Clinical trial registration: ACTRN12611000497909 at the Australian New Zealand Clinical Trials Registry.Despite recommendations to administer paracetamol for symptom relief in influenza and influenzalike-illnesses, this study has found that regular administration of paracetamol has no effect on viral or clinical outcomes in this setting. bs_bs_banner Editor's Choice Abstract A male ferret, which was purchased from abroad at 9 months of age, had shown significant weight loss starting at 13 months of age. The ferret subsequently showed decreasing motor activity and recumbency and was euthanized at 14 months of age. At necropsy, a white, quail egg-sized mass was found in the mesentery. Histopathologically, multifocal granulomas consisting of necrotic foci, macrophages, fibroblasts and plentiful fibrous connective tissues were observed in the mesenteric mass. Surrounding the granulomas, inflammatory cell infiltration consisting of neutrophils, lymphocytes and plasmacytes was observed diffusely and significantly. Immunohistochemistry revealed small numbers of macrophages around necrotic foci that were positively stained for antimouse feline coronavirus. Electron microscopically, the cytoplasm of the macrophages contained viral particles, which were identified as coronavirus. The histopathological features in this ferret were similar to those in cats with feline infectious peritonitis (FIP). This was the first case in ferrets in Japan. (J Toxicol Pathol 2010; 23: 99-101) Abstract A total of 1,320 tracheal samples from 66 broiler flocks sent to slaughterhouses and 42 tracheal samples from 42 flocks of local chickens in the field were collected for infectious bronchitis virus (IBV) gene detection by reverse transcription polymerase chain reaction using nucleocapsid-specific primers and spike-specific primers. Prevalence in broiler flocks was 39.4% (26/66) and in local chicken flocks was 11.9% (5/42). Several IBVs similar to Japan were detected in Taiwan. One-direction neutralization revealed that the reference antisera did not offer protection against the IBVs similar to those from Japan. Abstract Background: Canine adenovirus 2, parainfluenza, and Bordetella bronchiseptica cause respiratory disease in dogs, and each has a modified live intranasal vaccine available. Molecular diagnostic assays to amplify specific nucleic acids are available for each of these agents. If positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays for disease would be decreased.Objective: To determine the impact of administration of commercially available modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza vaccine has on the results of a commercially available PCR panel.Animals: Eight puppies from a research breeding facility negative for these pathogens.Methods: Blinded prospective pilot study. Puppies were vaccinated with a single dose of modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza and parenteral dose of adenovirus 2, canine distemper virus, and parvovirus. Nasal and pharyngeal swabs were collected on multiple days and submitted for PCR assay.Results: Nucleic acids of all 3 organisms contained in the topical vaccine were detected from both samples multiple times through 28 days after vaccination with higher numbers of positive samples detected between days 3 and 10 after vaccination.Conclusions and Clinical Importance: Vaccine status should be considered when interpreting respiratory agent PCR results if modified live vaccines have been used. Development of quantitative PCR and wild-type sequencing are necessary to improve positive predictive value of these assays by distinguishing vaccinate from natural infection. Abstract Context: Morphine is an alkaloid isolated from the poppy plants. The addiction of morphine is a very serious social issue. Some long non-coding RNAs (lncRNAs) have been proposed to engage in drug addiction. Objective: Whether lncRNA maternally expressed gene 3 (MEG3) attended to morphine-mediated autophagy of mouse hippocampal neuronal HT22 cells was probed. Materials and methods: HT22 cells were subjected to 10 mM morphine for 24 h. Cell autophagy was assessed by measuring LC3-II/LC3-I and Beclin-1 expression. qRT-PCR was carried out to measure MEG3 expression. SiRNA oligoribonucleotides targeting MEG3 (si-MEG3) was transfected to silence MEG3. The orexin1 receptor (OX1R), c-fos, p/t-ERK and p/t-PKC expressions were tested by western blotting. SCH772984 was used as an inhibitor of ERK pathway. Results: Morphine elevated OX1R (2.92 times), c-fos (2.06 times), p/t-ERK (2.04 times) and p/t-PKC (2.4 times), Beclin-1 (3.2 times) and LC3-II/LC3-I (3.96 times) expression in HT22 cells. Moreover, followed by morphine exposure, the MEG3 expression was also elevated in HT22 cells (3.03 times). The silence of MEG3 lowered the Beclin-1 (1.85 times), LC3-II/LC3-I (2.12 times), c-fos (1.39 times) and p/t-ERK (1.44 times) expressions in morphine-treated HT22 cells. Inhibitor of ERK pathway SCH772984 further promoted the influence of MEG3 silence on morphine-caused Beclin-1 (1.97 times) and LC3-II/LC3-I (1.92 times) expressions decreases. Conclusions: Up-regulation of MEG3 attended to the morphine-caused autophagy of HT22 cells might be through elevating c-fos expression and promoting ERK pathway activation. More experiments are also needed in the future to analyse the influence of other lncRNAs in drug addiction.ARTICLE HISTORY Abstract Nucleoside analogs represent the largest class of small molecule-based antivirals, which currently form the backbone of chemotherapy of chronic infections caused by HIV, hepatitis B or C viruses, and herpes viruses. High antiviral potency and favorable pharmacokinetics parameters make some nucleoside analogs suitable also for the treatment of acute infections caused by other medically important RNA and DNA viruses. This review summarizes available information on antiviral research of nucleoside analogs against arthropod-borne members of the genus Flavivirus within the family Flaviviridae, being primarily focused on description of nucleoside inhibitors of flaviviral RNA-dependent RNA polymerase, methyltransferase, and helicase/NTPase. Inhibitors of intracellular nucleoside synthesis and newly discovered nucleoside derivatives with high antiflavivirus potency, whose modes of action are currently not completely understood, have drawn attention. Moreover, this review highlights important challenges and complications in nucleoside analog development and suggests possible strategies to overcome these limitations. Abstract Original article Highlights The serial interval of novel coronavirus infections was estimated from a total of 28 infector-infectee pairs. J o u r n a l P r e -p r o o f -2 - The median serial interval is shorter than the median incubation period, suggesting a substantial proportion of pre-symptomatic transmission. A short serial interval makes it difficult to trace contacts due to the rapid turnover of case generations.Objective: To estimate the serial interval of novel coronavirus from information on 28 infector-infectee pairs.We collected dates of illness onset for primary cases (infectors) and secondary cases (infectees) from published research articles and case investigation reports. We subjectively ranked the credibility of the data and performed analyses on both the full dataset (n=28) and a subset of pairs with highest certainty in reporting (n=18). In addition, we adjust for right truncation of the data as the epidemic is still in its growth phase.Results: Accounting for right truncation and analyzing all pairs, we estimated the median serial interval at 4.0 days (95% credible interval [CrI]: 3.1, 4.9).Limiting our data to only the most certain pairs, the median serial interval was estimated at 4.6 days (95% CrI: 3.5, 5.9).The serial interval of COVID-19 is close to or shorter than its median incubation period. This suggests that a substantial proportion of secondary transmission may occur prior to illness onset. The COVID-19 serial interval is also shorter than the serial interval of severe acute respiratory syndrome (SARS), indicating that calculations made using the SARS serial interval may introduce bias.J o u r n a l P r e -p r o o f -3 -If the transmission takes place during the symptomatic period of the primary case, the serial interval is longer than the incubation period. However, this relationship can be reversed when pre-symptomatic transmission takes place.Furthermore, it is possible that the secondary case may even experience illness onset prior to onset in their infector.J o u r n a l P r e -p r o o f Abstract This study investigated the co-localization of the Middle East respiratory syndrome coronavirus (MERS-CoV) and its receptor dipeptidyl peptidase-4 (DPP4) by immunohistochemistry (IHC) across respiratory and lymphoid organs of experimentally MERS-CoV infected pigs and llamas. Also, scanning electron microscopy was performed to assess the ciliary integrity of respiratory epithelial cells in both species. In pigs, on day 2 post-inoculation (p.i.), DPP4-MERS-CoV co-localization was detected in medial turbinate epithelium. On day 4 p.i., the virus/receptor co-localized in frontal and medial turbinate epithelial cells in pigs, and epithelial cells distributed unevenly through the whole nasal cavity and in the cervical lymph node in llamas. Abstract Thank you for the transfer of your research manuscript to EMBO reports. I now went through the referee reports from The EMBO Journal. Both referees acknowledge the potential interest of the findings. Nevertheless, both referees have raised a number of concerns and suggestions to improve the manuscript, or to strengthen the data and the conclusions drawn. As the reports are below, I will not detail them here.As EMBO reports emphasizes novel functional over detailed mechanistic insight, we will not require to address points regarding more refined mechanistic details. However, all concerns regarding technical and experimental limitations, and data presentation need to be addressed. Moreover, as EMBO reports publishes robustly documented, novel major findings of physiological or clinical relevance and wide interest, we think that the biological relevance of the findings (as indicated by referee #1) should be strengthened. Further, as stated by referee #2, the novelty and advance of the findings should be increased by adding more data, addressing the questions raised by this referee.Given the constructive referee comments, we would like to invite you to revise your manuscript, with the understanding that all referee concerns must be fully addressed in the revised manuscript (as outlined above), and a complete point-by-point response. Acceptance of your manuscript will depend on a positive outcome of a second round of review, using the same referees as at The EMBO Journal (indicating, that the paper is now revised and submitted to EMBO reports). It is EMBO reports policy to allow a single round of revision only and acceptance or rejection of the manuscript will therefore depend on the completeness of your responses included in the next, final version of the manuscript. EMBO reports -Peer Review Process File © European Molecular Biology Organization 2Referee #1:In their work, Sousa et al. argue that the homeostatic functions of microglia are lost under inflammatory conditions mimicked in this work by LPS treatment in vivo and in vitro. The authors isolated homogeneous microglia populations and show by single cell RNA Sequencing segregation of microglia in different subgroups apparently triggered by inflammatory processes. This topic is therefore of interest to the microglia community as it highlights the microglial response to an acute inflammatory stimulus and compares it to chronic stimuli such as neurodegeneration.The authors argue that microglial subpopulations exist that show differential responses to acute inflammation. By selecting population-specific cell surface markers, they were able to effectively isolate only brain-resident microglia, based on Cd11b+/Cd45int expression, as opposed to other intrinsic or peripheral cells (Fig. 1) . The authors show that upon an acute LPS injection or upon in vitro LPS stimulation, microglia lose their homeostatic and phagocytosing gene expression profile, whilst pro-inflammatory genes are upregulated (Fig. 2) . This data was confirmed using a single-cell sequencing approach, showing that microglia exposed to LPS show a distinct reactive phenotype compared to saline-injected control mice (Fig. 3) . By analysing the sequencing data more closely, the group could identify a specific microglia sub-population ("subset LPS") that showed an intermediate phenotype compared to control and reactive microglia (Fig. 4) . They postulate based on differential gene analysis that these cells might either be damaged cells, recovering from their activation status or a cell population with specific DNA repair properties. Lastly, in Fig. 5 the authors compared the gene signatures of LPS-exposed microglia to that of "disease-associated microglia" (DAM) that has been described by Keren-Shaul et al. (2017) in response to neurodegeneration and describe high inflammatory reactivity upon LPS treatment and a phagocytic gene signature in DAM.The paper analyses reactive microglia on a single-cell level, which adds to the growing amount of research dissecting specific microglial responses and subtypes to varying stimuli using highresolution approaches. Yet, the relevance the particular knowledge of LPS induced microglia subgroups to advance treatment options for diseases of the CNS involving neuroinflammation remains questionable and is not addressed in this study. The work lacks clinical relevance, which should be increased by adding further experiments. Abstract In this study, we document a case of phenobarbital-induced anticonvulsant hypersensitivity syndrome (AHS), which has been rarely reported in veterinary medicine. A 2-yearold, 5.4 kg, neutered male Russian Blue cat was diagnosed with idiopathic epilepsy and started on phenobarbital treatment. Eight days after initiation of phenobarbital treatment, the cat showed tachypnea and hyperthermia. CBC and serum biochemistry were unremarkable. However, the patient showed high serum amyloid A (SAA). On abdominal ultrasonography, generalized enlargement of abdominal lymph nodes and splenic multiple hypo-echoic nodules, which were consistent with reactive lymphadenopathy were found. The cat was diagnosed with AHS, and phenobarbital was discontinued. After 10 days of cessation, the patient had normal SAA, and clinical signs were resolved. Abstract Ethical considerations are essential in planning for and responding to outbreaks of infectious diseases. During the outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in the Republic of Korea in 2015, serious challenges emerged regarding important ethical issues, such as transparency and the protection of privacy. The development of bioethics in Korea has been influenced by individualistic perspectives applied in clinical contexts, leading to a paucity of ethical perspectives relevant to population-level phenomena such as outbreaks. Alternative theories of public health ethics include the perspectives of relational autonomy and the patient as victim and vector. Public health actions need to incorporate clear and systematic procedures founded upon ethical principles. The MERS-CoV epidemic in Korea created significant public support for more aggressive early interventions in future outbreaks. This trend makes it all the more imperative for ethical principles and procedures to be implemented in future planning and responses to outbreaks in order to promote perceptions of legitimacy and civic participation. Abstract Objectives: The present study aimed to investigate the prevalence of and factors associated with H1N1 preventive behaviors in a community-based population. Methods: A cross-sectional study was conducted in three urban and two rural communities in Korea. Interviews were conducted with 3462 individuals (1608 men and 1854 women) aged ! 19 years during FebruaryeMarch 2010. Influenzarelated information including anxiety, preventive behaviors and their perceived effectiveness, vaccination status, past influenza-like illness symptoms, and sources of and trust in information was obtained. Results: Among 3462 participants, 173 reported experiencing influenza-like illness symptoms within the past 12 months. The mean H1N1 preventive behavior score was 25.5 AE 5.5 (out of a possible 40). The percent of participants reporting high perceived effectiveness and high anxiety was 46.2% and 21.4%, respectively. After controlling for potential confounders, H1N1 preventive behavior scores were predicted by a high (b Z 3.577, p < 0.001) or moderate (b Z 2.529, p < 0.001) perception of their effectiveness. Similarly, moderate (b Z 1.516, p < 0.001) and high (b Z 4.103, p < 0.001) anxiety scores predicted high preventive behavior scores. Conclusion: Effective methods of promoting population behavior change may be nationwide campaigns through mass media, as well as education and promotion by health care providers and broadcasters. Abstract Studies reporting chest images of respiratory syncytial virus (RSV)-induced lower respiratory tract infection (LRTI) in an outbreak setting and their relationship to the clinical manifestation are limited. During a genetically confirmed RSV outbreak, eight patients underwent both chest X-ray and computed tomography (CT). Among these, 5 cases had newly appearing abnormalities on CT, although chest X-ray was able to detect abnormalities in only 2 cases (40%). Although bronchial wall thickening was common, other findings and their distribution were variable, even in an outbreak setting. All patients with both a history of anticancer chemotherapy against hematological cancer and lower respiratory symptoms, such as wheezing, sputum, and hypoxemia, had abnormalities on CT, suggesting that these two factors might be important for predicting the existence of LRTI in RSV-infected patients.During the 2014 outbreak, RSV infection was diagnosed by reverse transcriptase polymerase chain reaction using respiratory specimens, and genetic proximity was confirmed by a phylogenetical analysis (15). Eight RSV-infected patients Abstract Respiratory viruses can cause a wide spectrum of pulmonary diseases, ranging from mild, upper respiratory tract infections to severe and life-threatening lower respiratory tract infections, including the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Viral clearance and subsequent recovery from infection require activation of an effective host immune response; however, many immune effector cells may also cause injury to host tissues. Severe acute respiratory syndrome (SARS) coronavirus and Middle East respiratory syndrome (MERS) coronavirus cause severe infection of the lower respiratory tract, with 10% and 35% overall mortality rates, respectively; however, >50% mortality rates are seen in the aged and immunosuppressed populations. While these viruses are susceptible to interferon treatment in vitro, they both encode numerous genes that allow for successful evasion of the host immune system until after high virus titres have been achieved. In this review, we discuss the importance of the innate immune response and the development of lung pathology following human coronavirus infection. Abstract 1 . Abbreviations used in this paper: BiP, immunoglobulin heavy chain binding protein; CgB, chromogranin B ; GAG chain, glycosaminoglycan chain ; hsPG, heparan sulfate proteoglycan; PDI, protein disulfide isomerase ; SgH, secretogranin II ; TGN, trans-Golgi network . Abstract Hepatitis C virus (HCV) is thought to be involved in the pathogenesis of autoimmune hepatitis (AIH) type 2, which is defined by the presence of type I antiliver kidney microsome autoantibodies directed mainly against cytochrome P450 (CYP)2D6 and by autoreactive liver infiltrating T cells. Virus-specific CD8 ϩ cytotoxic T lymphocytes (CTLs) that recognize infected cells and contribute to viral clearance and tissue injury during HCV infection could be involved in the induction of AIH. To explore whether the antiviral cellular immunity may turn against self-antigens, we characterized the primary CTL response against an HLA-A * 0201-restricted HCV-derived epitope, i.e., HCV core 178-187, which shows sequence homology with human CYP2A6 and CYP2A7 8-17. To determine the relevance of these homologies for the pathogenesis of HCV-associated AIH, we used synthetic peptides to induce primary CTL responses in peripheral blood mononuclear cells of healthy blood donors and patients with chronic HCV infection. We found that the naive CTL repertoire of both groups contains cross-reactive CTLs inducible by the HCV peptide recognizing both CYP2A6 and CYP2A7 peptides as well as endogenously processed CYP2A6 protein. Importantly, we failed to induce CTLs with the CYPderived peptides that showed a lower capacity to form stable complexes with the HLA-A2 molecule. These findings demonstrate the potential of HCV to induce autoreactive CD8 ϩ CTLs by molecular mimicry, possibly contributing to virus-associated autoimmunity. Abstract 16S rRNA gene is one of the preferred targets for resolving species phylogenesis issues in microbiological-related contexts. However, the identification of single-nucleotide variations capable of distinguishing a sequence among a set of homologous ones can be problematic. Here we present ORMA (Oligonucleotide Retrieving for Molecular Applications), a set of scripts for discriminating positions search and for performing the selection of high-quality oligonucleotide probes to be used in molecular applications. Two assays based on Ligase Detection Reaction (LDR) are presented. First, a new set of probe pairs on cyanobacteria 16S rRNA sequences of 18 different species was compared to that of a previous study. Then, a set of LDR probe pairs for the discrimination of 13 pathogens contaminating bovine milk was evaluated. The software determined more than 100 candidate probe pairs per dataset, from more than 300 16S rRNA sequences, in less than 5 min. Results demonstrated how ORMA improved the performance of the LDR assay on cyanobacteria, correctly identifying 12 out of 14 samples, and allowed the perfect discrimination among the 13 milk pathogenic-related species. ORMA represents a significant improvement from other contexts where enzyme-based techniques have been employed on already known mutations of a single base or on entire subsequences. Abstract Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus. Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors. In this study we established a 293T cell line that constitutively synthesizes a virus-based negative strand RNA, which expresses Gaussia luciferase upon influenza A virus infection. Using this cell line, an assay was developed and optimized to search for inhibitors of influenza virus replication. Biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format (Z 0 factor value40.8). A pilot screening provides further evidence for validation of the assay. Taken together, this work provides a simple, convenient, and reliable HTS assay to identify compounds with anti-influenza activity. Abstract Case summary Herein we describe an unusual metastatic pattern and paraneoplastic manifestation of a bronchial carcinoma in a cat. An 8 year-old cat presented with a diminished appetite, dysphagia, weight loss, lethargy and coughing. Thoracic radiographs revealed a lung mass. Bronchial carcinoma was diagnosed on the basis of histology and was associated with a lymphoplasmocytic infiltration of the fibrovascular stroma. Biochemistry showed hyperproteinaemia. Serum protein electrophoresis showed a narrow spike in the gamma region. Bone marrow cytology revealed an infiltrate with numerous clustered epithelial cells. The cat was euthanased 2 months later because of anorexia and poor general condition. Relevance and novel information To the best of our knowledge, this is the first clinical description of primary bronchial carcinoma associated with bone marrow metastases and paraneoplastic monoclonal gammopathy in a cat. Abstract Effects of ubiquitin-proteasome system (UPS) inhibitor MG-132 on the expression levels of tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 in mice with viral myocarditis were investigated to analyze the correlation of myocardial tissue score of mice between TNF-α and TGF-β1. Eighty healthy male SPF mice aged 6 weeks were selected and 20 mice were randomly selected as the blank group. The blank group did not receive any intervention. Mortality rates of each group were recorded and compared on day 8 of modeling, and heart specimens from the remaining mice were histopathologically examined and the expression of mRNA and protein of TNF-α and TGF-β1 in myocardial tissues were detected by western blot analysis. Correlation between mouse myocardial histopathologic scores and expression of protein of TNF-α and TGF-β1 in myocardial tissues, as well as the expression of TNF-α and TGF-β1 in myocardial tissue in VMC mice was analyzed. The expression levels of myocardial histopathological scores, mRNA and protein of TNF-α and TGF-β1 in the blank and control group were significantly lower than those in the VMC and the MG-132 group. The myocardial histopathological scores, mRNA and TNF-α and TGF-β1 protein in the MG-132 group were significantly lower than those in the VMC group (P<0.05). The expression of TNF-α and TGF-β1 protein in myocardial tissues was positively correlated with the pathological score in myocardial tissue of mice (r=0.843, P<0.05; r=0.763, P<0.05), and there was a positive correlation between the expression of TNF-α and TGF-β1 protein in myocardial tissues of VMC mice (r= 0.672, P<0.05). UPS inhibitor MG-132, which can significantly alleviate the myocardial injury of VMC mice, reduced the expression of inflammatory factors in myocardial tissues, and improved the survival rate of mice, thus it is a potential new treatment for VMC. Abstract Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs)/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. Abstract Objectives: To synthesize new transitional metal complexes derived from 3-aryl-azo-4-hydroxy coumarin analogues and to evaluate their antimicrobial activities.Methods: The syntheses of complexes of coumarin analogues were accomplished by mixing a hydro-alcoholic solution of 3-aryl-azo-4-hydroxy coumarin analogues with transition metal chlorides. The structural environment of the synthesized molecules was characterized using different instrumental methods. The antimicrobial activity of the compounds was determined by the agar well diffusion method.Results: The cobalt complexes of (E)-3-((4-chlorophenyl) diazenyl)-4-hydroxy-2H-chromen-2-one (HL 1 ): (4a) and (E)-3-((4-methoxyphenyl) diazenyl)-4-hydroxy-2H-chromen-2-one (HL 2 ): (4e) showed excellent antimicrobial activities compared with their ligands.The reports of the antimicrobial investigation showed that the cobalt complexes of 3-aryl-azo-4hydroxy coumarin analogues exhibited potential antimicrobial activity that was stronger than that of their ligands. Abstract Rubella virus (RV) has been reported to Abstract Dhh1 and Pat1 in yeast are mRNA decapping activators/translational repressors thought to play key roles in the transition of mRNAs from translation to degradation. However, little is known about the physical and functional relationships between these proteins and the translation machinery. We describe a previously unknown type of diauxic shiftdependent modulation of the intracellular locations of Dhh1 and Pat1. Like the formation of P bodies, this phenomenon changes the spatial relationship between components involved in translation and mRNA degradation. We report significant spatial separation of Dhh1 and Pat1 from ribosomes in exponentially growing cells. Moreover, biochemical analyses reveal that these proteins are excluded from polysomal complexes in exponentially growing cells, indicating that they may not be associated with active states of the translation machinery. In contrast, under diauxic growth shift conditions, Dhh1 and Pat1 are found to co-localize with polysomal complexes. This work suggests that Dhh1 and Pat1 functions are modulated by a relocalization mechanism that involves eIF4A. Pulldown experiments reveal that the intracellular binding partners of Dhh1 and Pat1 change as cells undergo the diauxic growth shift. This reveals a new dimension to the relationship between translation activity and interactions between mRNA, the translation machinery and decapping activator proteins. Abstract The present study aimed to explore the mechanisms underlying sepsis-induced acute lung injury (ALI) and identify more effective therapeutic strategies to treat it. The gene expression data set GSE10474 was downloaded and assessed to identify differentially expressed genes (DEGs). Principal component analysis, functional enrichment analysis and differential co-expression analysis of DEGs were performed. Furthermore, potential target drugs for key DEGs were assessed. A total of 209 DEGs, including 107 upregulated and 102 downregulated genes were screened. A number of DEGs, including zinc finger and BTB domain containing 17 (ZBTB17), heat shock protein 90 kDa β, member 1 (HSP90B1) and major histocompatibility complex, class II, DR α were identified. Furthermore, gene ontology terms including antigen processing and presentation, glycerophospholipid metabolism, transcriptional misregulation in cancer, thyroid hormone synthesis and pathways associated with diseases, such as asthma were identified. In addition, a differential co-expression network containing ubiquitin-conjugating enzyme E2 D4, putative and tubulin, γ complex associated protein 3 was constructed. Furthermore, a number of gene-drug interactions, including between HSP90B1 and adenosine-5'-diphosphate and radicicol, were identified. Therefore, DEGs, including ZBTB17 and HSP90B1, may be important in the pathogenesis of sepsis-induced ALI. Furthermore, drugs including adenosine-5'-diphosphate may be novel drug candidates to treat patients with ALI. Abstract Zoonoses represent a public health risk recently pointed out by the spreading of previously unknown human infectious diseases emerging from animal reservoirs such as severe acute respiratory syndrome and avian influenza caused by H5N1-virus. These outbreaks have shown that animal breeding activities can pose a significant public health risk. Until now, the risk of zoonoses has probably been underestimated, particularly in occupational settings. The emergence or re-emergence of bacterial (Mycobacterium bovis and Brucella spp) or viral (hepatitis E virus) infections shows that zoonoses should be considered as emerging risks in agricultural and animal breeding and should be addressed by specific preventive interventions. Close cooperation and interaction between veterinarians, occupational health physicians and public health operators is necessary, for a worldwide strategy to ex pand interdisciplinary collaborations and communications in all aspects of health care for humans, animals and the environment. This is what the One Health Approach was intended to be. Abstract Macrophages, found in circulating blood as well as integrated into several tissues and organs throughout the body, represent an important first line of defense against disease and a necessary component of healthy tissue homeostasis. Additionally, macrophages that arise from the differentiation of monocytes recruited from the blood to inflamed tissues play a central role in regulating local inflammation. Studies of macrophage activation in the last decade or so have revealed that these cells adopt a staggering range of phenotypes that are finely tuned responses to a variety of different stimuli, and that the resulting subsets of activated macrophages play critical roles in both progression and resolution of disease. This review summarizes the current understanding of the contributions of differentially polarized macrophages to various infectious and inflammatory diseases and the ongoing effort to develop novel therapies that target this key aspect of macrophage biology. Abstract In March 2013, three fatal human cases of infection with influenza A virus (H7N9) were reported in China. Since then, human cases have been accumulating. Given the public health importance of this virus, we performed a pathogenicity study of the H7N9 virus in the cynomolgus macaque model, focusing on clinical aspects of disease, radiographic, histological, and gene expression profile changes in the upper and lower respiratory tracts, and changes in systemic cytokine and chemokine profiles during infection. Cynomolgus macaques developed transient, mild to severe disease with radiographic evidence of pulmonary infiltration. Virus replicated in the upper as well as lower respiratory tract, with sustained replication in the upper respiratory tract until the end of the experiment at 6 days after inoculation. Virus shedding occurred mainly via the throat. Histopathological changes in the lungs were similar to those observed in humans, albeit less severe, with diffuse alveolar damage, infiltration of polymorphonuclear cells, formation of hyaline membranes, pneumocyte hyperplasia, and fibroproliferative changes. Analysis of gene expression profiles in lung lesions identified pathways involved in tissue damage during H7N9 infection as well as leads for development of therapeutics targeting host responses rather than virus replication. Overall, H7N9 infection was not as severe in cynomolgus macaques as in humans, supporting the possible role of underlying medical complications in disease severity as discussed for human H7N9 infection (H. N. Gao et al., N. Engl.H7N9 virus-induced disease in humans is often very severe and even lethal, the majority of reported H7N9 cases occurred in older people and people with underlying medical conditions. To better understand the pathogenicity of this virus, healthy cynomolgus macaques were inoculated with influenza A virus H7N9. Cynomolgus macaques were used as a model because the receptor distribution for H7N9 virus in macaques was recently shown to be more similar to that in humans than that of other frequently used animal models. From comparison with previous studies, we conclude that the emerging H7N9 influenza virus was more pathogenic in cynomolgus macaques than seasonal influenza A viruses and most isolates of the pandemic H1N1 virus but less pathogenic than the 1918 Spanish influenza virus or highly pathogenic avian influenza (HPAI) H5N1 virus. Abstract A major focus of systems biology is to characterize interactions between cellular components, in order to develop an accurate picture of the intricate networks within biological systems. Over the past decade, protein microarrays have greatly contributed to advances in proteomics and are becoming an important platform for systems biology. Protein microarrays are highly flexible, ranging from large-scale proteome microarrays to smaller customizable microarrays, making the technology amenable for detection of a broad spectrum of biochemical properties of proteins. In this article, we will focus on the numerous studies that have utilized protein microarrays to reconstruct biological networks including protein-DNA interactions, posttranslational protein modifications (PTMs), lectin-glycan recognition, pathogen-host interactions and hierarchical signaling cascades. The diversity in applications allows for integration of interaction data from numerous molecular classes and cellular states, providing insight into the structure of complex biological systems. We will also discuss emerging applications and future directions of protein microarray technology in the global frontier. Abstract To assess the effects of hepatitis B virus (HBV) on the expression of host α-1,2-mannosidases and determine the underlying mechanisms.We measured the expression levels of MAN1A1, MAN1A2, MAN1B1, and MAN1C1 in cell lines HepG2.2.15, HepN10, HepAD38 and HepG2 by Western blot. Viral antigens (HBsAg and HBeAg) in the culture medium were measured using the chemiluminescence method. HBV DNA quantification assays were performed using a commercial real-time PCR kit. Protein levels of human liver tissue α-1,2-mannosidases were also evaluated by Western blot. Plasmids containing seven individual viral genes of HBV (PTT22-HBx, PTT22-HBs, PTT22-preS2, PTT22-preS1, PTT22-HBc, PTT22-HBe, and PTT22-HBp) or control plasmids (PTT22-vector) were transfected into HepG2 cells. MK886 (PPARα) and GW9662 (PPARγ) inhibitors were used to explore the effects of HBV on α-1,2-mannosidase expression after the PPARα and PPARγ pathways were blocked.We showed that the expression of α-1,2-mannosidases was higher in stably transfected HBV cells than in controls. The expression levels of α-1,2-mannosidase were higher in AD38 cells than those in ND10 cells, which were in turn greater than those in G2.2.15 cells, and positively correlated with the expression of HBsAg Submit a Manuscriptin all the cell lines. Levels of α-1,2-mannosidase in nontumorous liver tissues of HBV-related HCC patients were also higher than in the tissues from non-HBVrelated HCC patients. Moreover, transfecting HepG2 cells with a component of the HBV viral envelope also increased the expression of α-1,2-mannosidases. However, this envelope protein component could not induce MAN1C1 expression in the presence of a PPARα inhibitor, MK886. We also found that MK886 did not affect the expression of MAN1C1 in AD38 cells without tetracycline in the culture medium. This phenomenon was not observed in the case of GW9662.Our results indicate that HBV increases the expression of α-mannosidases both in vitro and in vivo via activation of the PPARα pathway by its envelope protein.To date, few studies have investigated whether hepatitis B virus (HBV) hijacks the host hepatocyte's demannosylation system to trim the mannose oligosaccharides found on its viral envelope, thereby evading DC-SIGN recognition. Our study showed that HBV could increase the expression of α-mannosidase I both in vitro and in vivo . Moreover, the HBV envelope protein increases the expression of α-mannosidase I via the PPARα pathway. Therefore, α-mannosidase I may be a novel drug target to inhibit the demannosylation of HBV, and prevent viral escape. Hu S, Jiang LB, Zou XJ, Yi W, Tian DY. Hepatitis B virus upregulates host expression of α-1,2-mannosidases via the PPARα pathway. World J Gastroenterol 2016; 22(43): 9534-9543 Available from: URL Abstract Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase. Abstract Background. Umbilical cord blood transplant (UCBT) is used for patients who do not have a matched donor, but engraftment often takes longer than with a standard allogeneic transplant, likely increasing the risk for infection. We characterized specific infections and outcomes in adults undergoing UCBT at our 2 centers.Methods. All adults who underwent UCBT between January 1, 2006 and December 31, 2015 were included. Infectious episodes from 6 months before to 2 years after UCBT were reviewed.Results. Fifty-seven patients underwent UCBT; 47 had neutrophil engraftment. A total of 179 infectious episodes occurred in 55 patients, 73 (41%) within 30 days post-UCBT. Viruses caused 85 (47%) infections. Cytomegalovirus caused 32 infectious episodes and was most common from day 30 to 100. Human herpesvirus 6 occurred in 28 episodes, was most common within 30 days, and caused 1 death. Bacteria were responsible for 82 (46%) infections, most commonly bacteremias due to Staphylococcus spp, Enterococcus spp, and Enterobacteriaceae. Of 11 invasive fungal infections, 9 were aspergillosis, 4 of which were fatal. Overall mortality was 56% in the first year. Thirteen deaths were from infection; 11 occurred in the first 100 days and 7 in the first 30 days post-UCBT. Of 10 patients who never engrafted, 9 died, 6 from infection, within 100 days post-UCBT.Conclusions. Infectious complications were common after UCBT, especially in the first 30 days. Deaths from viral infections were fewer than expected. Delayed engraftment and nonengraftment continue to convey increased risk for fatal bacterial and fungal infections post-UCBT. Abstract The aim of the study was to comprehensively examine the efficacy and safety of noninvasive ventilation used at the pulmonary infection control (PIC) window for acute respiratory failure (ARF) in patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD).Seven electronic databases and relevant resources were searched to identify randomized controlled trials (RCTs) comparing patients using noninvasive ventilation at PIC window with those continuing receiving invasive ventilation. Retrieved citations were screened, risk of bias was assessed, and data were extracted by 2 independent review authors. Overall effect sizes were synthesized by using meta-analyses. Quality of evidence was rated by using Grading of Recommendations, Assessment, Development and Evaluation approach.A total of 17 trials involving 959 participants were included for this review. Compared with continuous invasive ventilation, noninvasive ventilation used at PIC window significantly reduced mortality, ventilator-associated pneumonia, weaning failures, reintubations, duration of invasive ventilation, total duration of mechanical ventilation, length of stay (LOS) in intensive care unit, and LOS in hospital as well as hospital costs. Of these, mortality significantly decreased (risk ratio = 0.27, 95% confidence interval: 0.17-0.42, P < 0.001) without significant heterogeneity (I 2 = 0%, P = 0.99). Quality of evidence regarding the 9 outcomes across the included studies was rated from moderate to low.Use of noninvasive ventilation at PIC window showed beneficial effects across identified trials for ARF in AECOPD patients. Considering the absence of high quality of available evidence and the uncertainty of long-term effect of this intervention, a weak recommendation for clinical practice was generated, and further well-designed and adequately powered RCTs are required to validate this conclusion.Abbreviations: AECOPD = acute exacerbation of chronic obstructive pulmonary disease, ARF = acute respiratory failure, CI = confidence interval, COPD = chronic obstructive pulmonary disease, GRADE = Grading of Recommendations, Assessment, Development and Evaluation, LOS = length of stay, MD = mean difference, OIS = optimal information size, PIC = pulmonary infection control, RCTs = randomized controlled trials, RR = risk ratio, SD = standard deviation, VAP = ventilator-associated pneumonia. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) is continuously spreading and causing severe and fatal acute respiratory disease in humans. Prophylactic and therapeutic strategies are therefore urgently needed to control MERS-CoV infection. Here, we generated a humanized monoclonal antibody (mAb), designated hMS-1, which targeted the MERS-CoV receptor-binding domain (RBD) with high affinity. hMS-1 significantly blocked MERS-CoV RBD binding to its viral receptor, human dipeptidyl peptidase 4 (hDPP4), potently neutralized infection by a prototype MERS-CoV, and effectively crossneutralized evolved MERS-CoV isolates through recognizing highly conserved RBD epitopes. Notably, single-dose treatment with hMS-1 completely protected hDPP4 transgenic (hDPP4-Tg) mice from lethal infection with MERS-CoV. Taken together, our data suggest that hMS-1 might be developed as an effective immunotherapeutic agent to treat patients infected with MERS-CoV, particularly in emergent cases. Abstract Chemokines have been demonstrated to serve an important role in a variety of diseases, particularly in tumor progression. There have been numerous studies that have reported that T cells serve major roles in tumor progression. However, the function of CXC motif chemokine ligand 9 (CXCL9) in prostate cancer remains unknown. The present study aimed to investigate the role of CXCL9 in prostate cancer. A prostate cancer mouse model was generated by treating C57/BL-6 and B6.Cg-Selplgtm1Fur/J mice with 3,2'-dimethyl 4-aminobiphenyl (DMAB). Hematoxylin and eosin staining detected the histopathological alterations of mouse prostate tissues. Immunohistochemistry (IHC) staining determined cell proliferation of the mice. Flow cytometry was used to detect the alterations of T cells in C57+DMAB or CXCL9+DMAB mice. Immunofluorescence revealed that there was positive expression of interleukin-6 (IL-6) and transforming growth factor (TGF)-β in the mouse tissues. The survival rates of C57+DMAB and CXCL9+DMAB mice was analyzed. The association of CXCL9 expression and clinical stages was also evaluated. Results revealed that prostate cancer pathology and cell proliferation in CXCL9+DMAB mice were significantly greater compared with the C57+DMAB mice. Compared with C57+DMAB mice, the number of T cells in peripheral blood and spleen of CXCL9+DMAB mice was significantly reduced. IHC demonstrated that the expression of IL-6 and TGF-β was significantly downregulated in the CXCL9+DMAB mice. The survival rate of CXCL9+DMAB mice was significantly decreased compared with the C57+DMAB mice. In addition, reverse transcription-quantitative polymerase chain reaction analysis demonstrated that CXCL9 mRNA expression in clinical samples was positively associated with clinical pathological stages of prostate cancer. In conclusion, CXCL9 may promote prostate cancer progression via inhibition of cytokines from T cells.ST and YG conceived and designed the experiments; ST, FS, YL and YG performed the experiments; ST, KW and YG analyzed the data; ST, KW, FS and YG contributed reagents/materials/analysis tools and ST and YG wrote the manuscript.Animal experiments were approved by the Medical Ethics Committee of Linyi People's Hospital.These tissue samples were collected with the consent of the patients and the study was approved by the Medical Ethics Committee of the Linyi People's Hospital. Abstract Bats are reservoirs for emerging zoonotic viruses that can have a profound impact on human and animal health, including lyssaviruses, filoviruses, paramyxoviruses, and severe acute respiratory syndrome coronaviruses (SARS-CoVs). In the course of a project focused on pathogen discovery in contexts where human-bat contact might facilitate more efficient interspecies transmission of viruses, we surveyed gastrointestinal tissue obtained from bats collected in caves in Nigeria that are frequented by humans. , et al. 2010. Identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in Nigeria. mBio 1(4):Coronavirus consensus PCR and unbiased high-throughput pyrosequencing revealed the presence of coronavirus sequences related to those of SARS-CoV in a Commerson's leaf-nosed bat (Hipposideros commersoni). Additional genomic sequencing indicated that this virus, unlike subgroup 2b CoVs, which includes SARS-CoV, is unique, comprising three overlapping open reading frames between the M and N genes and two conserved stem-loop II motifs. Phylogenetic analyses in conjunction with these features suggest that this virus represents a new subgroup within group 2 CoVs.IMPORTANCE Bats (order Chiroptera, suborders Megachiroptera and Microchiroptera) are reservoirs for a wide range of viruses that cause diseases in humans and livestock, including the severe acute respiratory syndrome coronavirus (SARS-CoV), responsible for the global SARS outbreak in 2003. The diversity of viruses harbored by bats is only just beginning to be understood because of expanded wildlife surveillance and the development and application of new tools for pathogen discovery. This paper describes a new coronavirus, one with a distinctive genomic organization that may provide insights into coronavirus evolution and biology. Abstract Waddlia chondrophila, an obligate intracellular bacterium of the Chlamydiales order, is considered as an agent of bovine abortion and a likely cause of miscarriage in humans. Its role in respiratory diseases was questioned after the detection of its DNA in clinical samples taken from patients suffering from pneumonia or bronchiolitis. To better define the role of Waddlia in both miscarriage and pneumonia, a tool allowing large-scale serological investigations of Waddlia seropositivity is needed. Therefore, enriched outer membrane proteins of W.chondrophila were used as antigens to develop a specific ELISA.After thorough analytical optimization, the ELISA was validated by comparison with micro-immunofluorescence and it showed a sensitivity above 85% with 100% specificity. The ELISA was subsequently applied to human sera to specify the role of W. chondrophila in pneumonia. Overall, 3.6% of children showed antibody reactivity against W. chondrophila but no significant difference was observed between children with and without pneumonia. Proteomic analyses were then performed using mass spectrometry, highlighting members of the outer membrane protein family as the dominant proteins. The major Waddlia putative immunogenic proteins were identified by immunoblot using positive and negative human sera. The new ELISA represents an efficient tool with high throughput applications. Although no association with pneumonia and Waddlia seropositivity was observed, this ELISA could be used to specify the role of W. chondrophila in miscarriage and in other diseases. Abstract Environmental changes have an undoubted influence on the appearance, distribution, and evolution of infectious diseases, and notably on those transmitted by vectors. Global change refers to environmental changes arising from human activities affecting the fundamental mechanisms operating in the biosphere. This paper discusses the changes observed in recent times with regard to some important arboviral (arthropod-borne viral) diseases of animals, and the role global change could have played in these variations. Two of the most important arboviral diseases of animals, bluetongue (BT) and West Nile fever/encephalitis (WNF), have been selected as models. In both cases, in the last 15 years an important leap forward has been observed, which has lead to considering them emerging diseases in different parts of the world. BT, affecting domestic ruminants, has recently afflicted livestock in Europe in an unprecedented epizootic, causing enormous economic losses. WNF affects wildlife (birds), domestic animals (equines), and humans, thus, beyond the economic consequences of its occurrence, as a zoonotic disease, it poses an important public health threat. West Nile virus (WNV) has expanded in the last 12 years worldwide, and particularly in the Americas, where it first occurred in 1999, extending throughout the Americas relentlessly since then, causing a severe epidemic of disastrous consequences for public health, wildlife, and livestock. In Europe, WNV is known long time ago, but it is since the last years of the twentieth century that its incidence has risen substantially. Circumstances such as global warming, changes in land use and water management, increase in travel, trade of animals, and others, can have an important influence in the observed changes in both diseases. The following question is raised: What is the contribution of global changes to the current increase of these diseases in the world? Abstract Middle East respiratory syndrome (MERS) is an acute infectious disease of the respiratory system caused by the new betacoronavirus (MERS coronavirus, MERS-CoV), which shows high mortality rates. The typical symptoms of MERS are fever, cough, and shortness of breath, and it is often accompanied by pneumonia. The MERS-CoV was introduced to Republic of Korea in May 2015 by a patient returning from Saudi Arabia. The disease spread mostly through hospital infections, and by the time the epidemic ended in August, the total number of confirmed diagnoses was 186, among which 36 patients died. Reflecting the latest evidence for antiviral drugs in the treatment of MERS-CoV infection and the experiences of treating MERS patients in Republic of Korea, these guidelines focus on antiviral drugs to achieve effective treatment of MERS-CoV infections. Abstract Li Wenliang, a face to the frontline healthcare worker. The first doctor to notify the emergence of the SARS-CoV-2, (COVID-19), outbreak Abstract Background: The prevalence and risk factors for infection with enteropathogens in dogs frequenting dog parks have been poorly documented, and infected dogs can pose a potential zoonotic risk for owners.Hypothesis/Objectives: To determine the prevalence and risk factors of infection with enteropathogens and zoonotic Giardia strains in dogs attending dog parks in Northern California and to compare results of fecal flotation procedures performed at a commercial and university parasitology laboratory.Animals: Three-hundred dogs attending 3 regional dog parks in Northern California.Methods: Prospective study. Fresh fecal specimens were collected from all dogs, scored for consistency, and owners completed a questionnaire. Specimens were analyzed by fecal centrifugation flotation, DFA, and PCR for detection of 11 enteropathogens. Giardia genotyping was performed for assemblage determination.Results: Enteropathogens were detected in 114/300 dogs (38%), of which 62 (54%) did not have diarrhea. Frequency of dog park attendance correlated significantly with fecal consistency (P = .0039), but did not correlate with enteropathogen detection. Twenty-seven dogs (9%) were infected with Giardia, and genotyping revealed nonzoonotic assemblages C and D. The frequency of Giardia detection on fecal flotation was significantly lower at the commercial laboratory versus the university laboratory (P = .013), and PCR for Giardia was negative in 11/27 dogs (41%) that were positive on fecal flotation or DFA.Conclusions and Clinical Importance: Enteropathogens were commonly detected in dogs frequenting dog parks, and infection with Giardia correlated with fecal consistency. PCR detection of Giardia had limited diagnostic utility, and detection of Giardia cysts by microscopic technique can vary among laboratories. Abstract The initial cluster of severe pneumonia cases that triggered the COVID-19 epidemic was identified in Wuhan, China in December 2019. While early cases of the disease were linked to a wet market, human-to-human transmission has driven the rapid spread of the virus throughout China. The Chinese government has implemented containment strategies of city-wide lockdowns, screening at airports and train stations, and isolation of suspected patients; however, the cumulative case count keeps growing every day. The ongoing outbreak presents a challenge for modelers, as limited data are available on the early growth trajectory, and the epidemiological characteristics of the novel coronavirus are yet to be fully elucidated. We use phenomenological models that have been validated during previous outbreaks to generate and assess short-term forecasts of the cumulative number of confirmed reported cases in Hubei province, the epicenter of the epidemic, and for the overall trajectory in China, excluding the province of Hubei. We collect daily reported cumulative confirmed cases for the 2019-nCoV outbreak for each Chinese province from the National Health Commission of China. Here, we provide 5, 10, and 15 day forecasts for five consecutive days, February 5th through February 9th, with quantified uncertainty based on a generalized logistic growth model, the Richards growth model, and a sub-epidemic wave model. Our most recent forecasts reported here, based on data up until February 9, 2020, largely agree across the three models presented and suggest an average range of 7409e7496 additional confirmed cases in Hubei and 1128e1929 additional cases in other provinces within the next five days. Models also predict an average total cumulative case count between 37,415 and 38,028 in Hubei and 11,588e13,499 in other provinces by February 24, 2020. Mean estimates and uncertainty bounds for both Hubei and other provinces have remained relatively stable in the last three reporting dates (February 7th e 9th). We also observe that each of the models predicts that the epidemic has reached saturation in both Hubei and other provinces. Our findings suggest that the containment strategies implemented in China are successfully reducing transmission and that the epidemic growth has slowed in recent days. Abstract Infectious disease during an emergency condition can raise the death rate 60 times in comparison to other causes including trauma. An epidemic, or outbreak, can occur when several aspects of the agent (pathogen), population (hosts), and the environment create an ideal situation for spread. Overcrowding, poor regional design and hygiene due to poverty, dirty drinking water, rapid climate changes, and natural disasters, can lead to conditions that allow easier transmission of disease. Once it has been established that an emergency condition exists, there must be a prompt and thorough response for communicable disease control. A camp should be created, and the disease managed rapidly. The overall goals are rapid assessment, prevention, surveillance, outbreak control, and disease management.Other safeguards such as airborne droplet, regular droplet, and contact precautions are necessary to prevent spread of unique vectors. Airborne particles are small (less than or Turk J Emerg Med 2015;15(Suppl 1):20-26 Abstract The Middle East respiratory syndrome coronavirus (MERS-CoV) has been recognized as a highly pathogenic virus that infects the human respiratory tract and has high morbidity and mortality. The MERS-CoV is a huge burden on Saudi Arabian health-care facilities, causing approximately 40% mortality. The transmission mechanism of the virus is still not well understood. Therefore, the prevention of any route of transmission is the best measure to arrest the spread of this disease. Using the real time polymerase chain reaction (RT-PCR), MERS-CoV was detected in the nasal secretions of a human cadaver. Full precautions should be applied and carefully followed to prevent the transmission of the virus, especially among health care workers. Abstract Introduction The implementation of evidence-based clinical practice guidelines is one of the most effective interventions for improving quality of care. A gap between guidelines and clinical practice often exists, which may result in patients not receiving appropriate care. This project aimed at improving adherence to lung cancer guidelines at our institution.Method The records of patients with lung cancer were evaluated for adherence to guidelines by using an auditing tool that was developed to capture pertinent information. The study team collected data about the following variables: compliance with documentation of pathological diagnosis, documentation of disease stage prior to treatment initiation, presentation at thoracic tumour board within 30 days of diagnosis, management course, and management of end of life in terms of early 'no code' initiation, stopping chemotherapy and referral to palliative care prior to 2 weeks of death. Annual audits were performed from 2012 to 2015. Education and discussion with team members to address the deviations were the main interventions to improve adherence. Results The baseline measurements were taken in 2012 (49 patients). Histological subtype identification improved from 94% to 100%. Presentation of new cases at the tumour board improved from 35% to 82%. Testing for epidermal growth factor receptor mutation for nonsquamous cell lung cancer improved from 77% to 100%. The staging was documented in 100% of the cases. Conclusion Running audits to monitor adherence to guidelines and discussions with the team have a positive effect on providing consistent evidence-based care for patients with lung cancer. Abstract Background: Fungi contribute to the inflammatory response of lungs in horses with recurrent airway obstruction and in some forms of asthma in humans. The role of fungi in inflammatory airway disease (IAD) has not been assessed.Objectives: Evaluate the prevalence of fungi in the respiratory samples of horses diagnosed with IAD, describe clinical signs associated with the presence of fungi in respiratory samples, and assess the risk factors associated with IAD and with the presence of fungi in the airways.Animals: Seven-hundred thirty-one active horses referred to a specialized ambulatory practice for signs of respiratory disease or poor performance.Methods: A prospective observational study was performed, collecting clinical data, environmental conditions, and results of a tracheal wash (TW; cytology, fungal culture, and bacterial culture), and bronchoalveolar lavage (cytology).Results: A positive fungal culture was obtained in 55% (402/731) of horses. Horses with fungal elements observed on the TW cytology had 2 times greater chance of having IAD than horses without fungi (odds ratio [OR] = 2.1; 95% CI 1.08-3.33; P = .0003). Risks of being diagnosed with IAD and likelihood of fungi in TW were higher when horses were bedded on straw (OR = 2.0; 95% CI 1.2-3.2 and OR = 1.9; 95% CI 1.3-2.6, respectively) or fed dry hay (OR = 2.7; 95% CI 1.7-4.4 and OR = 2.6; 95% CI 1.6-3.4, respectively).Conclusions and Clinical Importance: Horses inhaling aerosolized fungal particles are at a significantly higher risk of developing IAD. The type of bedding and forage represent significant risk factors for IAD and fungal contamination of equine airways.cough, environment, equine asthma, inflammatory airway disease, mold, poor performance, tracheal wash Abstract A number of agents are now available for use in protecting against ionizing radiation. These radiationprotective agents, however, have many adverse effects. Efforts have been made to develop new radiationprotective agents for medical application. Here, we investigated whether a compound, polyethylenimine (PEI), which activates Toll-like receptor 5 (TLR5)-mediated NF-kB signaling pathways, could have an antiradiation effect on a mouse model. First, a cell-based screening model for an agonist of TLR5-mediated NF-kB pathway was established and then validated by activation of TLR5-mediated NF-kB luciferase reporter activity with a known TLR5 agonist, flagellin. We found that PEI induced dose-dependent activation of the TLR5-mediated NF-kB pathway, indicating that PEI is indeed a TLR5 agonist. Furthermore, the anti-radiation effect of polyethylenimine was assessed using a γ-ray total body irradiation (TBI) mouse model. Compared with the irradiation control, both survival time and survival rate were significantly improved in mice that received either a low dose of polyethylenimine (P = 0.019) or a high dose of polyethylenimine (P < 0.001). We also observed a positive correlation between animal body weight and survival time in mice that received a low dose of polyethylenimine, a high dose of polyethylenimine and amifostine, over a period of 30 days, r = 0.42 (P < 0.02), 0.72 (P < 0.0001) and 0.95 (P < 0.0001), respectively, while a negative correlation between animal body weight and survival time was observed in the irradiation control (r = -0.89; P < 0.0001). These results indicate that polyethylenimine is a new TLR5 agonist with potential application in offering protection for patients receiving radiotherapy or in radiation-related accidents. Abstract Background: Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis.Methods: Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis.The lower detection limits ranged from 10 1 copies/μL to 5 × 10 1 copies/μL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent.Conclusions: The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens. Abstract Recent evidence has confirmed that a mutation of the isocitrate dehydrogenase (IDH) gene occurs early in gliomagenesis and contributes to suppressed immunity. The present study aimed to determine the candidate genes associated with IDH mutation status that could serve as biomarkers of immune suppression for improved prognosis prediction. Clinical information and RNA-seq gene expression data were collected for 932 glioma samples from the CGGA and TCGA databases, and differentially expressed genes in both lower-grade glioma (LGG) and glioblastoma (GBM) samples were identified according to IDH mutation status. Only one gene, interferon-stimulated exonuclease gene 20 (ISG20), with reduced expression in IDH mutant tumors, demonstrated significant prognostic value. ISG20 expression level significantly increased with increasing tumor grade, and its high expression was associated with a poor clinical outcome. Moreover, increased ISG20 expression was associated with increased infiltration of monocyte-derived macrophages and neutrophils, and suppressed adaptive immune response. ISG20 expression was also positively correlated with PD-1, PD-L1, and CTLA4 expression, along with the levels of several chemokines. We conclude that ISG20 is a useful biomarker to identify IDH-mediated immune processes in glioma and may serve as a potential therapeutic target.ARTICLE HISTORY Abstract The spike gene of porcine epidemic diarrhea virus (PEDV) was sequenced from 55 South China field strains isolated from pigs with symptoms of diarrhea. The sequences were compared within the set of field strains as well as with reference strains available in GenBank. Within the 55 South China PEDV field strains, the deduced amino acid sequence identities ranged from 93.8% to 99.9 % and ranged from 90.7% to 99.5% when compared with the foreign reference strains in GenBank. Our phylogenetic analysis showed that 10 of the 55 South China PEDV strains belonged to G1b and 45 belonged to G2b. Abstract Background: Systemic lupus erythematosus (SLE) is associated with immunogenetic factors. This study was planned to test for the association of TNF-α − 308 and IFN-γ +874 gene polymorphisms with susceptibility and severity of SLE in Egyptian cases. Subjects and methods: This is a case controlled study including 125 Egyptian cases with SLE in addition to 112 healthy unrelated individuals from the same locality. For all participants, TNF-α − 308 G NA and IFN-γ + 874 ANT genetic polymorphisms were characterized using the PCR technique. Results: Cases with SLE showed a significantly higher TNF-α −308 A allele carriage rate (AA + GA genotypes) compared to controls (26.4% vs. 12.5%, p = 0.009, OR = 2.51, 95% CI = 1.26-4.99). These cases showed also a significantly higher carriage rate for the IFN-γ +874 T allele (AT + TT genotypes) compared to controls (47.2% vs. 32.1%, p = 0.02, OR = 1.89, 95% CI = 1.11-3.21). Comparing age, gender, and disease severity presented by nephritis class, activity and chronicity indices in cases carrying the TNF-α −308 A allele and in cases carrying IFN-γ +874 T allele versus others showed no significant difference (p N 0.05). Conclusions: TNF-α −308 A and IFN-γ +874 T allele carriage are associated with susceptibility but not severity of SLE in Egyptian subjects. Abstract Background. Hospital-acquired respiratory viral infections can result in morbidity and mortality of hospitalized patients. This study was undertaken to better understand the magnitude of the problem of nosocomial respiratory viral infections in adult and pediatric patients.Methods. This was a retrospective study at a tertiary care adult and pediatric teaching hospital. Study patients met a priori criteria for definite or possible nosocomial respiratory viral infection.Results. From April 1, 2015 to April 1, 2016, we identified 40 nosocomial respiratory viral infections in 38 patients involving 14 definite and 3 possible cases in our adult hospital and 18 definite and 5 possible cases in our pediatric hospital. The incidence was 5 cases/10 000 admissions and 44 cases/10 000 admissions to our adult and pediatric hospitals, respectively. Only 6.8% of cases were due to influenza. Although 63% of cases occurred during the fall and winter, such infections were identified throughout the year. Five (13%) nosocomial respiratory viral infections occurred in 2 adult and 3 pediatric patients who died during the hospitalization.Conclusions. Nosocomial respiratory viral infections are an underappreciated cause of morbidity and mortality in hospitalized adult and pediatric patients. The incidence was nearly 10-fold higher in our pediatric hospital. We estimate there are approximately 18 955 pediatric and adult cases of nosocomial respiratory viral infections in US acute care hospitals each year. Abstract Background: Acute-phase proteins (APPs) are sensitive markers of inflammation, and serum C-reactive protein (CRP) recently has been shown to be a useful diagnostic marker in dogs with bacterial pneumonia (BP). In humans with community-acquired pneumonia, APPs also have great utility as follow-up markers aiding in the assessment of treatment response.Objectives: The aim of our study was to investigate the applicability of APPs as markers of treatment response in dogs with BP.Animals: Nineteen dogs diagnosed with BP and 64 healthy dogs. Methods: The study was conducted as a prospective longitudinal observational study. Serum CRP, serum amyloid A (SAA), and haptoglobin concentrations were followed during a natural course of BP. Normalization of serum CRP was used to guide the duration of antibiotic treatment (treatment was stopped 5-7 days after CRP normalized) in 8 of 17 dogs surviving to discharge; 9 of 17 dogs were treated according to conventional recommendations.Results: All measured APPs initially were significantly increased, but the magnitude of increase was not correlated to disease severity. C-reactive protein and SAA concentrations decreased rapidly after initiation of antimicrobial treatment. When normalization of serum CRP was used to guide the duration of antibiotic treatment, treatment duration was significantly (P = .015) decreased without increasing the number of relapses.Conclusions and Clinical Importance: Serum CRP and SAA reflected the recovery process well and therefore may be used as markers of treatment response. According to the results, the normalization of serum CRP may be used to guide the duration of antibiotic treatment in dogs with BP. Abstract To reduce the use of antibiotics in animal agriculture, a number of effective or commercially viable alternatives have been implemented by food animal producers or are under development. Perhaps the most well-established strategies are flock and herd management practices to mitigate disease introduction and spread, and, subsequently, reduce the need for antibiotic use. While vaccines in food animal production have been used to prevent both bacterial and viral diseases, but historically, most vaccines have targeted viral diseases. Though vaccines against viral diseases can help reduce the need for antibiotic use by controlling the spread of secondary bacterial infections, more recent vaccines under development specifically target bacteria. New developments in selecting and potentially tailoring bacteriophages provide a promising avenue for controlling pathogenic bacteria without the need for traditional small-molecule antibiotics. In this article we discuss these established and emerging strategies, which are anticipated to reduce the reliance on antibiotics in food animal production and should reduce the prevalence and transmission to humans of antimicrobial resistant bacteria from these systems. Abstract 253 protein, C-reactive protein (CRPt), varies markedly under differing physiologic conditions (47). CRP, a homopentamer composed of nonglycosylated 24-kD subunits associated by noncovalent forces (24, 29), is a plasma protein whose rate of synthesis by the hepatocyte increases by several hundredfold or more during the systemic acute phase response to tissue injury (39). In studies of the synthesis and secretion of CRP by rabbit primary hepatocyte cultures prepared from animals stimulated in vivo to undergo the acute phase response, the half-time for secretion of newly synthesized CRP was found to decrease markedly from as much as 18 h in control hepatocytes to as little as 75 min in cells from acute phase animals (47), while the kinetics of albumin secretion determined in the same cultures, were rapid (half-time of 30-45 min) and did not vary during the acute phase response. Results of studies in which a fusion gene consisting of the rabbit CRP gene linked to the mouse metallothionein promoter was transfected into HeLa cells (29) indicated that the default rate of rabbit CRP secretion was rapid and suggested that changes in its transit time were due to its specific retention within unstimulated rabbit hepatocytes, rather than to facilitated export in cells from stimulated animals. However, CRP does not contain carboxy-terminal sequences reported to result in ER retention (29). Further, assembly of CRP does not appear to be rate-limiting, since in control as well as stimulated cells >90% of antigenically detectable CRP labeled in a 10-min pulse is pentameric as judged by its size on gel filtration, its differential reactivity with structure-specific antibodies, and its ability to bind phosphocholine (29, 46, 47, and our unpublished observations).In the present report, pulse-chase subcellular fractionation experiments identify the ER as the compartment in which CRP is retained. Both biochemical and immunohistochemical methods demonstrate specific binding of CRP to the lumenal face of detergent-permeabilized rough microsomes, but not to Golgi subfractions. Kinetic and equilibrium binding studies identify a high affinity CRP binding site which is present in hepatic rough microsomes from normal rabbits, but is not detected in microsomes from animals undergoing the acute phase response. Finally, nitrocellulose blots probed with radiolabeled CRP demonstrate a 60-kD band, distinct from BiP, which is detected in extracts of control rough microsomes, but not in Golgi fractions or rough microsomes from stimulated animals. Together, these findings are consistent with the hypothesis that CRP is specifically retained within the ER by a novel mechanism which is downregulated during the acute phase response. Abstract Infection of respiratory mucosa with viral pathogens triggers complex immunologic events in the affected host. We sought to characterize this response through proteomic analysis of nasopharyngeal lavage in human subjects experimentally challenged with influenza A/H3N2 or human rhinovirus, and to develop targeted assays measuring peptides involved in this host response allowing classification of acute respiratory virus infection. Unbiased proteomic discovery analysis identified 3285 peptides corresponding to 438 unique proteins, and revealed that infection with H3N2 induces significant alterations in protein expression. These include proteins involved in acute inflammatory response, innate immune response, and the complement cascade. These data provide insights into the nature of the biological response to viral infection of the upper respiratory tract, and the proteins that are dysregulated by viral infection form the basis of signature that accurately classifies the infected state. Verification of this signature using targeted mass spectrometry in independent cohorts of subjects challenged with influenza or rhinovirus demonstrates that it performs with high accuracy (0.8623 AUROC, 75% TPR, 97.46% TNR). With further development as a clinical diagnostic, this signature may have utility in rapid screening for emerging infections, avoidance of inappropriate antibacterial therapy, and more rapid implementation of appropriate therapeutic and public health strategies. Abstract We have investigated the localization of Kexlp, a type I transmembrane carboxypeptidase involved in precursor processing within the yeast secretory pathway. Indirect immunofluorescence demonstrated the presence of Kexlp in a punctate organelle resembling the yeast Golgi apparatus as identified by Kex2p and Sec7p (Franzusoff, A., K. Redding, J. Crosby, R. S. Fuller, and R. Schekman. 1991. J. Cell Biol. 112:27-37). Glycosylation studies of Kexlp were consistent with a Golgi location, as Kexlp was progressively N-glycosylated in an MNNl-dependent manner.To address the basis of Kexlp targeting to the Golgi apparatus, we examined the cellular location of a series of carboxy-terminal truncations of the protein.The results indicate that a cytoplasmically exposed carboxy-terminal domain is required for retention of this membrane protein within the Golgi apparatus. Deletions of the retention region or overproduction of wild-type Kexlp led to mislocalization of Kexlp to the vacuolar membrane. This unexpected finding is discussed in terms of models involving either the vacuole as a default destination for membrane proteins, or by endocytosis to the vacuole following their default localization to the plasma membrane. Abstract A three-year-old spayed domestic short-haired cat presented for evaluation of weight loss, cardiomegaly and pleural effusion. Echocardiographic examination demonstrated a thickened pericardium with mild pericardial effusion and a large volume of pleural effusion characterized by exudate. Although the cat was treated with antibiotics, the clinical symptoms did not improve. The cat developed dyspnea and died on day 7. Necropsy revealed a large amount of modified transudates ascites, pleural effusion and markedly dilated pericardium. Histopathological examination revealed severe exudation of fibrin and granulation tissue in a thick layer of the epicardium. The cat was diagnosed with fibrinous pericarditis secondary to bacterial infection. Abstract Quaternary prevention should be implemented to minimize harm to patients because the ultimate goal of medicine is to prevent disease and promote health. Primary care physicians have a major responsibility in quaternary prevention, and the establishment of clinical epidemiology as a distinct field of study would create a role charged with minimizing patient harm arising from over-medicalization. Abstract The high degree of phenotypic similarity among Trichophyton species makes their identification difficult. Objectives: The current study aims to establish the use of rolling circle amplification (RCA) based on internal transcribed spacer ribosomal DNA (ITS rDNA) as a powerful, simple, and rapid procedure for distinguishing closely related organisms, and specifically to identify Trichophyton species, which cause human and animal disorders. Materials and Methods: A total of sixty-one isolates belonging to three species of Trichophyton were identified to the species level based on microscopic and macroscopic examinations and their ITS rDNA regions were sequenced. Three specific circular oligonucleotide probes targeting the ITS1 and ITS2 regions were designed to differentiate Trichophyton rubrum, T. mentagrophytes, and T. tonsurans. Results: Of the 61 putative Trichophyton clinical isolates, 52 were identified to the species level. The most common species was T. mentagrophytes var. interdigitale (31 isolates), followed by T. rubrum (11 isolates), T. tonsurans (9 isolates), and T. violaceum (1 isolates); moreover, 9 isolates were identified as non-Trichophyton species. The RCA method correctly identified four Trichophyton species and was 100% specific for each species. Neither cross-reaction between the examined species of Trichophyton nor false positive or false negative results were observed. Conclusions: Species identification of Trichophyton is crucially important for epidemiological and phylogenetic purposes and for genotype delineation. RCA based on ITS polymorphisms can be used to generate identification barcodes and as an alternative to DNA sequencing; it is a very fast, specific, and economical tool for species identification. Abstract Aerial spraying of products to kill larvae or adult mosquitoes is a public health measure used to control vectorborne diseases. In some outbreaks, the intervention has evoked controversy and community resistance. This study evaluated how local opinion leaders in US localities affected by Zika think about community engagement in public health policies for outbreak response. In December 2017 through March 2018, 4 focus groups were convened in Houston, TX, New Orleans, LA, Miami, FL, and Brooklyn, NY. They discussed a hypothetical scenario that featured vector control by aerial spraying. Participants (N = 20) more readily accepted this vector control method under 4 conditions: They were informed of alternatives, benefits, and risks for human health and the environment. Public health claims were backed by objective evidence and an authority figure genuinely working in the community's interests. They received timely notice about how to mitigate toxin exposure. And, aerial spraying helped to protect vulnerable individuals. The community engagement requirements of the local opinion leaders resonate with core principles of recent public health ethics frameworks: namely, personal autonomy, transparency, reasonableness, and solidarity. Participants foresaw problems with community consent in an era of growing social media use and mistrust in governmental and scientific authority. They also debated whether health authorities should use moral-based arguments, in addition to science-based ones, to communicate aerial spraying's risks and benefits. Abstract Background: Zinc deficiency-like (ZDL) syndrome is an inherited defect of Fleckvieh calves, with striking similarity to bovine hereditary zinc deficiency (BHZD). However, the causative mutation in a phospholipase D4 encoding gene (PLD4) shows no connection to zinc metabolism.Objectives: To describe clinical signs, laboratory variables, and pathological findings of ZDL syndrome and their utility to differentiate ZDL from BHZD and infectious diseases with similar phenotype.Animals: Nine hospitalized calves with crusting dermatitis and confirmed mutation in PLD4 and medical records from 25 calves with crusting dermatitis or suspected zinc deficiency.Methods: Prospective and retrospective case series. Results: The 9 calves (age: 5-53 weeks) displayed a moderate to severe crusting dermatitis mainly on the head, ventrum, and joints. Respiratory and digestive tract inflammations were frequently observed. Zinc supplementation did not lead to remission of clinical signs in 4 calves. Laboratory variables revealed slight anemia in 8 calves, hypoalbuminemia in 6 calves, but reduced serum zinc concentrations in only 3 calves. Mucosal erosions/ulcerations were present in 7 calves and thymus atrophy or reduced thymic weights in 8 calves. Histologically, skin lesions were indistinguishable from BHZD. Retrospective analysis of medical records revealed the presence of this phenotype since 1988 and pedigree analysis revealed a common ancestor of several affected calves.Conclusions and Clinical Importance: ZDL syndrome should be suspected in Fleckvieh calves with crusting dermatitis together with diarrhea or respiratory tract inflammations without response to oral zinc supplementation. Definite diagnosis requires molecular genetic confirmation of the PLD4 mutation. Abstract The identification of a novel b coronavirus, nCoV, as the causative agent of severe respiratory illness in humans originating in Saudi Arabia, Qatar and Jordan has raised concerns about the possibility of a coronavirus pandemic similar to that of SARS-CoV. As a definitive treatment regimen has never been thoroughly evaluated for coronavirus infections, there is an urgent need to rapidly identify potential therapeutics to address future cases of nCoV. To determine an intervention strategy, the effect of interferon-a2b and ribavirin on nCoV isolate hCoV-EMC/2012 replication in Vero and LLC-MK2 cells was evaluated. hCoV-EMC/2012 was sensitive to both interferon-a2b and ribavirin alone in Vero and LLC-MK2 cells, but only at relatively high concentrations; however, when combined, lower concentrations of interferon-a2b and ribavirin achieved comparable endpoints. Thus, a combination of interferon-a2b and ribavirin, which are already commonly used in the clinic, may be useful for patient management in the event of future nCoV infections.A novel b coronavirus (nCoV), has been identified as the etiological agent of 17 confirmed cases (11 deaths) of a severe respiratory illness with occasional renal failure from patients in Saudi Arabia 1 , Qatar 2 , Jordan and the United Kingdom 3,4 . Complete genome sequencing 1,5 determined that this new virus is closely related to two Asian bat betacoronaviruses (HKU4 and HKU5) 6 in lineage C. This makes nCoV the first lineage C betacoronavirus known to infect humans 5 . While human-to-human transmission is assumed to be less extensive as compared to SARS-CoV, three of the cases in Saudi Arabia were within one family and several healthcare workers who cared for two of the cases in Jordan have been classified as probable cases 5 . Moreover, in the most recent cluster of cases 7 , two of the three cases did not have a history of travel to the Middle East, but are suspected to have resulted from human-to-human transmission within the UK from a family member with a travel history to Saudi Arabia and Pakistan. This would suggest that human-to-human transmission can occur in close contact settings. Presumably, the sporadic nature of the apparently unlinked index cases in three different, albeit geographically proximal countries, and the close relationship to Asian bat coronaviruses, suggests that the source of nCoV is zoonotic 8 .Despite limited information on this new virus, it has been determined that in contrast to SARS-CoV, which uses angiotensin-converting enzyme 2 (ACE2) to gain entry into cells 9,10 , nCoV uses dipeptidyl peptidase 4 (DPP4 or CD26) as a functional receptor 11 . This finding may be important as the requirement for ACE2 was thought to be partially responsible for the pathogenicity of SARS-CoV, while also serving as one of the factors that may have limited spread from human-to-human. As the pathogenesis of nCoV could be significantly different from previously studied coronaviruses, the ability to predict whether this virus is likely to result in a larger epidemic or even pandemic, such as occurred with SARS-CoV, is unknown.The rapid identification of therapeutics is a high priority as there is currently no specific therapy or vaccine for nCoV and the resulting disease has been severe with a high case-fatality rate. The clinical experience from SARS suggests that a number of interventions including ribavirin with and without corticosteroids 12-14 , ribavirin with protease inhibitors 15,16 and interferon (alfacon-1) with corticosteroids 17 may improve outcome, but a definitive treatment regimen was not clearly established 18 . Here we address the effectiveness in vitro of two antiviral drugs, interferon-a2b (IFN-a2b) and ribavirin, in an attempt to identify a therapeutic approach that can be immediately utilized in the clinic to benefit future cases. SUBJECT AREAS: ANTIVIRALS SARS VIRUS ANTIVIRAL AGENTS VIRAL INFECTION Abstract Transcription of hepatitis B virus (HBV) from the covalently closed circular DNA (cccDNA) template is essential for its replication. Suppressing the level and transcriptional activity of cccDNA might have anti-HBV effect. Although cellular transcription factors, such as CREB, which mediate HBV transcription, have been well described, transcriptional coactivators that facilitate this process are incompletely understood. In this study we showed that CREBregulated transcriptional coactivator 1 (CRTC1) is required for HBV transcription and replication. The steady-state levels of CRTC1 protein were elevated in HBV-positive hepatoma cells and liver tissues. Ectopic expression of CRTC1 or its homolog CRTC2 or CRTC3 in hepatoma cells stimulated the activity of the preS2/S promoter of HBV, whereas overexpression of a dominant inactive form of CRTC1 inhibited HBV transcription. CRTC1 interacts with CREB and they are mutually required for the recruitment to the preS2/S promoter on cccDNA and for the activation of HBV transcription. Accumulation of pregenomic RNA (pgRNA) and cccDNA was observed when CRTC1 or its homologs were overexpressed, whereas the levels of pgRNA, cccDNA and secreted HBsAg were diminished when CRTC1 was compromised. In addition, HBV transactivator protein HBx stabilized CRTC1 and promoted its activity on HBV transcription. Our work reveals an essential role of CRTC1 coactivator in facilitating and supporting HBV transcription and replication. Abstract Programmed -1 ribosomal frameshifting (−1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. −1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTninducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules. Abstract Although the influenza vaccine is relatively safe and effective, serious complications can develop in rare cases. We encountered two cases of interstitial pneumonia that developed after vaccination during the 2014-2015 influenza season. Overall, nine cases, including the two presented here, have been recorded in PubMed and the Cochrane library; eight patients were treated with corticosteroids, and all nine survived, suggesting a good prognosis. Interstitial pneumonia is rare; however, we found an increase in its incidence after 2009. Therefore, clinicians must be aware of the possibility of this complication and duly educate all patients in advance. Abstract Background. The clinical presentation and management of human metapneumovirus (hMPV) infections in immunocompromised children is not well understood. Methods. We performed a retrospective evaluation of pediatric patients with laboratory-confirmed hMPV infections and underlying hematologic malignancy, solid tumors, solid organ transplant, rheumatologic disease, and/or receipt of chronic immunosuppressants. Data were analyzed using t tests and Fisher's exact tests. Results. Overall, 55 patients (median age: 5 years; range: 5 months-19 years) with hMPV infection documented between 2006 and 2010 were identified, including 24 (44%) with hematologic malignancy, 9 (16%) undergoing hematopoietic stem cell transplant, 9 (16%) with solid tumors, and 8 (15%) with solid organ transplants. Three (5%) presented with fever alone, 35 (64%) presented with upper respiratory tract infections, and 16 (29%) presented with lower respiratory tract infections (LRTI). Twelve (23%) patients required intensive care unit admission and/or supplemental oxygen 28% FiO 2 . Those with severe disease were more likely to be neutropenic (P = .02), but otherwise did not differ by age (P = .27), hematopoietic stem cell transplant recipient status (P = .19), or presence of lymphopenia (P = .09). Nine (16%) patients received treatment with ribavirin, intravenous immunoglobulin, or both. Three children (5%) died of hMPV pneumonia. Conclusions. Immunocompromised pediatric patients with hMPV infection have high rates of LRTI and mortality. The benefits of treatment with ribavirin and intravenous immunoglobulin in this patient population require further evaluation. Abstract Redirecting the splicing machinery through the hybridization of high affinity, RNase H-incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R) 4 (with Ahx standing for 6-aminohexanoic acid) leads to sequencespecific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R) 4 -PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structureactivity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs. Abstract The aim of this study was to investigate the experiences of medical transportation of Korean travelers who suffered accidents abroad and then transferred home by our aeromedical team.We collected demographic and clinical data on patients injured while traveling abroad from January 2013 to July 2017. Descriptive analyses based on 4 different transportation methods and transport time since hospitalization were performed.A total of 33 patients were repatriated during the study period. Of these, 28 (84.8%) were trauma cases with pedestrian injuries being the most common (11 cases; 39.3%). Twenty patients were repatriated by flight-stretchers, 6 by flight-prestige, 2 by ship, and 5 by air ambulance. The air ambulance was the most expensive (average 61,124 US Dollars) mode of transportation (P = .001) and the ship took the longest time (14 hours) to transport patients back to Korea from regions with similar distance (P = .0023).We experienced medical repatriation of 33 seriously injured Korean travelers back to South Korea. Transfer time should be an important considering factor and directly contacting and communicating with the specialized staff of foreign hospitals could also be very important to reduce unnecessary overseas hospital stay and cost incidence.Abbreviation: USD = US Dollars. Abstract A genome space is a moduli space of genomes. In this space, each point corresponds to a genome. The natural distance between two points in the genome space reflects the biological distance between these two genomes. Currently, there is no method to represent genomes by a point in a space without losing biological information. Here, we propose a new graphical representation for DNA sequences. The breakthrough of the subject is that we can construct the moment vectors from DNA sequences using this new graphical method and prove that the correspondence between moment vectors and DNA sequences is one-to-one. Using these moment vectors, we have constructed a novel genome space as a subspace in R N . It allows us to show that the SARS-CoV is most closely related to a coronavirus from the palm civet not from a bird as initially suspected, and the newly discovered human coronavirus HCoV-HKU1 is more closely related to SARS than to any other known member of group 2 coronavirus. Furthermore, we reconstructed the phylogenetic tree for 34 lentiviruses (including human immunodeficiency virus) based on their whole genome sequences. Our genome space will provide a new powerful tool for analyzing the classification of genomes and their phylogenetic relationships. Abstract The clinical evaluation of convalescent plasma (CP) for the treatment of Ebola virus disease (EVD) in the current outbreak, predominantly affecting Guinea, Sierra Leone, and Liberia, was prioritized by the World Health Organization in September 2014. In each of these countries, nonrandomized comparative clinical trials were initiated. The Ebola-Tx trial in Conakry, Guinea, enrolled 102 patients by 7 July 2015; no severe adverse reactions were noted. The Ebola-CP trial in Sierra Leone and the EVD001 trial in Liberia have included few patients. Although no efficacy data are available yet, current field experience supports the safety, acceptability, and feasibility of CP as EVD treatment. Longer-term follow-up as well as data from nontrial settings and evidence on the scalability of the intervention are required. CP sourced from within the outbreak is the most readily available source of anti-EVD antibodies. Until the advent of effective antivirals or monoclonal antibodies, CP merits further evaluation. Abstract Cell culture Buffalo green monkey (BGM) kidney cells, HeLa R19 cells, and rhabdomyosarcoma RD cells were grown at 37°C, 5% CO 2 in DMEM Ready Mix (PAA) containing 10% fetal bovine serum or in DMEM (Lonza) supplemented with 10% fetal bovine serum. Vero, HeLa H and BGM cells used in the multicycle antiviral assays were grown in MEM supplemented with 10% FCS (Integro), 2 mM Lglutamine and 20 mM HEPES. HAP1 cells (Carette et al., 2011) (provided by Thijn Brummelkamp, NKI, Amsterdam, The Netherlands) were grown in IMDM supplemented with 10% fetal bovine serum. Abstract Imatinib mesylate is a targeted therapy that acts by inhibiting tyrosine kinase of the bcr-abl fusion oncoprotein, which is specific to chronic myeloid leukemia (CML), and the c-transmembrane receptor, which is specific to gastrointestinal stromal tumors. Interstitial pneumonitis is a rare adverse event of imatinib therapy. It is clinically difficult to distinguish from infectious pneumonia, which can frequently occur due to the underlying disease. The standard treatment for imatinib-induced pneumonitis is to discontinue the medication and optionally administer corticosteroids. However, there are a few cases of successful retrial with imatinib. We describe a case of successful rechallenge of imatinib in a patient with imatinib-induced interstitial pneumonitis and CML without a recurrence of the underlying disease after 3 months of follow-up. Abstract The present study describes the association between inflammatory cell types and feline infectious peritonitis virus (FIPV) antigen in the brain of 4 cats diagnosed as feline infectious peritonitis (FIP). Immunohistochemically, FIPV antigens were detected in the inflammatory foci of the leptomeninges, choroid plexus and ventricles in 3 of the 4 cats. In 3 cases, inflammatory foci mainly consisted of CD204-and Iba1-positive macrophages, and the FIPV antigens were found in the macrophages. In the other case which was negative for FIPV antigen, severe inflammation predominantly consisting of CD20-positive B lymphocytes was observed in the leptomeninges and subventricles, accompanied with diffuse proliferation of gemistocytic astrocytes. The difference in histopathology may reflect the inflammatory process or the strain variation of FIP virus.Feline infectious peritonitis (FIP) is a widely distributed infectious disease caused by feline coronavirus (FCoV) in domestic and wild cat species [1, 2, 11, 13] . FCoV consists of two biotypes including feline enteric coronavirus (FECV) and FIP virus (FIPV). FECV infection is asymptomatic or exclusively causes mild gastrointestinal clinical symptons, while FIPV causes fatal symptoms such as fever, anorexia, weight loss, abdominal distention and neurological symptoms in young cats [2, 13] . The clinical symptoms differ depending on the form and organ distribution of the lesion, and in cases with lesions in central nervous system, neurological symptoms such as depression, ataxia and seizures appear in 85% of cases [15] . It was assumed that FIPV originating from FECV acquired its monocyte-or macrophage-tropism responsible for viral dissemination and developed specific FIP lesions due to vasculitis [17, 18] .The FIP lesions are characterized by localized or disseminated fibrinous, pyogranulomatous, or granulomatous inflammation with protein-rich effusion in the body cavities [9, 10]. These lesions are often accompanied with granulomatous or necrotizing phlebitis and/or periphlebitis, which have been considered as a typical hallmark of FIP [3, 10] . Lesions in the central nervous system (CNS) are frequently observed mainly in the leptomeninges, ventricles, choroid plexus and sometimes neuroparenchyma [5, 12, 16] . Although histopathological features of FIP lesions in the CNS have been well documented [6, 14, 15] , the association between inflammatory cell types and the distribution of viral antigens remains to be clarified. The present study describes the inflammatory cell types and viral antigen distribution in the brain lesions of 4 cats diagnosed as FIP. Also, considering that the FIPV has monocyte and macrophage tropism, immunohistochemical examinations were performed using two kinds of macrophage markers: CD204 (monocyte and macrophage marker) and Iba-1 (microglia and macrophage marker).Four necropsy cases of cats diagnosed as FIP were obtained from the specimen archives of the Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, the University of Tokyo. Diagnoses of FIP were mainly based on clinical history, physical examination findings, general blood chemistry and antibody titer for FCoV antigen. Final diagnoses were based on gross findings at necropsy and histopathological findings. Information of the 4 cases, including age, sex, major clinical signs, the antibody titer of FCoV and organs with histologically severe inflammatory lesions is summarized in Table 1. Abstract Rationale: Imatinib mesylate (imatinib) is a classic tyrosine kinase inhibitor used to treat chronic myeloid leukemia. Although it is well tolerated by most patients and helps in the achievement of complete remission, a few rare imatinib-associated adverse effects such as pulmonary interstitial fibrosis have been reported. Because of its rareity, the clinical features of imatinib-induced interstitial lung disease (ILD) remain unclear.Patient concerns: A 49-year-old Chinese man with chronic myeloid leukemia received oral treatment with imatinib and initially exhibited a good response. However, he presented with cough and fever 9 months after treatment initiation.Diagnoses: Pulmonary computed tomography indicated diffuse interstitial fibrosis in both lungs. All tests for possible infectious pathologies provided negative results.Interventions: The patient was diagnosed with interstitial pneumonia and treated with antibiotics; however, there was no improvement. On the basis of a suspicion of imatinib-induced ILD, imatinib was discontinued and prednisone treatment was initiated.Outcomes: The patient's symptoms ameliorated with treatment, and imatinib was reintroduced. However, he developed cough and dyspnea again, and his treatment was switched to nilotinib as a second-line regimen. He was regularly monitored, and although his clinical symptoms ameliorated, computed tomography performed 29 months after he was diagnosed with ILD showed irreversible pulmonary interstitial fibrosis without progression.Lessons: Clinicians should consider the possibility of severe irreversible ILD and carefully monitor patients receiving imatinib treatment.Abbreviations: CML = chronic myeloid leukemia, CMV = cytomegalovirus, CT = computed tomography, DLCO = diffusing capacity of the lungs for carbon monoxide, EBV = Epstein-Barr virus, FEV 1 = forced expiratory volume in 1 s, HSV = herpes simplex virus, ILD = interstitial lung disease, IP = interstitial pneumonia, Ph+ = Philadelphia chromosome-positive, RT-PCR = reverse transcription polymerase chain reaction, TKI = tyrosine kinase inhibitor, WBC = white blood cell. Abstract Objectives: Emergency medical personnel (EMPs) are pre-hospital emergency responders who are at risk of exposure to infections and may also serve as a source for the transmission of infections. However, few studies of infection control have specifically addressed EMPs in the Republic of Korea (hereafter Korea). The goal of this study was to assess the current status of infection prevention and control programs (IPCPs) for EMPs in Korea.A cross-sectional survey was conducted to quantitatively assess the resources and activities of IPCPs. A total of 907 EMPs in five metropolitan cities completed a structured questionnaire from September 2014 to January 2015. The data were analyzed using descriptive statistics, multi-response analysis, and the chi-square test.The mean age of the participants was 34.8±15.1 years. IPCPs were found to have weaknesses with regard to the following resources: the assignment of infection control personnel (ICP) (79.5%), hand hygiene resources such as waterless antiseptics (79.3%), the use of paper towels (38.9%), personal protective equipment such as face shields (46.9%), and safety containers for sharps and a separated space for the disposal of infectious waste (10.1%). Likewise, the following activities were found to be inadequately incorporated into the workflow of EMPs: education about infection control (77.5%), post-exposure management (35.9%), and the decontamination of items and spaces after use (88.4%). ICP were found to have a significant effect on the resources and activities of IPCPs (p<0.001). The resources and activities of IPCPs were found to be significantly different among the five cities (p<0.001).IPCPs for EMPs showed some limitations in their resources and activities. IPCPs should be actively supported, and specific IPCP activities for EMPs should be developed. Abstract Middle East Respiratory Syndrome coronavirus (MERS-CoV) was first isolated from a patient with severe pneumonia in 2012. The 2015 Korea outbreak of MERS-CoV involved 186 cases, including 38 fatalities. A total of 83% of transmission events were due to five superspreaders, and 44% of the 186 MERS cases were the patients who had been exposed in nosocomial transmission at 16 hospitals. The epidemic lasted for 2 months and the government quarantined 16,993 individuals for 14 days to control the outbreak. This outbreak provides a unique opportunity to fill the gap in our knowledge of MERS-CoV infection. Therefore, in this paper, we review the literature on epidemiology, virology, clinical features, and prevention of MERS-CoV, which were acquired from the 2015 Korea outbreak of MERS-CoV. Abstract Aspirin is the oldest non-steroidal anti-inflammatory drug (NSAID), and it sometimes causes asthma-like symptoms known as aspirin-exacerbated respiratory disease (AERD), which can be serious. Unwanted effects of aspirin (aspirin intolerance) are also observed in patients with food-dependent exercise-induced anaphylaxis, a type I allergy disease, and aspirin-induced urticaria (AIU). However the target and the mechanism of the aspirin intolerance are still unknown. There is no animal or cellular model of AERD, because its pathophysiological mechanism is still unknown, but it is thought that inhibition of cyclooxygenase by causative agents leads to an increase of free arachidonic acid, which is metabolized into cysteinyl leukotrienes (cysLTs) that provoke airway smooth muscle constriction and asthma symptoms. As the bed-tobench approach, to confirm the clinical discussion in experimental cellular models, we have tried to develop a cellular model of AERD using activated RBL-2H3 cells, a rat mast cell like cell line. Indomethacin (another NSAID and also causes AERD), enhances in vitro cysLTs production by RBL-2H3 cells, while there is no induction of cysLTs production in the absence of inflammatory activation. Since this suggests that all inflammatory cells with activation of prostaglandin and cysLT metabolism should respond to NSAIDs, and then I have concluded that aspirin intolerance should be separated from subsequent bronchoconstriction. Evidence about the cellular mechanisms of NSAIDs may be employed for development of in vitro AERD models as the approach from bench-to-bed. Abstract We estimated the impact on the US export economy of an illustrative infectious disease outbreak scenario in Southeast Asia that has 3 stages starting in 1 country and, if uncontained, spreads to 9 countries. We used 2014-2016 West Africa Ebola epidemic-related World Bank estimates of 3.3% and 16.1% reductions in gross domestic product (GDP). We also used US Department of Commerce job data to calculate export-related jobs at risk to any outbreak-related disruption in US exports. Assuming a direct correlation between GDP reductions and reduced demand for US exports, we estimated that the illustrative outbreak would cost from approximately $13 million to approximately $64 million (1 country) to $8 billion to $41 billion (9 countries) and place 1,500 to almost 1.4 million export-related US jobs at risk. Our analysis illustrates how global health security is enhanced, and the US economy is protected, when public health threats are rapidly detected and contained at their source. Abstract To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 × 10 9 and 0.86 × 10 9 , respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 × 10 4 IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats. Abstract The Ebola virus disease outbreak in West Africa and the Middle East Respiratory Syndrome outbreak in the Republic of Korea have given huge impacts in different aspects. Health security is no more a new coinage. Global health security became more realistic in its practical application. In the perspective of global health, it will be helpful to peruse lessons learned from the Ebola outbreak in West Africa and MERS outbreak in Korea.Global Health Security S27 Abstract Dengue virus is a major human pathogen responsible for 400 million infections yearly. As with other RNA viruses, daunting challenges to antiviral design exist due to the high error rates of RNA-dependent RNA synthesis. Indeed, treatment of dengue virus infection with a nucleoside analog resulted in the expected genetic selection of resistant viruses in tissue culture and in mice. However, when the function of the oligomeric core protein was inhibited, no detectable selection of drug resistance in tissue culture or in mice was detected, despite the presence of drug-resistant variants in the population. Suppressed selection of drug-resistant virus correlated with cooligomerization of the targeted drug-susceptible and drug-resistant core proteins. The concept of "dominant drug targets," in which inhibition of oligomeric viral assemblages leads to the formation of drugsusceptible chimeras, can therefore be used to prevent the outgrowth of drug resistance during dengue virus infection. IMPORTANCE Drug resistance is a major hurdle in the development of effective antivirals, especially those directed at RNA viruses. We have found that one can use the concept of the genetic dominance of defective subunits to "turn cousins into enemies," i.e., to thwart the outgrowth of drug-resistant viral genomes as soon as they are generated. This requires deliberate targeting of larger assemblages, which would otherwise rarely be considered by antiviral researchers.Citation Mateo R, Nagamine CM, Kirkegaard K. 2015. Suppression of drug resistance in dengue virus. mBio 6(6):e01960-15. Abstract Feline infectious peritonitis virus (FIPV) causes a severe, immune-mediated disease called FIP in domestic and wild cats. It is unclear whether FIP transmits from cat to cat through the oral route of FIPV infection, and the reason for this includes that FIP is caused by oral inoculation with some FIPV strains (e.g., type II FIPV WSU 79-1146), but is not caused by other FIPV (e.g., type I FIPV KU-2 strain: FIPV-I KU-2). In this study, when cats passively immunized with anti-FIPV-I KU-2 antibodies were orally inoculated with FIPV-I KU-2, FIP was caused at a 50% probability, i.e., FIPV not causing FIP through oral infection caused FIP by inducing antibody-dependent enhancement. Many strains of type I FIPV do not cause FIP by inoculation through the oral route in cats. Based on the findings of this study, type I FIPV which orally infected cats may cause FIP depending on the condition. Abstract Since the first imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection was reported on May 20, 2015 in Korea, there have been 186 laboratoryconfirmed cases of MERS-CoV infection with 36 fatalities. Ninety-seven percent (181/186) of the cases had exposure to the health care facilities. We are reporting a superspreading event that transmitted MERS-CoV to 81 persons at a hospital emergency room (ER) during the Korean outbreak in 2015. The index case was a 35-yr-old man who had vigorous coughing while staying at the ER for 58 hr. As in severe acute respiratory syndrome outbreaks, superspreading events can cause a large outbreak of MERS in healthcare facilities with severe consequences. All healthcare facilities should establish and implement infection prevention and control measure as well as triage policies and procedures for early detection and isolation of suspected MERS-CoV cases. Abstract Citation Furmanski M. 2015. The 1977 H1N1 influenza virus reemergence demonstrated gain-of-function hazards. mBio 6(5):e01434-15. Abstract Background. Chronic lung allograft dysfunction (CLAD) is the major limiting factor for long-term survival in lung transplant recipients. Viral respiratory tract infection (VRTI) has been previously associated with CLAD development. The main purpose of this study was to evaluate the long-term effects of VRTI during the first year after lung transplantation in relation to CLAD development. Method. Ninety-eight patients undergoing lung transplantation were prospectively enrolled between 2009 and 2012. They were monitored for infections with predefined intervals and on extra visits during the first year, the total follow-up period ranged between 5 and 8 years. Nasopharyngeal swab and bronchoalveolar lavage samples were analyzed using a multiplex polymerase chain reaction panel for respiratory pathogens. Data regarding clinical characteristics and infectious events were recorded. Results. Viral respiratory tract infection during the first year was identified as a risk factor for long-term CLAD development (P = 0.041, hazard ratio 1.94 [1.03-3.66]) in a time-dependent multivariate Cox regression analysis. We also found that coronavirus in particular was associated with increased risk for CLAD development. Other identified risk factors were acute rejection and cyclosporine treatment. Conclusions. This study suggests that VRTI during the first year after lung transplantation is associated with longterm CLAD development and that coronavirus infections in particular might be a risk factor. Abstract Introduction: The aim of the study was to demonstrate interleukin-18 (IL-18) expression in keratinocytes from psoriatic lesions in comparison to keratinocytes from uninvolved skin and to study the change of expression after therapeutic interventions. Material and methods: This study included 16 patients of different clinical subtypes of psoriasis. Interleukin-18 gene expression analysis was performed using real time quantitative PCR. Three biopsies were obtained from each patient. Two were taken from the lesional psoriatic skin and from uninvolved skin before starting treatment. A third lesional skin biopsy was taken at the end of 2 months of treatment. The treatment was in the form of topical steroids or oral systemic methotrexate. Results: Of all 16 studied patients, significantly increased IL-18 expression was noted in keratinocytes from psoriatic lesions before and after treatment when compared to keratinocytes from uninvolved skin (p = 0.001 and p = 0.002 respectively). The IL-18 expression in the skin lesions after treatment was significantly lower than lesional skin before treatment (p = 0.023). In psoriatic skin lesions of all studied patients IL-18 expression was significantly correlated with disease duration (r = 0.40 and p = 0.01) and clinical severity of psoriasis (r = 0.72 and p = 0.001). Conclusions: Increased IL-18 expression in keratinocytes from psoriatic lesions of our patients and its correlation with disease duration and severity supported the concept of psoriasis as a T cell mediated autoimmune disease. This could establish therapeutic and preventive approaches for psoriasis that ultimately lead to improved outcomes for patients. Abstract Emerging infectious diseases continue to impose unpredictable burdens on global health and economy. Infectious disease surveillance and pandemic preparedness are essential to mitigate the impact of future threats. Global surveillance networks provide unprecedented monitoring data on plant, animal and human infectious diseases. Using such sources, we report on current major One Health threats and update on their epidemiological status. Abstract One of the important fields to apply computational tools for domain boundaries prediction is structural biology. They can be used to design protein constructs that must be expressed in a stable and functional form and must produce diffraction-quality crystals. However, prediction of protein domain boundaries on the basis of amino acid sequences is still very problematical. In present study the performance of several computational approaches are compared. It is observed that the statistical significance of most of the predictions is rather poor. Nevertheless, when the right number of domains is correctly predicted, domain boundaries are predicted within very few residues from their real location. It can be concluded that prediction methods cannot be used yet as routine tools in structural biology, though some of them are rather promising.NNput Abstract We sought to examine the relationship between the clinical manifestations of nonspecific reactive hepatitis and respiratory virus infection in pediatric patients. Methods: Patients admitted to the pediatric unit of Konyang University Hospital for lower respiratory tract disease between January 1, 2014 and December 31, 2014 and who underwent reverse transcriptase polymerase chain reaction tests were examined. The patients were divided into those with increased levels of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) and those with normal ALT or AST levels. Further, patients with increased ALT and AST levels were individually compared with patients in the normal group, and the blood test results were compared according to the type of respiratory virus. Results: Patients with increased ALT or AST levels had one more day of hospital stay, on average, compared with patients in the normal group (5.3±3.1 days vs. 4.4±3.0 days, p=0.019). Patients in the increased ALT level group were younger and had a longer mean hospital stay, compared with patients in the normal group (p=0.022 and 0.003, respectively). The incidences of increased ALT or AST were the highest in adenovirus infections (6/24, 25.0%), followed by enterovirus (2/11, 18.2%) and respiratory syncytial virus A (21/131, 16.0%) infections. Conclusion: Nonspecific reactive hepatitis is more common among patients with adenovirus, enterovirus and respiratory syncytial virus infection, as well as among those infected at a younger age. Compared with AST levels, ALT levels are better indicators of the severity of nonspecific reactive hepatitis. Abstract Emerging infectious diseases (EID) threaten public health and are sustained by increasing global commerce, travel and disruption of ecological systems. Travelers could play a role in importing EIDs and could be a sentinel of major epidemics. In connection with the extension of poverty, urbanization, extensive livestock rearing and globalization, we could be exposed to a third epidemiological transition characterized by zoonotic diseases and infections with multidrugresistant bacteria. The risk appears low for emerging infectious diseases, or very low for high-risk emerging infectious diseases, but higher for multidrug-resistant enterobacteriaceae carriage with possibly limited consequences. The role played by migrants is weaker than imagined. Immigrants don't play the role of sentinel epidemic so far. They could play a role in importing multidrug-resistant enterobacteriaceae, but it is poorly evaluated. Abstract This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Despite asthma being the most common chronic childhood ailment, there is still much to learn about the disease. Early childhood infections with well-known or emerging viruses can lay the pathophysiologic framework for asthma development and exacerbation later in life, which may be due partly to alteration of the airway microbiome. Once asthma is established, acute exacerbations are usually associated with infections with respiratory viruses, such as rhinoviruses (RVs). Once again, there are bidirectional interactions between viruses and airway bacteria that appear to influence the severity of illness and the likelihood of exacerbation. Studies employing recent advances in viral and bacterial identification analytic techniques will clarify these new concepts and may provide the basis for new treatments or prevention or respiratory infection-associated exacerbation. This paper is a review of the associations among respiratory viruses, bacteria, inflammatory mechanisms, and asthma exacerbation. Abstract In this study, we describe the detection of novel paramyxoviruses from the Eidolon helvum species of fruit bats. We extracted RNA from 312 spleen samples from bats captured in Zambia over a period of 4 years (2008)(2009)(2010)(2011). Semi-nested RT-PCR detected a total of 25 (8%) positive samples for paramyxoviruses which were then directly sequenced and analyzed using phylogenetic analysis. Among the positive samples, seven novel paramyxoviruses were detected. Five viruses were closely related to the genus Henipavirus, while two viruses were related to the unclassified Bat paramyxoviruses from Ghana and Congo Brazzaville. Our study identified novel Henipavirus-related and unrelated viruses using RT-PCR in fruit bats from Kansaka National Park and indicated the presence of similar Bat paramyxoviruses originating from wide geographic areas, suggesting the ability of bats to harbor and transmit viruses. The presence of these viruses in fruit bats might pose a public health risk. Abstract Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis-and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends on cis cleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses. Citation Romero-Brey I, Berger C, Kallis S, Kolovou A, Paul D, Lohmann V, Bartenschlager R. 2015. NS5A domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis C virus replication. mBio 6(4):e00759-15. Abstract A serological survey of Middle East respiratory syndrome coronavirus (MERS-CoV) was conducted among dromedary camels and herbivorous animals sharing the same pasturage in Ethiopia. The pseudotyped vesicular stomatitis virus coated with the spike protein of MERS-CoV was used in virus neutralization (VN) tests performed in a biosafety level (BSL)-2 laboratory. The results were similar to those obtained from the VN test using live MERS-CoV and were more sensitive than the ELISA performed using synthetic MERS S1 fragment as the antigen as well as the competitive ELISA performed using a monoclonal antibody against MERS-CoV. According to the comprehensive results of the four types of serodiagnosis methods, positive antibodies were detected only in dromedary camels and the remaining herbivorous animals were not infected with the virus. Moreover, using the present procedure, serological tests for MERS-CoV can be conducted even in BSL 2 laboratory. Abstract The Golgi complex is composed of at least four distinct compartments, termed the cis-, medial, and trans-Golgi cisternae and the trans-Golgi network Abstract Objective: This study investigated the sources of stress, corresponding symptoms, and stress relief among nurses of the first Chinese anti-Ebola medical team during the Sierra Leone aid mission. Method: A purposive sampling method was used and 10 nurses were selected from the first Chinese anti-Ebola medical team that was dispatched to Sierra Leone. Data were collected via phone and semistructured interviews, then analyzed using Colaizzi's seven-step method. Results: The data showed three major themes: (1) The causes of stress during the Sierra Leone aid mission mainly related to unsafety, responsibility, and unfamiliarity; (2) Physical, cognitive, emotional, and behavioral symptoms were documented; (3) Nurses experienced relief from stress after the mission. Conclusion: Targeted measures, proper responses and good community support can effectively lower stress among nurses on anti-Ebola missions. Abstract Numerous viral infections have arisen and affected global healthcare facilities. Millions of people are at severe risk of acquiring several evolving viral infections through several factors. In the present article we have described about risk factors, chance of infection, and prevention methods of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV), human coronaviruses (CoVs) frequently cause a normal cold which is mild and self-restricting. Zoonotic transmission of CoVs such as the newly discovered MERS-CoV and SARS-CoV, may be associated with severe lower respiratory tract infection. The present review provides the recent clinical and pathological information on MERS and SARS. The task is to transform these discoveries about MERS and SARS pathogenesis and to develop intervention methods that will eventually allow the effective control of these recently arising severe viral infections. Global health sector has learnt many lessons through the recent outbreak of MERS and SARS, but the need for identifying new antiviral treatment was not learned. In the present article we have reviewed the literature on the several facets like transmission, precautions and effectiveness of treatments used in patients with MERS-CoV and SARS infections. Abstract Background: Bacterial pneumonia (BP) is an inflammation of the lower airways and lung parenchyma secondary to bacterial infection. The pathogenesis of BP in dogs is complex and the role of canine respiratory viruses has not been fully evaluated.Objectives: The aim of this study was to investigate the occurrence of viral co-infections in dogs with BP and to assess demographic or clinical variables as well as disease severity associated with viral co-infections.Animals: Twenty household dogs with BP caused by opportunistic bacteria and 13 dogs with chronic (>30 days) tracheobronchitis caused by Bordetella bronchiseptica (BBTB).Methods: Prospective cross-sectional observational study. Diagnosis was confirmed by clinical and laboratory findings, diagnostic imaging, and cytologic and microbiologic analysis of bronchoalveolar lavage or transtracheal wash fluid. Canine parainfluenza virus (CPIV), canine adenovirus, canine herpes virus, canine influenzavirus, canine distemper virus, canine respiratory coronavirus (CRCoV) and canine pneumovirus, as well as B. bronchiseptica and Mycoplasma spp. were analyzed in respiratory samples using PCR assays.Results: CPIV was detected in 7/20 and CRCoV in 1/20 dogs with BP. Respiratory viruses were not detected in dogs with BBTB. There were no significant differences in clinical variables between BP dogs with and without a viral co-infection.Conclusion and Clinical Importance: Respiratory viruses were found frequently in dogs with BP and may therefore play an important role in the etiology and pathogenesis of BP. Clinical variables and disease severity did not differ between BP dogs with and without viral co-infection. Abstract Citation Pfeiffer JK. 2015. Is the debate and "pause" on experiments that alter pathogens with pandemic potential influencing future plans of graduate students and postdoctoral fellows? mBio 6(1):e02525-14. Abstract Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSVhost cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Addition-ally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions. Abstract Hand, foot and mouth disease (HFMD) is an important public health problem that has emerged over the past several years. HFMD predominantly infects children under seven years old and occasionally causes severe disease in adults. Among the enteroviruses, enterovirus 71 (EV71) and coxsackievirus 16 (CA16) are the major causative agents of HFMD. In addition, adenovirus cocirculates with enterovirus and has become a possible additional pathogenic factor for HFMD in some cases. Here, we have investigated the neutralizing antibody responses to both enterovirus and adenovirus in adults, with the aim of exploring the prevalence trends of these viruses and the nature of protective immunity in humans to these viral infections. Sera from 391 healthy adults from 21 provinces and cities in China were tested for the presence of antibodies against EV71, CA16, adenovirus human serotype 5 (AdHu5) and chimpanzee adenovirus pan7 (AdC7) using neutralization tests. High seroprevalence rates of EV71, CA16 and AdHu5 were found in the population (85.7%, 58.8% and 74.2%, respectively). The coseropositivity rate of these three viruses was 39.4% (154 of 391), with median neutralizing antibody titers of 80, 40 and 640, respectively, and the neutralizing antibody titer for EV71 was found to be correlated with those of CA16 and AdHu5. AdC7 was found to be a rare adenovirus serotype in the human population, with a seropositivity rate of 11.8%, suggesting that it could be a good choice for a vaccine carrier that could be used in vaccine development. Abstract Zika virus (ZIKV) has become a global public health emergency due to its rapidly expanding range and its ability to cause severe congenital defects such as microcephaly. However, there are no FDA-approved therapies or vaccines against ZIKV infection. Through our screening of viral entry inhibitors, we found that chloroquine (CQ), a commonly used antimalarial and a FDA-approved drug that has also been repurposed against other pathogens, could significantly inhibit ZIKV infection in vitro, by blocking virus internalization. We also demonstrated that CQ attenuates ZIKV-associated morbidity and mortality in mice. Finally, we proved that CQ protects fetal mice from microcephaly caused by ZIKV infection. Our methodology of focusing on previously identified antivirals in screens for effectiveness against ZIKV proved to be a rapid and efficient means of discovering new ZIKV therapeutics. Selecting drugs that were previously FDA-approved, such as CQ, also improves the likelihood that they may more quickly reach stages of clinical testing and use by the public. Abstract Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. Abstract Multiple choice question Objective structured clinical examination Postgraduate training Standardized patient a b s t r a c t Background: Postgraduate year training programs play an important role in the development of a comprehensive medical education. The goal of these training programs is to inculcate in physicians the expected level of skill in patient care. After the initiation of such programs in the USA, Europe, and Japan, studies were conducted in Taiwan to investigate relevant training methods, and a training system was established in 2003. Beginning with 3-month programs, followed by 6-month programs, the programs were constantly modified and enhanced by the establishment of the 1-year training program in 2011. This year was the transition period from the 6-month programs to the 1-year programs. Methods: We used a 50-item multiple choice question (MCQ) test and six 10-min stations for objective structured clinical examination (OSCE), which was composed of four stations relating to standardized patients and two stations concerning the clinical skill evaluation, to evaluate the learning results of the trainees. The trainees were divided into four groups according to the training program. Results: There was no significant difference between the performance of the 6 months and 1-year groups. The p values were 0.424 in the MCQ test and 0.082 in the OSCE evaluation. Conclusion: A well-designed postgraduate training program should develop trainees' competencies. The results of this study may provide useful insight for ways to improve the design of training programs. Further investigation to better understand the impact of different programs is warranted. Abstract Epidemiology and virologyEvaluation of combined interventions to increase HIV diagnosis among men who have sex with men (MSM) in Curitiba, Brazil Introduction: Prior studies in Brazil have shown that the group of men who have sex with men (MSM) is the most severely affected by HIV. In 2015, a multi-disciplinary team launched a community-wide intervention in the city of Curitiba to evaluate the acceptability, uptake and cost-effectiveness of multiple HIV testing services (HTS). The current study aims to evaluate HIV testing coverage among MSM after 6 months of intervention.Methods: A probabilistic sample of 3,272 men was selected in Curitiba. The sample was stratified by place of residence with higher selection probabilities in areas with greater MSM concentration. Questions about history of HIV testing, frequency of testing in the last 12 months, place and result of last test, as well as knowledge and acceptability of expanding HTS were made.Results: The proportion of MSM was 4.7%. In the total sample, HIV testing coverage at least once in lifetime was 46.0%; 22.9% in the last 6 months; and 15.6% between 7 and 12 months prior to the interview. Among MSM, the proportions were much higher: 79%; 43.1%; and 19.3% respectively. Approximately one third of MSM had already had an oral fluid test at 4 times higher proportion greater than the total sample (8.1%). However, most MSM (41.3%) tested for the last time in primary care services; 40.4% in private services; and only 8.3% used the other testing services provided by the project. The HIV prevalence among men who tested at least once in lifetime was 0.6% and among MSM was 3.8%, with a prevalence ratio greater than 6.The results indicate that the intervention to increase HIV diagnosis has substantially expanded MSM access to HIV testing after only 6 months of implementation. The target of testing 90% MSM in Curitiba is likely to be exceeded in less than 2 years. Follow-up of MSM testing via the web platform, and other service evaluations will inform program improvements for Curitiba and will facilitate expansion to larger Brazilian cities.Introduction: Though the percentage of HIV positive MSM is low among the registered cases in Armenia, taking into account the behavioral characteristics of MSM, the prevention of HIV among this group of population is a priority in Armenia.Prevention programs for MSM are delivered in four cities in Armenia, and MSM receive the full package of services: information on HIV, provision of condoms and lubricants, counseling and testing on HIV.Aim: To identify key behavioral characteristics due to which MSM acquire HIV, the level of knowledge on HIV and the prevalence of HIV.Methods: The study was done using RDS method in 2012 and 2014 among MSM in the capitol of Armenia-Yerevan. Behavioral and biological surveillance was conducted. 300 MSM answered to the questionnaire each year and the blood test on HIV antibodies was done. The knowledge was measured by the answers to the five key questions according to the WHO guidelines. Data were analyzed by RDSAT method. The study was conducted by National Center for AIDS Prevention according to the national guideline approved by the MoH of Armenia in 2010.: The prevalence of HIV among MSM was 2.6% in 2012 and 0.4% in 2014. Knowledge on HIV was 54.3% in 2012 and 78.9% in 2014. Indicator of condom use in last anal sex was 71.6% in 2012 and 65.3% in 2014. The percentage of MSM used condoms in the last sex with casual partners was 73.1% in 2012 and 87.4% in 2014. The percentage of MSM used condoms in the last one year was 36.7% in 2012 and 35.4% in 2014. Conclusion: The prevalence of HIV decreased in 2012-2014, there is an increase in knowledge on HIV, MSM still practice risky behavior.There is a need to continue the prevention programs focusing on transforming safe sexual behavior, increasing their self-esteem, helping them to change their attitude towards their health and making healthy, non-stigmatized environment, where MSM will feel free to express their problems and receive adequate solutions to them. Abstract has claimed more than 2600 lives as of 24 February 2020 and posed a huge threat to global public health. The Chinese government has implemented control measures including setting up special hospitals and travel restriction to mitigate the spread. We propose conceptual models for the COVID-19 outbreak in Wuhan with the consideration of individual behavioural reaction and governmental actions, e.g., holiday extension, travel restriction, hospitalisation and quarantine. We employe the estimates of these two key components from the 1918 influenza pandemic in London, United Kingdom, incorporated zoonotic introductions and the emigration, and then compute future trends and the reporting ratio. The model is concise in structure, and it successfully captures the course of the COVID-19 outbreak, and thus sheds light on understanding the trends of the outbreak. Abstract Background: Due to its great sensitivity, the nucleic acid amplification test (NAAT) is widely used for detection of respiratory viruses (RV). However, few reports have described a direct comparison between multiplex RT-PCR assays for RV. The objective of this study was to perform a direct comparison of three multiplex RT-PCR assays for the detection of respiratory viruses.A total of 201 respiratory samples (161 nasopharyngeal swab samples and 40 sputum samples) were tested with three commercial RV assays: Seegene Anyplex II RV16 (AP), LG AdvanSure RV (AD), and Biosewoom Real-Q RV (RQ). The additional tests for the discrepant results were conducted by repeat RV assay or monoplex PCR coupled direct sequencing. Data analysis using percent agreement, kappa, and prevalence-adjusted and bias-adjusted kappa (PABAK) values was performed for comparisons among the three RV assays. Results: Of the 201 samples, AP, AD, and RQ detected 105 (52.2%), 99 (49.3%), and 95 (47.3%) positive cases respectively. The overall agreement, kappa, and PABAK values for the three assays ranged between 97%-98%, 0.76-0.86, and 0.93-0.96 respectively. The performance of the three assays was very similar, with 94%-100% agreement for all comparisons, each virus types. The additional testing of samplesshowed discrepant results demonstrating that AD assay had the highest rate of concordance with original results.We suggest that all multiplex assay would be suitable for the detection of for respiratory viruses in clinical setting.agreement, nucleic acid amplification test, respiratory virus Abstract We describe futures of ICU admission, demographic characteristics, treatment and outcome for critically ill patients with laboratory-confirmed and suspected infection with the H1N1 virus admitted to the three different critical care departments in Turkey. Methods: Retrospective study of critically ill patients with 2009 influenza A(H1N1) at ICU. Demographic data, symptoms, comorbid conditions, and clinical outcomes were collected using a case report form. Results: Critical illness occurred in 61 patients admitted to an ICU with confirmed (n=45) or probable and suspected 2009 influenza A(H1N1). Patients were young (mean, 41.5 years), were female (54%). Fifty-six patients, required mechanical ventilation (14 invasive, 27 noninvasive, 15 both) during the course of ICU. On admission, mean APACHE II score was 18.7±6.3 and median PaO 2 /FIO 2 was 127.9±70.4. 31 patients (50.8%) was die. There were no significant differences in baseline PaO 2 /FIO 2 and ventilation strategies between survivors and nonsurvivors. Patients who survived were more likely to have NIMV use at the time of admission to the ICU. Conclusion: Critical illness from 2009 influenza A(H1N1) in ICU predominantly affects young patients with little major comorbidity and had a high case-fatality rate. NIMV could be used in 2009 influenza A (H1N1) infection-related hypoxemic respiratory failure. Abstract The first membrane-spanning domain (ml) of the M glycoprotein of avian coronavirus (formerly called El) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with ml, the resulting chimera (Gml) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gml formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDSpolyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gml mutants with single amino acid substitutions in the ml domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gml, but occurred gradually with a lag of ,',,10 rain, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomer-1. Abbreviations used in this paper: CCCP, carbonyl cyanide m-cldorophenylhydrazone; hCG, human chorionic gonadotropin; IBV, infectious bronchitis virus; VSV, vesicular stomatitis virus. Abstract Clinical application of siRNA-based therapeutics outside of the liver has been hindered by the inefficient delivery of siRNA effector molecules into extra-hepatic organs and cells of interest. To understand the parameters that enable RNAi activity in vivo, it is necessary to develop a systematic approach to identify which cells within a tissue are permissive to oligonucleotide internalization and activity. In the present study, we evaluate the distribution and activity within the lung of chemically stabilized siRNA to characterize cell-type tropism and structure-activity relationship. We demonstrate intratracheal delivery of fully modified siRNA for RNAi-mediated target knockdown in lung CD11c + cells (dendritic cells, alveolar macrophages) and alveolar epithelial cells. Finally, we use an allergen-induced model of lung inflammation to demonstrate the capacity of inhaled siRNA to induce target knockdown in dendritic cells and ameliorate lung pathology. Abstract Aims: To better inform medical practitioners on the role of antiseptics in oropharyngeal health and disease, this article focuses on povidone-iodine (PVP-I), an established and widely-available antiseptic agent. Methodology: Review of the anti-infective profile, efficacy and safety of PVP-I in managing common upper respiratory tract infections such as the common cold, influenza and tonsillo-pharyngitis, as well as oral complications resulting from cancer treatment (oral mucositis), and dental conditions (periodontitis, caries). Results: Antiseptics with broad-spectrum anti-infective activity and low resistance potential offer an attractive option in both infection control and prevention. While there is some evidence of benefit of antiseptics in a variety of clinical settings that include dental and oral hygiene, dermatology, oncology, and pulmonology, there appears to be discordance between the evidence-base and practice. This is especially apparent in the management and prevention of oropharyngeal infections, for which the use of antiseptics varies considerably between clinical practices, and is in marked contrast to their dermal application, where they are extensively used as both a prophylaxis and a treatment of skin and wound infections, thus minimising the use of antibiotics. Conclusion: The link between oral and oropharyngeal health status and susceptibility to infection has long been recognised. The high rates of antibiotic misuse and subsequent development of bacterial resistance (e.g. increasing vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA)) in large parts of the world, especially across Asia Pacific, highlight the need for identifying alternative antimicrobials that would minimise the use of these medications. This, together with recent large-scale outbreaks of, for example, avian and swine influenza virus, further underline the importance of an increasing armamentarium for infection prevention and control.Data were collected through PubMed using specified search criteria based on efficacy, safety and microbicidal activity of PVP-I. Other health-based search engines that included the Cochrane Library were also used to search for reviews on antiseptic use in defined clinical settings. General searches using Google for non-English articles on PVP-I were also conducted. Expert opinion was sought in areas of limited published information such as optimal duration of PVP-I treatment.While the current evidence-base for antiseptic use in the oropharyngeal setting per se is limited compared with other settings, studies of PVP-I demonstrated effectiveness against a broad spectrum of common mouth and throat pathogens and minimised the risk of upper respiratory tract infections, certain dental conditions, and severity of cancer therapy-associated oral complications. Use of PVP-I may thereby assist in the rationalisation of antibiotic prescription.ª Abstract Cloning and Site-Directed Mutagenesis. The expression vector pET11d/FMDV (1) was used as a template for all site-directed Lb pro mutants (amino acids . Human ISG15 (Cys78Ser) and Met1 diubiquitin constructs were cloned into the pOPIN-B His-tagged expression vector (2). A codon-optimized ISG15 was obtained from GeneArt Gene Synthesis (Invitrogen) and cloned in frame to the intein/chitin binding domain pTXB1 vector (New England BioLabs) using conventional cloning methods. Site-directed mutagenesis was performed according to the QuikChange method. Plasmids for cell transfection assays (FLAG-ISG15, UBE1L, UBE2L6, and HERC5 of human origin) were cloned into the pcDNA3.1 mammalian expression vector.Protein Expression and Purification. Lb pro was expressed and purified according to ref. 3. His-tagged ISG15 and Met1 diubiquitin were expressed in Escherichia coli Rosetta2 (DE3) pLacI (Novagen). Cultures were grown to OD 600 (0.6-0.8) and induced with 0.2 mM Isopropyl β-D-1-thiogalactopyranoside for 16 h at 18°C. Cell pellets were resuspended in 30 mL of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM β-mercaptoethanol) and frozen at −80°C. Thawed pellets were supplemented with an EDTA-free Complete Protease Inhibitor tablet (Roche), 1 mg/mL lysozyme, and 0.1 mg/mL DNaseI and incubated on ice for 10 min. The cell suspension was sonicated, and cell debris was cleared by centrifugation at 50,000 × g for 30 min. The cleared lysate was incubated with TALON Superflow resin (GE Healthcare) for 10 min and washed with 1 L of lysis buffer by gravity flow. Protein was eluted with lysis buffer supplemented with 250 mM imidazole. Purified proteins were dialyzed for 16 h into lysis buffer to remove excess imidazole. ISG15-intein protein was expressed identically to His-tagged proteins but purified with chitin resin (New England BioLabs) as described previously (4, 5).AMC Fluorescence Measurements. ISG15-AMC cleavage assays were performed as described previously (6) with minor modifications. In each reaction, 10 μL of 2 μM ISG15-AMC (2× concentration) was mixed with 10 μL enzyme (2× concentration) in AMC assay buffer (50 mM Tris, pH 8.0, 10 mM DTT). The reactions were monitored using λ ex = 350 nm and λ em = 450 nm.Fluorescence Polarization Assays. All ubiquitin/ubiquitin-like TAMRA assays were performed according to ref. 7. Briefly, enzymes and substrates were diluted in dilution buffer (50 mM Tris, pH 8.0, 10 mM DTT). Substrate concentrations remained constant at 150 nM, while enzyme concentrations varied based on cleavage efficiency. Cleavage assays were performed in a black 384-well plate (Corning), and fluorescence polarization (FP) measurements were made with a 550-nm excitation filter and a 590-nm emission filter using a Pherastar plate reader (BMG Labtech). All measurements were compared with substrate only (negative) and 25 nM KG-TAMRA peptide (positive) reference standards.Kinetics. ISG15-TAMRA was used as a substrate for determination of Lb pro Michaelis-Menten kinetics. The change in FP on Lb pro hydrolysis was measured in a black 384-well plate using a PheraStar plate reader (BMG Labtech): λ ex = 550 nm and λ em = 590 nm. Reactions were incubated at 25°C in a total volume of 20 μL in FP kinetics buffer (25 mM Tris, pH 8.5, 2 mM DTT, 0.1 mg/mL BSA). In each well, 10 μL ISG15-TAMRA (2× final concentration) was mixed with 10 μL of 40 nM enzyme (2× final concentration). The ISG15-TAMRA concentration was variedSwatek et al. www.pnas.org/cgi/content/short/1710617115 Abstract Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5 0 -untranslated region (5 0 -UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5 0 -UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem-loop I of IBV 5 0 -UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBVinfected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis. Abstract b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 790-794 h t t p : / / w w w . b j m i c r o b i o l . c o m . b r / Distemper Parvovirus Diarrhea Dog Co-infection a b s t r a c t Although the use of vaccines has controlled enteric diseases in dogs in many developed countries, vaccine coverage is still under optimal situation in Brazil. There is a large population of nonimmunized dogs and few studies about the identification of the viruses associated with diarrhea. To address this situation, stool samples from 325 dogs were analyzed by polymerase chain reaction for the detection of common enteric viruses such as Canine adenovirus (CAdV), Canine coronavirus (CCoV), Canine distemper virus (CDV), Canine rotavirus (CRV)and Carnivorous protoparvovirus 1 (canine parvovirus 2; CPV-2). At least one of these species was detected in 56.6% (184/325) of the samples. The viruses detected most frequently in either diarrheic or nondiarrheic dog feces were CPV-2 (54.3% of the positive samples), CDV (45.1%) and CCoV (30.4%), followed by CRV (8.2%) and CAdV (4.9%). Only one agent was detected in the majority of the positive samples (63%), but co-infections were present in 37% of the positive samples and mainly included CDV and CPV-2. The data presented herein can improve the clinical knowledge in regions with low vaccine coverage and highlight the need to improve the methods used to control these infectious diseases in domestic dogs. Abstract Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe pulmonary infection, with ∼35 % mortality. Spike glycoprotein (S) of MERS-CoV is a key target for vaccines and therapeutics because S mediates viral entry and membrane-fusion to host cells. Here, four different S subunit proteins, receptor-binding domain (RBD; 358-606 aa), S1 (1-751 aa), S2 (752-1296 aa), and SΔTM (1-1296 aa), were generated using the baculoviral system and immunized in mice to develop neutralizing antibodies. We developed 77 hybridomas and selected five neutralizing mAbs by immunization with SΔTM against MERS-CoV EMC/2012 strain S-pseudotyped lentivirus. However, all five monoclonal antibodies (mAb) did not neutralize the pseudotyped V534A mutation. Additionally, one mAb RBD-14F8 did not show neutralizing activity against pseudoviruses with amino acid substitution of L506 F or D509 G (England1 strain, EMC/2012 L506 F, and EMC/2012 D509 G), and RBD-43E4 mAb could not neutralize the pseudotyped I529 T mutation, while three other neutralizing mAbs showed broad neutralizing activity. This implies that the mutation in residue 506-509, 529, and 534 of S is critical to generate neutralization escape variants of MERS-CoV. Interestingly, all five neutralizing mAbs have binding affinity to RBD, although most mAbs generated by RBD did not have neutralizing activity. Additionally, chimeric antibodies of RBD-14F8 and RBD-43E4 with human Fc and light chain showed neutralizing effect against wild type MERS-CoV KOR/KNIH/002, similar to the original mouse mAbs. Thus, our mAbs can be utilized for the identification of specific mutations of MERS-CoV. Abstract Targeting of MHC class II molecules to the endocytic compartment where they encounter processed antigen is determined by the invariant chain 0i). By analysis of Ii-transferrin receptor (TR) chimera trafficking, we have identified sorting signals in the Ii cytoplasmic tail and transmembrane region that mediate this process. Two non-tyrosine-based sorting signals in the Ii cytoplasmic tail were identified that mediate localization to plasma membrane clathrincoated pits and promote rapid endocytosis. Leu 7 and Ile s were required for the activity of the signal most distal to the cell membrane whereas pro15 Met16 L~ul7 were important for the membrane-proximal signal.The same or overlapping non-tyrosine-based sorting signals are essential for delivery of Ii-TR chimeras, either by an intracellular route or via the plasma membrane, to an endoeytic compartment where they are rapidly degraded. The Ii transmembrane region is also required for efficient delivery to this endocytic processing compartment and contains a signal distinct from the Ii cytoplasmic tail. More than 80 % of the Abstract MBoC | ARTICLE In wound repair vimentin mediates the transition of mesenchymal leader cells to a myofibroblast phenotype ABSTRACT Following injury, mesenchymal repair cells are activated to function as leader cells that modulate wound healing. These cells have the potential to differentiate to myofibroblasts, resulting in fibrosis and scarring. The signals underlying these differing pathways are complex and incompletely understood. The ex vivo mock cataract surgery cultures are an attractive model with which to address this question. With this model we study, concurrently, the mechanisms that control mesenchymal leader cell function in injury repair within their native microenvironment and the signals that induce this same cell population to acquire a myofibroblast phenotype when these cells encounter the environment of the adjacent tissue culture platform. Here we show that on injury, the cytoskeletal protein vimentin is released into the extracellular space, binds to the cell surface of the mesenchymal leader cells located at the wound edge in the native matrix environment, and supports wound closure. In profibrotic environments, the extracellular vimentin pool also links specifically to the mesenchymal leader cells and has an essential role in signaling their fate change to a myofibroblast. These findings suggest a novel role for extracellular, cell-surface-associated vimentin in mediating repair-cell function in wound repair and in transitioning these cells to a myofibroblast phenotype. Abstract An oligonucleotide array (microarray) incorporating 13,000 elements representing selected strains of hepatitis A virus (HAV), human coxsackieviruses A and B (CVA and CVB), genogroups I and II of Norovirus (NV), and human rotavirus (RV) gene segments 3,4,10, and 11 was designed based on the principle of tiling. Each oligonucleotide was 29 bases long, starting at every 5 th base of every sequence, resulting in an overlap of 24 bases in two consecutive oligonucleotides. The applicability of the array for virus identification was examined using PCR amplified products from multiple HAV and CV strains. PCR products labeled with biotin were hybridized to the array, and the biotin was detected using a brief reaction with Cy3-labeled streptavidin, the array subjected to laser scanning, and the hybridization data plotted as fluorescence intensity against each oligonucleotide in the array. The combined signal intensities of all probes representing a particular strain of virus were calculated and plotted against all virus strains identified on a linear representation of the array. The profile of the total signal intensity identified the strain that is most likely represented in the amplified cDNA target. The results obtained with HAV and CV indicated that the hybridization profile thus generated can be used to identify closely related viral strains. This represents a significant improvement over current methods for virus identification using PCR amplification and amplicon sequencing. Abstract Clinical progression over time and cytokine profiles have not been well defined in patients with Middle East respiratory syndrome coronavirus (MERS-CoV) infection. We included 17 patients with laboratory-confirmed MERS-CoV during the 2015 outbreak in Korea. Clinical and laboratory parameters were collected prospectively. Serum cytokine and chemokine levels in serial serum samples were measured using enzyme-linked immunosorbent assay. All patients presented with fever. The median time to defervescence was 18 days. Nine patients required oxygen supplementation and classified into severe group. In the severe group, chest infiltrates suddenly began to worsen around day 7 of illness, and dyspnea developed at the end of the first week and became apparent in the second week. Median time from symptom onset to oxygen supplementation was 8 days. The severe group had higher neutrophil counts during week 1 than the mild group (4,500 vs. 2,200/µL, P = 0.026). In the second week of illness, the severe group had higher serum levels of IL-6 (54 vs. 4 pg/mL, P = 0.006) and CXCL-10 (2,642 vs. 382 pg/mL, P < 0.001). IFN-α response was not observed in mild cases. Our data shows that clinical condition may suddenly deteriorate around 7 days of illness and the serum levels of IL-6 and CXCL-10 was significantly elevated in MERS-CoV patients who developed severe diseases.During the outbreak of MERS-CoV in Korea, all laboratory-confirmed cases were admitted to the government-designated hospitals regardless of their severity of illness. They were discharged from the hospitals when two consecutive daily real-time reverse transcription (rRT)-PCR were all negative after 48 hours of the resolution of all clinical symptoms. The laboratory diagnosis was made by WHO guideline (12). We included all patients admitted to three Seoul National University (SNU) affiliated hospitals (SNU Hospital, SNU Bundang Hospital, and SNU Boramae Medical Center). We excluded those patients who were admitted 14 days after symptom onset. These patients were the same as those of our studies reported previously (10,11).Board certified infectious disease specialists in each hospital prospectively documented clinical symptoms, physical examinations, and laboratory values using standardized case recording form. For the patients who were transferred from other hospitals, the medical records and chest radiographs of the patients at the hospital were also reviewed. Serum samples were collect-ed at serial interval and stored at -70°C.The patients were categorized into severe or mild group depending upon oxygen supplementation requirements. The severe group required oxygen supplementation to maintain arterial saturation above 90%. Abstract Tripartite motif containing 55 (TRIM55) plays a regulatory role in assembly of sarcomeres, but few studies have assessed its function in hepatocellular carcinoma (HCC).Immunohistochemistry (IHC) was used to detect expression of TRIM55 in tissues samples of HCC patients.Transwell assay was used to study migration and invasion ability of HCC cells. Western blot and immunofluorescence (IF) were used to analyze mechanism of TRIM55 in cell migration and invasion.We found TRIM55 was downregulated in HCC tissues and was associated with prognosis of HCC patients.Cox regression analysis showed that TRIM55 was an independent risk factor of prognosis of HCC patients. Overexpression of TRIM55 was associated with lower cell migration and invasion ability, and it led to high expression of E-cadherin and low expression of Vimentin and MMP2.Our study found TRIM55 is an independent factor affecting the prognosis of HCC patients, and overexpression of TRIM55 inhibits migration and invasion of HCC cells through epithelial-mesenchymal transition and MMP2. Abstract Myeloid cell leukemia-1 (Mcl-1) is often overexpressed in human cancer and is an important target for developing antineoplastic drugs. In this study, a data set containing 2.3 million lead-like molecules and a data set of all the US Food and Drug Administration (FDA)-approved drugs are virtually screened for potential Mcl-1 ligands using Protein Data Bank (PDB) ID 2MHS. The potential Mcl-1 ligands are evaluated and computationally docked on to three conformation ensembles generated by normal mode analysis (NMA), molecular dynamics (MD), and nuclear magnetic resonance (NMR), respectively. The evaluated potential Mcl-1 ligands are then compared with their clinical use. Remarkably, half of the top 30 potential drugs are used clinically to treat cancer, thus partially validating our virtual screen. The partial validation also favors the idea that the other half of the top 30 potential drugs could be used in the treatment of cancer. The normal mode-, MD-, and NMR-based conformation greatly expand the conformational sampling used herein for in silico identification of potential Mcl-1 inhibitors. Abstract We report a human parechovirus-3 (HPeV-3) infection in 2 neonates who had prolonged fever (>5 days) with palmar-plantar erythema. This distinctive rash was observed 4-5 days after fever onset, just before defervescence. Elevated aspartate aminotransferase, lactate dehydrogenase, and ferritin levels were characteristic laboratory findings in the 2 cases, suggesting tissue damage caused by hypercytokinemia. Case 1 was treated with intravenous immunoglobulin, considering the possibility of severe systemic inflammatory responses. The initial ferritin level was 385 ng/mL (range, 0-400 ng/mL); however, the level increased to 2,581 ng/dL on day 5 after fever onset. Case 2 presented with milder clinical symptoms, and the patient recovered spontaneously. HPeV-3 was detected in cerebrospinal fluid and/or blood samples, but no other causative agents were detected. The findings from our cases, in accordance with recent studies, suggest that clinical features such as palmarplantar erythema and/or hyperferritinemia might be indicators of HPeV-3 infection in neonates with sepsis-like illness. In clinical practice, where virology testing is not easily accessible, clinical features such as palmar-plantar erythema and/or hyperferritinemia might be helpful to diagnose HPeV-3 infection. Abstract The de novo and ancestral genes can be identified with the greatest reliability under three conditions: a) the de novo gene is restricted to a monophyletic clade (the focal clade), whereas the ancestral gene is found throughout this clade but also in an outgroup clade; b) all the de novo genes within the focal clade share significant sequence similarity; c) the overlapping regions of the ancestral genes share significant sequence similarity within the focal clade but also with the outgroup taxa. These conditions were met for most the overlaps used in our analysis. We explain below our approach to identify the de novo and ancestral genes in overlaps where these conditions were not met.Figure S1 presents a case in which some genes are demonstrably de novo and occur within the same clade but have no statistical significant similarity with each other. All of the ancestral genes (red arrows) share significant sequence similarity over the overlapping region. The two de novo genes represented by blue arrows have similar sequences. The de novo gene of taxon T3 (black arrow) does not have significant sequence similarity to the other two taxa (T1 and T2). Because the three de novo genes form a monophyletic clade and have the same genomic location, we infer that the black gene is homologous to the blue genes but that its sequence has diverged beyond recognition. Therefore, we included all three taxa in the clade and deduced the Last Common Ancestor (LCA) of the de novo gene at the branch leading to the three taxa (marked by a blue circle). The alternative scenario, i.e. that the blue and the black genes Abstract Rhinitis is a disease of the upper airway characterized by runny and/or blocked nose and/or sneezing. Though not viewed as a life threatening condition, it is also recognized to impose significant burden to the quality of life of sufferers and their caretakers and imposes an economic cost to society. Through a PubMed online search of the literature from 2006 to September 2011, this paper aims to review the published literature on rhinitis in young children below the age of 6 years. It is apparent from epidemiology studies that rhinitis in this age group is a relatively common problem. The condition has a heterogenous etiology with classification into allergic and non-allergic rhinitis. Respiratory viral infections may play a role in the pathogenesis of long standing rhinitis, but definitive studies are still lacking. Treatment guidelines for management are lacking for this age group, and is a significant unmet need. Although the consensus is that co-morbidities including otitis media with effusion, adenoidal hypertrophy and asthma, are important considerations of management of these children. Pharmacotherapy is limited for young children especially for those below the age of 2 years. This review underscores the lack of understanding of rhinitis in early childhood and therefore the need for further research in this area. This is an Open Access article distributed under the terms of the creative commons Attribution. Non-commercial License (http://creativecommons. org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Hardjojo A, et al. Asia Pacific allergy http://dx. Abstract Background. Seasonal influenza epidemics occur frequently. Rapid characterization of seasonal dynamics and forecasting of epidemic peaks and final sizes could help support real-time decision-making related to vaccination and other control measures. Real-time forecasting remains challenging.Methods. We used the previously described "incidence decay with exponential adjustment" (IDEA) model, a 2-parameter phenomenological model, to evaluate the characteristics of the 2015-2016 influenza season in 4 Canadian jurisdictions: the Provinces of Alberta, Nova Scotia and Ontario, and the City of Ottawa. Model fits were updated weekly with receipt of incident virologically confirmed case counts. Best-fit models were used to project seasonal influenza peaks and epidemic final sizes.Results. The 2015-2016 influenza season was mild and late-peaking. Parameter estimates generated through fitting were consistent in the 2 largest jurisdictions (Ontario and Alberta) and with pooled data including Nova Scotia counts (R 0 approximately 1.4 for all fits). Lower R 0 estimates were generated in Nova Scotia and Ottawa. Final size projections that made use of complete time series were accurate to within 6% of true final sizes, but final size was using pre-peak data. Projections of epidemic peaks stabilized before the true epidemic peak, but these were persistently early (~2 weeks) relative to the true peak.Conclusions. A simple, 2-parameter influenza model provided reasonably accurate real-time projections of influenza seasonal dynamics in an atypically late, mild influenza season. Challenges are similar to those seen with more complex forecasting methodologies. Future work includes identification of seasonal characteristics associated with variability in model performance. Abstract The nod-like receptor protein 3 (NLRP3) inflammasome drives inflammation in response to mitochondrial dysfunction. As metabolic powerhouses with prokaryotic ancestry, mitochondria are a cache for danger-associated molecular patterns and pathogen-associated molecular pattern-like molecules that elicit potent innate immune responses. Persistent mitochondrial damage caused by infection, or genetic or environmental factors, can lead to inappropriate or sustained inflammasome signalling. Here, we review the features of mitochondria that drive inflammatory signalling, with a particular focus on mitochondrial activation of the NLRP3 inflammasome. Given that mitochondrial network dynamics, metabolic activity and redox state are all intricately linked to each other and to NLRP3 inflammasome activity, we highlight the importance of a holistic approach to investigations of NLRP3 activation by dysfunctional mitochondria. Abstract Improved diagnostics are needed for children with musculoskeletal infections (MSKIs). We assessed the performance of target-enriched multiplex polymerase chain reaction (TEM-PCR) in children with MSKI. TEM-PCR was concordant with culture in pathogen identification and antibiotic susceptibility testing, while increasing the overall yield of pathogen detection. This technology has the potential to inform judicious antimicrobial use early in the disease course. Abstract Patrick Manson, a clinician-scientist serving in China (1866±1889), discovered that many tropical infectious diseases require a vector peculiar to warm climate for person to person transmission. He demonstrated the nocturnal periodicity of microfilariae in the blood of patients with elephantiasis. These microfilariae undergo metamorphosis when ingested by the mosquito acting as the vector for the completion of their life cycle. Furthermore, he demonstrated the linkage between the lung fluke and endemic haemoptysis by finding operculated eggs in patients' sputa. He predicted that the miracidium from hatched eggs uses crustaceans, such as fresh-water snails found at tropical conditions, as the intermediate hosts in the life cycle of many trematodes. His vector hypothesis leads to vector control which is now the cornerstone for the World Health Organization's programme for the elimination/control of lymphatic filariasis, dracunculiasis and malaria. Before leaving China, he established the Alice Memorial Hospital, the Hong Kong College of Medicine for Chinese (the forerunner of the University of Hong Kong), and the Hong Kong Medical Society for medical service and education. He also incepted the Hong Kong Dairy Farm for supplying hygienic milk affordable by pregnant women, children and patients. Abstract Contact patterns among hosts are considered as one of the most critical factors contributing to unequal pathogen transmission. Consequently, networks have been widely applied in infectious disease modeling. However most studies assume static network structure due to lack of accurate observation and appropriate analytic tools. In this study we used high temporal and spatial resolution animal position data to construct a high-resolution contact network relevant to infectious disease transmission. The animal contact network aggregated at hourly level was highly variable and dynamic within and between days, for both network structure (network degree distribution) and individual rank of degree distribution in the network (degree order). We integrated network degree distribution and degree order heterogeneities with a commonly used contact-based, directly transmitted disease model to quantify the effect of these two sources of heterogeneity on the infectious disease dynamics. Four conditions were simulated based on the combination of these two heterogeneities. Simulation results indicated that disease dynamics and individual contribution to new infections varied substantially among these four conditions under both parameter settings. Changes in the contact network had a greater effect on disease dynamics for pathogens with smaller basic reproduction number (i.e. R 0 , 2). Abstract More than 140 post-transcriptional modifications (PTMs) are known to decorate cellular RNAs, but their incidence, identity and significance in viral RNA are still largely unknown. We have developed an agnostic analytical approach to comprehensively survey PTMs on viral and cellular RNAs. Specifically, we used mass spectrometry to analyze PTMs on total RNA isolated from cells infected with Zika virus, Dengue virus, hepatitis C virus (HCV), poliovirus and human immunodeficiency virus type 1. All five RNA viruses significantly altered global PTM landscapes. Examination of PTM profiles of individual viral genomes isolated by affinity capture revealed a plethora of PTMs on viral RNAs, which far exceeds the handful of well-characterized modifications. Direct comparison of viral epitranscriptomes identified common and virus-specific PTMs. In particular, specific dimethylcytosine modifications were only present in total RNA from virus-infected cells, and in intracellular HCV RNA, and viral RNA from Zika and HCV virions. Moreover, dimethylcytosine abundance during viral infection was modulated by the cellular DEAD-box RNA helicase DDX6. By opening the Pandora's box on viral PTMs, this report presents numerous questions and hypotheses on PTM function and strongly supports PTMs as a new tier of regulation by which RNA viruses subvert the host and evade cellular surveillance systems. Abstract Houttuynia cordata Thunb. is a traditional herb used for clearing heat and eliminating toxins, and has also been used for the treatment of severe acute respiratory syndrome (SARS). In vitro, the crude H. cordata polysaccharides (CHCP) exhibited potent anti-complementary activity through both the classical and alternative pathways by acting on components C3 and C4 of the complement system without interfering with the coagulation system. This study was to investigate the preventive effects of CHCP on acute lung injury (ALI) induced by hemorrhagic shock plus lipopolysaccharide (LPS) instillation (two-hit) and LPS-induced fever in rats. CHCP significantly attenuated pulmonary injury in the "two-hit" ALI model by reducing pulmonary edema and protein exudation in bronchoalveolar lavage fluid (BALF). In addition, it reduced the deposit of complement activation products in the lung and improved oxidantantioxidant imbalance. Moreover, CHCP administration inhibited fever in rats, reduced the number of leukocytes and restored serum complement levels. The inhibition on the inappropriate activation of complement system by CHCP may play an important role in its beneficial effects on inflammatory diseases. The anti-complementary polysaccharides are likely to be among the key substances for the heatclearing function of H. cordata. Abstract A 27-month-old female cat was presented with septic peritonitis secondary to a ruptured pyometra and subsequent pyothorax. Vasopressor-refractory septic shock led to a suspicion of critical illness-related corticosteroid insufficiency, successfully treated with intravenous hydrocortisone. Previous megestrol acetate administration may have played a role in the development of adrenocortical dysfunction. Abstract The recent SARS epidemic has boosted interest in the discovery of novel human and animal coronaviruses. By July 2007, more than 3000 coronavirus sequence records, including 264 complete genomes, are available in GenBank. The number of coronavirus species with complete genomes available has increased from 9 in 2003 to 25 in 2007, of which six, including coronavirus HKU1, bat SARS coronavirus, group 1 bat coronavirus HKU2, groups 2c and 2d coronaviruses, were sequenced by our laboratory. To overcome the problems we encountered in the existing databases during comparative sequence analysis, we built a comprehensive database, CoVDB (http://covdb.microbiology. hku.hk), of annotated coronavirus genes and genomes. CoVDB provides a convenient platform for rapid and accurate batch sequence retrieval, the cornerstone and bottleneck for comparative gene or genome analysis. Sequences can be directly downloaded from the website in FASTA format. CoVDB also provides detailed annotation of all coronavirus sequences using a standardized nomenclature system, and overcomes the problems of duplicated and identical sequences in other databases. For complete genomes, a single representative sequence for each species is available for comparative analysis such as phylogenetic studies. With the annotated sequences in CoVDB, more specific blast search results can be generated for efficient downstream analysis. Abstract Shiga toxin-producing Escherichia coli (STEC) cause gastrointestinal illnesses including non-bloody or bloody diarrhoea, haemorrhagic colitis (HC), and the haemolytic uremic syndrome (HUS). To investigate the occurrence of STEC among grazing dromedaries from Kenya, E. coli isolated from fecal matter collected from 163 dromedaries on a large ranch were screened for the presence of stx1 and stx2. STEC strains were isolated and serotyped. Isolates were subjected to PCR for the subtyping of stx genes and for the detection of eae and ehx. In addition, whole genome sequencing (WGS) was carried out to detect further virulence genes and to determine the multilocus sequence types (MLST). Antimicrobial resistance profiles were determined by disk diffusion.STEC was isolated from 20 (12.3%) of the fecal samples. Thereof, nine (45%) isolates were STEC O156:H25, three (15%) isolates typed STEC O43:H2. The remaining isolates occurred as single serotypes or were O nontypeable. Eleven (55%) of the isolates harboured stx2a, nine (45%) eae, and 14 (70%) ehx, respectively. WGS revealed the presence of iss in 16 (80%), subAB in four (20%) and astA in two (10%) of the isolates, Furthermore, espA, tccP, nleA, nleB, tccP, and tir were found exclusively among STEC O156:H25.Eleven different sequence types (ST) were detected. The most prominent was ST300/ST5343, which comprised STEC O156:H25. All STEC isolates were pan susceptible to a panel of 16 antimicrobial agents. Overall, the results indicate that dromedary camels in Kenya may be reservoirs of STEC, including serotypes possessing virulence markers associated to disease in humans, such as STEC O156:H25. STEC in camels may represent a health hazard for humans with close contact to camels or to consumers of camel derived foodstuffs, such as unpasteurised camel milk. Abstract The aim of the current study was to investigate the effectiveness and clinicalfeasibilityofBiyeom-goforthetreatmentofnasalsymptomsassociatedwith rhinitis.Design: Prospectiveobservationalstudy. Setting: ThisstudywasconductedattheWoosukKoreanMedicineMedicalCenter inSouthKorea. Participants: Fifty-eightpatientswithrhinitisparticipatedinthisstudy.Allpatients receivedBiyeom-gotreatment>3timesdailyforatotalof4weeks. Main outcome measures: Theprimaryoutcomewasthetotalnasalsymptomscore. Mini-rhinoconjunctivitis quality of life questionnaire, nasal endoscopy index, total serumimmunoglobulinElevelsandimmunologicfactorsinnasallavagefluidwere alsomeasured. Results: Biyeom-goadministrationwasassociatedwithsignificantimprovementsin totalnasalsymptomsscores(P<.0001)andmini-rhinoconjunctivitisqualityoflife questionnairescores(P<.0001)inatime-dependentmanner.Thenasalendoscopy indexalsosignificantlyimprovedatweeks2(P=.0049),3(P<.0001)and4(P=.0001) afterBiyeom-gotreatment.Significantly,increasedinterleukin-2levels(P=.005)and decreased interleukin-8, chemokine (C-C motif) ligand (CCL) 5, chemokine (C-X-C motif)ligand(CXCL)9,CCL2andCXCL10levelswereobservedinthenasallavage fluid. Conclusions: ThepresentfindingssuggestthatBiyeom-gomaybebeneficialforthe managementofrhinitissymptomsandrhinitis-associatedqualityoflife.Furtherwelldesigned randomised controlled trials are needed to evaluate the effectiveness of Biyeom-goforrhinitis.ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercial-NoDerivsLicense,whichpermitsuseanddistributionin anymedium,providedtheoriginalworkisproperlycited,theuseisnon-commercialandnomodificationsoradaptationsaremade. Abstract Al ibrary of mannose-and fucose-based glycomimetics was synthesizeda nd screened in am icroarray format againstaset of C-typel ectin receptors (CLRs) that included DC-SIGN, DC-SIGNR, langerin, andd ectin-2. Glycomimetic li-gandsa ble to interact with dectin-2w ere identified for the first time. Comparative analysiso fb inding profiles allowed their selectivity against other CLRs to be probed. Abstract A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates.KEY WORDS: avian infectious bronchitis virus, Massachusetts, melting temperature analysis, RT-qPCR, S1 gene Avian infectious bronchitis (IB) is an acute and highly infect-contagious viral disease, responsible by significant economic loss to the poultry industry worldwide that is caused by avian infectious bronchitis virus (IBV) [6] . IBV genome is well known to be highly prone to undergo mutations, leading to changes on nucleotide and/or amino acids sequences of the major structural IBV genes/proteins, resulting in multiple variants of this virus. Most important target of mutations is the spike 1 (S1) glycoprotein gene, especially those located in HVR I, (region encoding amino acid residues 56-69), and HVR II (region encoding amino acid residues 117-131). These regions of S1 glycoprotein have sites involved on the virus attachment to the receptors in the target host cells and the most important neutralizing epitopes. Mutations in the S gene could result in antigenic changes and emergence of IBV variants. This constant arising of new IB variants in different countries [8, 18] is usually pointed as the main cause of vaccine failures in the field and constitutes a great challenge for the molecular direct diagnosis of IBV.Variants of IBV are mutants evolving from parental strains of this virus that differ from strains considered classic, such as those from Massachusetts group, regarding genotype, pathotype, antigenicity and/or immunogenicity. Additionally, IBV mutants are constantly subject to host immune selection and only strains with high antigenic variation are able to evade immune responses induced by vaccines and to survive in the population of hosts, leading to the development of clinical disease [25] .Protection studies between commercial vaccines and IBV variants, especially those using Massachusetts (Mass) vaccines, which is the most common attenuated available vaccine, have demonstrated at least partial cross-protection by some authors [9, 10, 12, 14, 34] , while another studies have not found cross-protection [7, 35] .Therefore, the rapid detection of IBV infection in poultry flocks and its classification between Mass and variants is considered as major challenge, which requires use of appropriate diagnostic methods. To overcome this issue, several rapid assays have been developed focusing on detection and/or characterization of S1 gene of IBV, including direct gene sequencing, RT-PCR methods with serotype-specific primers, and RT-PCR/RFLP, have become widely used, because their results correlated well with the virusserotyping [19, 21, 22, 33] . Beside these molecular methods, RT-qPCR methods using primers and probes for S1 gene or UTR region were developed and applied for IBV diagnosis, resulting in high sensitivity and specificity [5] . A SYBR Green RT-qPCR was tested on a wide range of coronaviruses and clinical samples, but it was not able to discriminate the different virus genus or Abstract The quarantine of people suspected of being exposed to an infectious agent is one of the most basic public health measure that has historically been used to combat the spread of communicable diseases in human communities. This study presents a new deterministic model for assessing the population-level impact of the quarantine of individuals suspected of being exposed to disease on the spread of the 2014e2015 outbreaks of Ebola viral disease. It is assumed that quarantine is imperfect (i.e., individuals can acquire infection during quarantine). In the absence of quarantine, the model is shown to exhibit global dynamics with respect to the disease-free and its unique endemic equilibrium when a certain epidemiological threshold (denoted byR 0 ) is either less than or greater than unity. Thus, unlike the full model with imperfect quarantine (which is known to exhibit the phenomenon of backward bifurcation), the version of the model with no quarantine does not undergo a backward bifurcation. Using data relevant to the 2014e2015 Ebola transmission dynamics in the three West African countries (Guinea, Liberia and Sierra Leone), uncertainty analysis of the model show that, although the current level and effectiveness of quarantine can lead to significant reduction in disease burden, they fail to bring the associated quarantine reproduction number (R Q 0 ) to a value less than unity (which is needed to make effective disease control or elimination feasible). This reduction of R Q 0 is, however, very possible with a modest increase in quarantine rate and effectiveness. It is further shown, via sensitivity analysis, that the parameters related to the effectiveness of quarantine (namely the parameter associated with the reduction in infectiousness of infected quarantined individuals and the contact rate during quarantine) are the main drivers of the disease transmission dynamics. Overall, this study shows that the singular implementation of a quarantine intervention strategy can lead to the effective control or elimination of Ebola viral disease in a community if its coverage and effectiveness levels are high enough. Abstract Purpose: Several markers for eosinophilic inflammation have been proposed to predict response to asthma treatment. However, definitive criteria for treatment decisions have not yet been established. We investigate a potentially useful relatively non-invasive biomarker, eosinophil-derived neurotoxin (EDN), to predict favorable responses to budesonide or montelukast, common treatment for children with asthma. Methods: Young children (1 to 6 years old) were enrolled in this randomized, parallel, 2-group, open-label trial. Criteria for eligibility included: 1) being symptomatic during the run-in period; and 2) having a serum EDN (sEDN) level ≥ 53 ng/mL, with positive specific immunoglobulin E to house dust mite. Eligible patients were randomly placed into 2 groups: the BIS group received budesonide inhalation suspension (BIS) 0.5 mg once daily; the MONT group received montelukast 4 mg once daily. Ineligible patients were invited to receive montelukast 4 mg once daily (OBS group). Treatment period was 12 weeks. Results: Asthma control days increased significantly in the BIS and MONT groups (P < 0.000) over the 12-week study period. There was no significant change in sEDN in the BIS group but there was a significant decrease in the MONT group (P < 0.000). Patients in the OBS group with high EDN levels (> 53 ng/mL) showed a significant decrease due to MONT treatment (P = 0.023). Rescue medication usage significantly decreased in the BIS and MONT groups (P < 0.000). Conclusions: EDN is a useful relatively non-invasive biomarker for predicting responses to montelukast and budesonide treatment of preschool children with beta2-agonist responsive recurrent wheeze and multiple-trigger wheeze (Trial registry at UMIN Clinical Trials Registry, UMIN000008335). Abstract To cite: Villar J, Blanco J, del Campo R, et al. Assessment of PaO 2 /FiO 2 for stratification of patients with moderate and severe acute respiratory distress syndrome. BMJ Open 2015;5:e006812.Objectives: A recent update of the definition of acute respiratory distress syndrome (ARDS) proposed an empirical classification based on ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO 2 /FiO 2 ) at ARDS onset. Since the proposal did not mandate PaO 2 /FiO 2 calculation under standardised ventilator settings (SVS), we hypothesised that a stratification based on baseline PaO 2 /FiO 2 would not provide accurate assessment of lung injury severity.Design: A prospective, multicentre, observational study. Setting: A network of teaching hospitals. Participants: 478 patients with eligible criteria for moderate (100300).Primary and secondary outcomes: Group severity and hospital mortality. Trial registration numbers: NCT00435110 and NCT00736892. Villar J, et al. BMJ Open 2015;5:e006812. Abstract The Hendra virus (HeV) poses a significant challenge to public health in Australia. Expanding migratory patterns observed among bats and the mutation of the virus to seek and successfully infect new hosts is a significant departure from the generalized epidemiological trend. The recent discovery of equine-related infections and deaths in addition to a canine infection demonstrates the inadequacy of the current equine vaccine developed in 2012. Traditional models for controlling the spread of the vector are futile given the rapid pace at which bats' habitats are eroded. Recent ongoing zoonotic epidemics, for example, Ebola and Middle East respiratory syndrome coronavirus, demonstrate that human-to-human transmission is a distinct reality rather than an obscure possibility. The development of a human HeV vaccine is essential for the biosecurity of Australia, as part of a multipronged strategy to control HeV in Australia. Abstract Avian leucosis virus subgroup J (ALV-J) can cause lifelong infection and can escape from the host immune defenses in chickens. Since macrophages act as the important defense line against invading pathogens in host innate immunity, we investigated the function and innate immune responses of chicken primary monocyte-derived macrophages (MDM) after ALV-J infection in this study. Our results indicated that ALV-J was stably maintained in MDM cells but that the viral growth rate was significantly lower than that in DF-1 cells. We also found that ALV-J infection significantly increased nitric oxide (NO) production, but had no effect on MDM phagocytic capacity. Interestingly, infection with ALV-J rapidly promoted the expression levels of Myxovirus resistance 1 (Mx) (3 h, 6 h), ISG12 (6 h), and interleukin-1β (IL-1β) (3 h, 12 h) at an early infection stage, whereas it sharply decreased the expression of Mx (24 h, 36 h), ISG12 (36 h), and made little change on IL-1β (24 h, 36 h) production at a late infection stage in MDM cells. Moreover, the protein levels of interferon-β (IFN-β) and interleukin-6 (IL-6) had sharply increased in infected MDM cells from 3 to 36 h post infection (hpi) of ALV-J. And, the protein level of interleukin-10 (IL-10) was dramatically decreased at 36 hpi in MDM cells infected with ALV-J. These results demonstrate that ALV-J can induce host innate immune responses and we hypothesize that macrophages play an important role in host innate immune attack and ALV-J immune escape. Abstract Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R 2 ) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID 50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay's clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea. Abstract Backgrounds: Up to February 16, 2020, 355 cases have been confirmed as having COVID-19 infection on the Diamond Princess cruise ship. It is of crucial importance to estimate the reproductive number (R0) of the novel virus in the early stage of outbreak and make a prediction of daily new cases on the ship. Method: We fitted the reported serial interval (mean and standard deviation) with a gamma distribution and applied "earlyR" package in R to estimate the R0 in the early stage of COVID-19 outbreak. We applied "projections" package in R to simulate the plausible cumulative epidemic trajectories and future daily incidence by fitting the data of existing daily incidence, a serial interval distribution, and the estimated R0 into a model based on the assumption that daily incidence obeys approximately Poisson distribution determined by daily infectiousness. Results: The Maximum-Likelihood (ML) value of R0 was 2.28 for COVID-19 outbreak at the early stage on the ship. The median with 95% confidence interval (CI) of R0 values was 2.28 (2.06-2.52) estimated by the bootstrap resampling method. The probable number of new cases for the next ten days would gradually increase, and the estimated cumulative cases would reach 1514 (1384-1656) at the tenth day in the future. However, if R0 value was reduced by 25% and 50%, the estimated total number of cumulative cases would be reduced to 1081 (981-1177) and 758 (697-817), respectively. Conclusion: The median with 95% CI of R0 of COVID-19 was about 2.28 (2.06-2.52) during the early stage experienced on the Diamond Princess cruise ship. The future daily incidence and probable outbreak size is largely dependent on the change of R0. Unless strict infection management and control are taken, our findings indicate the potential of COVID-19 to cause greater outbreak on the ship. Abstract Bats are unique mammals that are reservoirs of high levels of virus diversity. Although several of these viruses are zoonotic, the majority are not. Astroviruses, transmitted fecal-orally, are commonly detected in a wide diversity of bat species, are prevalent at high rates and are not thought to directly infect humans. These features make astroviruses useful in examining virus evolutionary history, epidemiology in the host, and temporal shedding trends. Our study screened for the presence of astroviruses in bats in Singapore, reconstructed the phylogenetic relations of the polymerase genes and tested for population characteristics associated with infection. Of the seven species screened, astroviruses were detected in Rhinolophus lepidus and Eonycteris spelaea. The R. lepidus sequences grouped with other Rhinolophus astrovirus sequences from China and Laos, while the Eoncyteris sequences formed a distinct clade with astroviruses from Rousettus spp. in Laos and Pteropus giganteus in Bangladesh, but not with other E. spelaea sequences. Longitudinal collections of Eonycteris feces demonstrated variable shedding. Juvenile status of bats was a risk factor for astroviruses. This study highlights the diversity of astroviruses in nectivorous and insectivorous bats in Singapore and provides a predictive framework for understanding astrovirus infection in these bats. It also suggests that in addition to host phylogenetic relatedness, host ecology, such as roosting behavior, may drive co-infections, virus maintenance and spillover. Abstract Introduction: The aim of the study was to demonstrate Interleukin-18 (IL-18) expression in keratinocytes from psoriatic lesions in comparison to keratinocytes from uninvolved skin and to study the change of expression after therapeutic interventions. Material and methods: This study included 16 patients of different clinical subtypes of psoriasis. IL-18 gene expression analysis was performed using realtime quantitative PCR. Three biopsies were obtained from each patient. Two were taken from the lesional psoriatic skin and from uninvolved skin before starting treatment. A third lesional skin biopsy was taken at the end of two months' treatment course. The treatment was in the form of topical steroids or oral systemic methotrexate. Results: Of all 16 studied patients significantly increased IL-18 expression was noted in keratinocytes from psoriatic lesions before and after treatment when compared to keratinocytes from uninvolved skin (P = 0.001 and 0.002 respectively). The IL-18 expression in the skin lesions after treatment was significantly lower than lesional skin before treatment (P = 0.023). In psoriatic skin lesions of all studied patients IL-18 expression was significantly correlated with disease duration (r = 0.40 and P = 0.01) and clinical severity of psoriasis (r = 0.72 and P = 0.001). Conclusions: Increased IL-18 expression in keratinocytes from psoriatic lesions of our patients and its correlation with disease duration and severity supported the concept which views psoriasis as a T-cell-mediated autoimmune disease. This could establish therapeutic and preventive approaches for psoriasis that ultimately lead to improved outcomes for patients. Abstract In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5 , a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5 against FIV-and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5 overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF). Abstract publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 769-773 h t t p : / / w w w . b j m i c r o b i o l . c o m . b r / Mexico Parvovirus Rotavirus aThis is the first report on circulating canine rotavirus in Mexico. Fifty samples from dogs with gastroenteritis were analyzed used polymerase chain reaction and reverse transcription polymerase chain reaction in order to identify parvovirus and rotavirus, respectively; 7% of dogs were infected with rotavirus exclusively, while 14% were co-infected with both rotavirus and parvovirus; clinical signs in co-infected dogs were more severe. Abstract Increased autoantibody reactivity in plasma from Myelodysplastic Syndromes (MDS) patients may provide novel disease signatures, and possible early detection. In a two-stage study we investigated Immunoglobulin G reactivity in plasma from MDS, Acute Myeloid Leukemia post MDS patients, and a healthy cohort. In exploratory Stage I we utilized high-throughput protein arrays to identify 35 high-interest proteins showing increased reactivity in patient subgroups compared to healthy controls. In validation Stage II we designed new arrays focusing on 25 of the proteins identified in Stage I and expanded the initial cohort. We validated increased antibody reactivity against AKT3, FCGR3A and ARL8B in patients, which enabled sample classification into stable MDS and healthy individuals. We also detected elevated AKT3 protein levels in MDS patient plasma. The discovery of increased specific autoantibody reactivity in MDS patients, provides molecular signatures for classification, supplementing existing risk categorizations, and may enhance diagnostic and prognostic capabilities for MDS. Abstract Introduction: The earthquake is one of the most natural catastrophic crises that can cause a lot of casualties. Considering an earthquake-prone country, Iran is ranked as one of the world's most dangerous countries Objective: In this article, we describe the actions taken by emergency medical service (EMS) after the earthquake in Kermanshah, Varzaghan, and Bam and compared the strengths and weaknesses of the emergency response program and the limitations and challenges of this system in dealing with these major crises. Method: This study is a cross-sectional study that compares some of the information and findings related to three earthquakes that occurred in Iran, including Bam, Varzaghan and Sarpol-e-Zahab earthquakes. The data reported in the present article is descriptive and is based on various independent sources such as National Emergency Operation Center, Local Emergency Operations Center (EOC), the EMS of the country, the World Health Organization, the United Nations, the statistics website, the Forensic Data website, the International Institute of Seismology and Earthquake Engineering, conferences and personal interviews. To ensure the credibility of the information, the authors reported data that had been verified by two or more sources. Results: The characteristics of the geographic area of the 3 earthquakes has been described. Post-earthquake response activities were described in details in subheadings including rapid warning and response, surge capacity plan, rapid response teams, emergency medical teams, increasing the capacity of health facilities, increasing transfer capacity, and handling, transportation and distribution of injuries. Conclusion: In the recent earthquake, had been occurred in Sarpol-e-Zahab, the health response of the country was largely satisfactory. The existence of structures such as EOC at various levels, the unified incident command system, emergency operations plan, and Medical Care Monitoring Center are among the most important reasons for satisfactory performance. Cite this article as: Saberian P, Kolivand PH, Hasani-Sharamin P, Dadashi M, Farhoud A. Iranian Emergency Medical Service Response in Disaster; Report of three Earthquakes in Iran. Adv J Emerg Med. 2019;3(2): e13. Abstract Liu et al. Meta-analysis of genome-wide association data of bipolar disorder and major depressive disorder. Mol Psychiatry 2011; 16: 2-6 (a)Gene Name Immune Function of Gene CACNA1C calcium channel, voltage-dependent, L type, alpha 1C subunit See article text for a discussion of immune and host defense functions of CACNA1C McMahon et al. Meta-analysis of genome-wide association data identifies a risk locus for major mood disorders on 3p21.1. Nat Genetics 2010; 4: 128-31 (a)Gene ID Gene Name Immune Function of Gene PBRM1 polybromo 1 aka BRG1-associated factor 180 (BAF180) PBRM1 codes for a protein component (BRG1-associated factor 180 [BAF 180]) of the SWI/SNF chromatin remodeling complex, which is crucial for activation and proliferation of T cells in response to antigen stimulation, 1 and which regulates induction and temporal sequencing of proinflammatory cytokines in response to an immune challenge, 2 as well as antigen receptor assembly (i.e. VDJ recombination) in B cell precursors; 3 following bacterial TLR stimulation, the BRG1/BRM subunits of the SWI/SNF complex are consistently required for the activation of secondary response genes and primary response genes induced with delayed kinetics within macrophages; 4 BRG1 and other BAF elements within the SWI/SNF complex has been identified as activators of transcription within the nuclear interactome for the HIV-1 virulence factor "Trans-Activator of Transcription" (Tat), 5-7 and the SWI/SNF complex more generally regulates retroviral gene integration into, and expression in, infected host cells, [8][9][10][11] and contributes to viral replication; 12 PBRM1 (BAF 180) is required for expression of the IFITM1 gene in response to interferon signaling, 13 and IFITM1 has been shown to restrict entry of a range of viruses, including HIV and SARS, into host cells, 14,15 consistent with fact that BAF complex within SWI/SNF is essential for IFN-target-gene dependent cellular antiviral activities; 16 BRG1 is essential for IFN-gamma induction of CIITA, the master regulator of major histocompatibility complex (MHC) class II expression, 17 and a switch from hBrm to BRG1 regulation of expression is an indicator of genes that are responsive to IFN-gamma signaling; 18 BRG1 contributes to MHC I immunity by upregulating enhancer A; 19 knock-down of BRG-1 activity blocks human papillomavirus E-2 driven transcriptional activation and DNA replication; 20 mice that constitutively express the SWI/SNF complex are highly susceptible to experimentally induced autoimmune encephalomyelitis; 1 GNL3 guanine nucleotide binding protein-like 3 GNL3 codes for a GTPase nucleostemin that is involved in stem cell proliferation and that upregulates the transcription factor CDX2, 21 which is a homeodomain transcription factor specific to the intestinal epithelium crucial for pathogenic E. coli to induce the di/tripeptide transporter PepT1 in gut and also to activate NFkB and MAPK leading to production of IL-8. 22 The chemokine CCL25 which plays an important role in recruiting lymphocytes to the intestinal epithelium is enhanced by CDX2, which regulates CDDL25 transcription. 23 CCL25, in turn, is believed to play a role in the development of T cells, 24 and is chemotactic for thymocytes, macrophages and dendritic cells. 25,26 1. Jeong SM, Lee C, Lee SK, Kim J, Seong RH: The SWI/SNF chromatin-remodeling complex modulates peripheral T cell activation and proliferation by controlling AP-1 expression. J Biol Chem 2010, 285:2340-2350. 2. Cullen SJ, Ponnappan S, Ponnappan U: Catalytic activity of the proteasome fine-tunes Brg1-mediated chromatin remodeling to regulate the expression of inflammatory genes. Mol Immunol 2009, 47:600-605. 3. Osipovich OA, Subrahmanyam R, Pierce S, Sen R, Oltz EM: Cutting edge: SWI/SNF mediates antisense Igh transcription and locus-wide accessibility in B cell precursors. J Immunol 2009, 183:1509-1513. 4. Ramirez-Carrozzi VR, Nazarian AA, Li CC, Gore SL, Sridharan R, Imbalzano AN, et al: Selective and antagonistic functions of SWI/SNF and Mi-2beta nucleosome remodeling complexes during an inflammatory response. Genes Dev 2006, 20:282-296. 5. Abstract The Eukaryotic Pathogen Genomics Database Resource (EuPathDB, http://eupathdb.org) is a collection of databases covering 170+ eukaryotic pathogens (protists & fungi), along with relevant free-living and non-pathogenic species, and select pathogen hosts. To facilitate the discovery of meaningful biological relationships, the databases couple preconfigured searches with visualization and analysis tools for comprehensive data mining via intuitive graphical interfaces and APIs. All data are analyzed with the same workflows, including creation of gene orthology profiles, so data are easily compared across data sets, data types and organisms. EuPathDB is updated with numerous new analysis tools, features, data sets and data types. New tools include GO, metabolic pathway and word enrichment analyses plus an online workspace for analysis of personal, non-public, large-scale data. Expanded data content is mostly genomic and functional genomic data while new data types include protein microarray, metabolic pathways, compounds, quantitative proteomics, copy number variation, and polyso-mal transcriptomics. New features include consistent categorization of searches, data sets and genome browser tracks; redesigned gene pages; effective integration of alternative transcripts; and a EuPathDB Galaxy instance for private analyses of a user's data. Abstract Feline coronavirus (FCoV) is classified into two biotypes based on its pathogenicity in cats: a feline enteric coronavirus of low pathogenicity and a highly virulent feline infectious peritonitis virus. It has been suspected that FCoV alters its biotype via mutations in the viral genome. The S and 3c genes of FCoV have been considered the candidates for viral pathogenicity conversion. In the present study, FCoVs were analyzed for the frequency and location of mutations in the S and 3c genes from faecal samples of cats in an animal shelter and the faeces, effusions, and tissues of cats that were referred to veterinary hospitals. Our results indicated that approximately 95% FCoVs in faeces did not carry mutations in the two genes. However, 80% FCoVs in effusion samples exhibited mutations in the S and 3c genes with remainder displaying a mutation in the S or 3c gene. It was also suggested that mutational analysis of the 3c gene could be useful for studying the horizontal transmission of FCoVs in multi-cat environments.The genome of feline coronavirus (FCoV), a member of the Alphacoronavirus 1 species of the genus Alphacoronavirus, comprises single-stranded positive-sense RNA [9] . FCoV infection is prevalent in cats worldwide and is divided into two biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). The former has low pathogenicity, causing mild enteritis or unapparent infection, and the latter is highly virulent and lethal. FIP is characterised by the accumulation of body cavity effusions (effusive or wet form) and the formation of granulomatous lesions affecting multiple organs (non-effusive or dry form) [10] . FIPVs are considered mutants of FECVs [11, 12, 14] .Although the viral genes responsible for biotype conversion have not been completely elucidated, the candidate genes have been identified. The S gene encodes spike protein on the viral membrane. It was reported that 95.8% of 118 serotype I FIPVs displayed missense mutations in codon 1,058 or 1,060 of the S gene, whereas none of the sample of 183 FECVs exhibited these mutations [6] . The non-synonymous mutations in codons 1,058 and 1,060 substituted methionine to leucine (M1,058L) and serine to alanine (S1,060A), respectively. The 3c gene encoding an accessory viral protein was also reported to be mutated in 60-100% of FIPVs, resulting in the loss or truncation of the 3c protein, whereas most FECVs carried an intact 3c gene [3-5, 8, 11, 13, 14]. Accordingly, it was considered that mutation of the S gene, 3c gene or both was involved in the acquisition or augmentation of lethal pathogenicity in the majorityof FIPV field strains. In the present study, we analyzed the S and 3c genes of FCoVs detected in faecal materials, effusion samples, and tissues that were obtained from cats in Japan to determine the frequency and location of the mutations. An analysis of the 3c gene suggested the horizontal infection of FCoVs, which were detected in effusions and tissues, among several housemate cats in a multi-cat environment.MATERIALS AND METHODSClinical specimens were obtained from 93 cats referred to private veterinary hospitals in Japan for suspected FIP based on clinical symptoms, including pyrexia, vomiting, diarrhoea, jaundice, emaciation, anaemia, ascites, pleural effusion, ophthalmologic abnormalities, neurological signs, and death. Some animals displayed an enlargement of abdominal organs that was noticed on palpation, radiography, or ultrasound. The samples of abdominal and pleural effusions, whole blood, serum, rectal swabs, faeces and tissues were sent to our laboratory under refrigeration. Tissues were obtained via autopsy of four cats that had been kept by the Abstract Work-related mental health impairment is recognized as a real problem in the context of helping responders, including health professionals, due to adverse health outcomes after a severe disaster. The Great East-Japan Earthquake, which occurred on 11 March 2011, was an unprecedented complex disaster that caused a nuclear accident at the Fukushima Daiichi Nuclear Power Plant (NPP). In addition to disaster stress and daily work, medical and health-care professionals, particularly nurses, provided counseling services to residents concerned about radiation health risks or mental health issues. This review focuses on the psychological aspects of the complex nuclear disaster, which was a combined artificial nuclear accident and natural disaster, and we investigated the psychological effects on hospital nurses associated with their experiences during the disaster. We looked at several investigations into the mental health of nurses after a nuclear disaster and in other situations. It was shown that mental health of nurses is impacted, not only after nuclear disasters but also in other circumstances. Furthermore, we noted the effects of extended periods of a heavy workload and daily life. Regarding anxiety about radiation exposure, nurses who had more knowledge of radiation tended to have better mental health, suggesting that education about the health risks of radiation exposure is important for health-care professionals. In summary, it is essential that nurses are provided with education about radiation exposure and its associated health risks, and also that there is a comprehensive approach to mental health care for nurses during the chronic phase of a disaster. Abstract Macrophages are a functionally heterogeneous group of cells with specialized functions depending not only on their subgroup but also on the function of the organ or tissue in which the cells are located. The concept of macrophage phenotypic heterogeneity has been investigated since the 1980s, and more recent studies have identified a diverse spectrum of phenotypic subpopulations. Several types of macrophages play a central role in the response to infectious agents and, along with other components of the immune system, determine the clinical outcome of major infectious diseases. Here, we review the functions of various macrophage phenotypic subpopulations, the concept of macrophage polarization, and the influence of these cells on the evolution of infections. In addition, we emphasize their role in the immune response in vivo and in situ, as well as the molecular effectors and signaling mechanisms used by these cells. Furthermore, we highlight the mechanisms of immune evasion triggered by infectious agents to counter the actions of macrophages and their consequences. Our aim here is to provide an overview of the role of macrophages in the pathogenesis of critical transmissible diseases and discuss how elucidation of this relationship could enhance our understanding of the host-pathogen association in organ-specific immune responses. Abstract Viral infection constitutes an unwanted intrusion that needs to be eradicated by host cells. On one hand, one of the first protective barriers set up to prevent viral replication, spread or persistence involves the induction of apoptotic cell death that aims to limit the availability of the cellular components for viral amplification. On the other hand, while they completely depend on the host molecular machinery, viruses also need to evade the cellular responses that are meant to destroy them. The existence of numerous antiapoptotic products within the viral kingdom proves that apoptosis constitutes a major threat that should better be bypassed. Among the different strategies developed to deal with apoptosis, one is based on what viruses do best: backfiring the cell on itself. Several unrelated viruses have been described to take advantage of apoptosis induction by expressing proteins targeted by caspases, the key effectors of apoptotic cell death. Caspase cleavage of these proteins results in various consequences, from logical apoptosis inhibition to more surprising enhancement or attenuation of viral replication. The present review aims at discussing the characterization and relevance of this post-translational modification that adds a new complexity in the already intricate host-apoptosis-virus triangle. Abstract Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry.MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions.Murine hepatitis virus macropinocytosis requires a fusogenic virus spike protein and signals through the epidermal growth factor receptor and the classical macropinocytosis pathway. These studies demonstrate CoV induction of macropinocytosis for a purpose other than entry and indicate that viruses likely exploit macropinocytosis at multiple steps in replication and pathogenesis. Abstract Citation Totura AL, Baric RS. 2015. Reply to "Statins may decrease the fatality rate of MERS infection." mBio 6(5):e01303-15. Abstract Rotavirus VP7 is a membrane-associated protein of the endoplasmic reticulum (ER). It is the product of rotavirus gene 9 which potentially encodes a protein of 326 amino acids that contains two amino terminal hydrophobic domains, hl and h2, each preceded by an initiation codon. Comparison of the size of products derived from altered genes containing coding sequences for both hl and h2 with those lacking the hl sequence ('dhl' mutants), indicates that initiation takes place at M30 immediately preceding h2 (residues F32 to 1-,48) and that h2 is cleaved, confirming the studies of others (Stirzaker, S. C., P. L. Whitfeld, D. L. Christie, A. R. Bellamy, and G. W. Both. 1987. J. Cell Biol. 105:2897-2903). Our previous work had shown that deletions in the carboxy end of h2, extending to amino acid 61 in the open reading frame, resulted in secretion of VP7. The region from amino acid number 51-61, present in wild-type VP7 but missing in the secreted mutant A47-61, was thus implicated to have a role in ER retention. To test this, a series of chimeric genes were constructed by fusing the first 63 codons of wild-type VP7, Al-14 or A51-61/ dhl, to the mouse salivary ~t-amylase gene, a secretory protein, such that the fusion junction was located at the exact mature terminus of amylase. The chimeric proteins VP763/amylase, Al-14Jamylase and A51-6163/dhl/amylase were secreted when expressed in cells and the h2 domain was cleaved when mRNA was translated in vitro. These results imply that the sequence 51-61 is necessary but not sufficient for ER retention. When a second series of VP7/amylase chimera were constructed extending the VP7 contribution to amino acid 111, the product expressed by AI-14HI/ amylase was not secreted whereas that of A47-61 ttl/ amylase was. Significantly, the intracellular Al-14m/amylase product exhibited an amylase enzymatic specific activity that was similar to that of the wild-type amylase product. We conclude that two regions of VP7 mediate its retention in the ER, the first lies within the sequence 51-61 and the second within the sequence 62-111, which contains the glycosylation site for VP7. Both regions are necessary for retention, though neither is sufficient alone. Abstract Ten years after the severe acute respiratory syndrome epidemic, a second coronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), has been identified as the cause of a highly lethal pneumonia in patients in the Middle East and in travelers from this region. Over the past 9 months, since the virus was first isolated, much has been learned about the biology of the virus. It is now clear that MERS-CoV is transmissible from person to person, and its close relationship with several bat coronaviruses suggests that these animals may be the ultimate source of the infection. However, many key issues need to be addressed, including identification of the proximate, presumably zoonotic, source of the infection, the prevalence of the infection in human populations, details regarding clinical and pathological features of the human infection, the establishment of a small rodent model for the infection, and the virological and immune basis for the severe disease observed in most patients. Most importantly, we do not know whether a MERS-CoV epidemic is likely or not. Infection with the virus has so far resulted in only 91 cases and 46 deaths (as of 29 July 2013), but it is nonetheless setting off alarm bells among public health officials, including Margaret Chan, Director-General of the World Health Organization, who called MERS-CoV "a threat to the entire world." This article reviews some of the progress that has been made and discusses some of the questions that need to be answered.Citation Perlman S. 2013. The Middle East respiratory syndrome-how worried should we be? mBio 4(4):e00531-13. Abstract Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only ∼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B-and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission. Abstract Background Respiratory viral (RV) outbreaks in rehabilitation facilities can jeopardise patient safety, interfere with patient rehabilitation goals and cause unit closures that impede patient flow in referring facilities. Problem Despite education about infection prevention practices, frequent RV outbreaks were declared each year at our rehabilitation facility. Methods Before and after study design. The primary outcome was the number of bed closure days due to outbreak per overall bed days. Process measures included delays in initiation of transmission-based precautions, RV testing and reporting of staff to occupational health and safety (OHS). Balancing measures included the number of isolation days and staff missed work hours.Interventions Based on comprehensive analysis of prior outbreaks, the following changes were implemented:(1) clear criteria for initiation of transmission-based precautions, (2) communication to visitors to avoid visitation if infectious symptoms were present, (3) exemption of staff absences if documented due to infectious illness, (4) development of an electronic programme providing guidance to staff about whether they should be excluded from work due to infectious illness. Results The number of bed closure days due to outbreak per overall bed days dropped from 2.8% to 0.5% during the intervention season and sustained at 0.6% during the postintervention season (p<0.001). There were fewer delays in initiation of droplet and contact precautions (28.8% to 15.5%, p=0.005) and collection of RV testing (42.9% to 20.3%, p<0.001), better reporting to OHS (9 vs 28.8 reports per 100 employees; p<0.001) and fewer isolation days (7.8% vs 7.3%; p=0.02) without a significant increase in missed work hours per 100 hours worked (4.0 vs 3.9; p=0.12). Conclusion This Quality Improvement study highlights the process changes that can prevent respiratory outbreaks in the rehabilitation setting. Abstract Emerging and re-emerging pathogens represent a substantial threat to public health, as demonstrated with numerous outbreaks over the past years, including the 2013-2016 outbreak of Ebola virus in western Africa. Coronaviruses are also a threat for humans, as evidenced in 2002/2003 with infection by the severe acute respiratory syndrome coronavirus (SARS-CoV), which caused more than 8000 human infections with 10% fatality rate in 37 countries. Ten years later, a novel human coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV), associated with severe pneumonia, arose in the Kingdom of Saudi Arabia. Until December 2016, MERS has accounted for more than 1800 cases and 35% fatality rate. Finding an animal model of disease is key to develop vaccines or antivirals against such emerging pathogens and to understand its pathogenesis. Knowledge of the potential role of domestic livestock and other animal species in the transmission of pathogens is of importance to understand the epidemiology of the disease. Little is known about MERS-CoV animal host range. In this paper, experimental data on potential hosts for MERS-CoV is reviewed. Advantages and limitations of different animal models are evaluated in relation to viral pathogenesis and transmission studies. Finally, the relevance of potential new target species is discussed. Abstract Background: Fecal calprotectin and immunoglobulin A (IgA) are markers of intestinal inflammation and immunity in adult dogs.Hypothesis: Fecal calprotectin and IgA concentrations in puppies are not influenced by fecal moisture in puppies but by enteropathogen shedding.Animals: Three hundred and twenty-four puppies. Methods: Fecal consistency was assessed by gross examination. Fecal moisture was evaluated before and after lyophilization. Canine parvovirus and coronavirus were detected in feces by qPCR and qRT-PCR respectively. Giardia intestinalis antigen was quantified by ELISA. The standard McMaster flotation technique was used to detect eggs and oocysts in feces. Fecal calprotectin and IgA concentrations were quantified by in-house radioimmunoassays.Results: For each marker (IgA and calprotectin), a strong positive correlation was observed between concentration in fresh feces and concentration in fecal dry matter. 75.6% of the puppies were found to be infected by at ≥1 of the enteropathogens evaluated. Fecal calprotectin concentration was significantly influenced by age (P = .001), with higher concentrations in younger puppies, but not by viral (P = .863) or parasitic infection (P = .791). Fecal IgA concentration was significantly influenced by enteropathogen shedding (P = .01), with a lower fecal IgA concentration in puppies shedding at ≥1 enteropathogen compared to puppies without any enteropathogen shedding, but not by age.Conclusions: Fecal calprotectin and IgA are of no diagnostic value to detect presence of enteropathogens in clinically healthy puppies or puppies with abnormal feces, but could help to better understand the maturation of digestive tract. Abstract Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management. Abstract Objective-This study aims to discuss the correlation between daily reported H7N9 cases and stock price indices in China.February 19, 2013 and March 31, 2014 were collected. A distributed lag non-linear model was used to describe the variation trend for the stock indices Abstract In-service training for health professionals to improve care of seriously ill newborns and children in low-income countries (Review) Abstract Surveillance of Middle East respiratory syndrome coronavirus (MERS-CoV) was conducted to explore the possible introduction and circulation of this novel virus in Catalonia, northeastern Spain. Five hundred and sixty-three samples from mild and severe respiratory infections collected between January 2012 and April 2013 were screened using real-time RT-PCR. All samples were negative, suggesting that MERS-CoV is not circulating silently in Catalonia. Abstract Background and purpose -There are limited data on risk factors for avascular necrosis of the hip, but cirrhosis has been proposed as a risk factor. We examined the association between cirrhosis and incidence of total hip arthroplasty for avascular necrosis.Methods -We used nationwide healthcare data to identify all Danish residents diagnosed with cirrhosis in 1994-2011, and matched them 1:5 by age and sex to non-cirrhotic reference individuals from the general population. We excluded people with a previous total hip arthroplasty, a previous hip fracture, or a previous diagnosis of avascular necrosis. We used stratifi ed Cox regression to estimate the hazard ratio (HR) for cirrhosis patients relative to reference individuals, adjusting for potential confounders. We used the cumulative incidence function to compute 5-year risks.Results -We included 25,421 cirrhosis patients and 114,052 reference individuals. Their median age was 57 years, and 65% were men. 45 cirrhosis patients and 44 reference individuals underwent total hip arthroplasty for avascular necrosis. Cirrhosis patients' HR for a total hip arthroplasty for avascular necrosis was 10 (95% CI: 6-17), yet their 5-year risk of avascular necrosis was only 0.2%. For the reference individuals, the 5-year risk was 0.02%.Interpretation -Cirrhosis is a strong risk factor for avascular necrosis of the hip, but it is rare even in cirrhosis patients. Abstract Microarray hybridization based identification of viral genotypes is increasingly assuming importance due to outbreaks of multiple pathogenic viruses affecting humans causing wide-spread morbidity and mortality. Surprisingly, microarray based identification of food-borne viruses, one of the largest groups of pathogenic viruses, causing more than 1.5 billion infections world-wide every year, has lagged behind. Cell-culture techniques are either unavailable or time consuming for routine application. Consequently, current detection methods for these pathogens largely depend on polymerase chain reaction (PCR) based techniques, generally requiring an investigator to preselect the target virus of interest. Here we describe the first attempt to use the microarray as an identification tool for these viruses. We have developed methodology to synthesize targets for virus identification without using PCR, making the process genuinely sequence independent. We show here that a tiling microarray can simultaneously detect and identify the genotype and strain of common food-borne viruses in a single experiment. Abstract OBJECTIVE: To explore the characteristics of seasonal distribution of active systemic lupus erythematosus (SLE) and the influences of meteorological factors including temperature and humidity on active systemic lupus erythematosus.The characteristics of seasonal distribution of active SLE and its correlation with meteorological factors were retrospectively analyzed in 640 patients living in the city of Zhanjiang, China and had active SLE between January 1997 and December 2006.In winter, when there are weaker ultraviolet (UV) rays, the ratio of patients with active SLE to total inpatients was 3.89 ø, which is significantly higher than in other seasons with stronger UV rays, including 2.17 ø in spring, 1.87 ø in summer and 2.12 ø in autumn. The number of patients with active SLE had significant negative correlation with mean temperature and was not significantly related to mean humidity.CONCLUSION: Active SLE has the characteristics of seasonal distribution and is associated with temperature. The mechanism remains to be further studied. Abstract Rationale: Acute fibrinous and organizing pneumonia (AFOP) is an uncommon type of acute lung injury associated with infection, connective tissue disorders, drug exposure, and hematologic malignancies.Patient concerns: A 53-year-old female presented with intermittent fever, chills, and dry cough since 10 days. Chest computed tomography scan showed multiple bilateral patchy infiltrates. PPD skin test was positive but tuberculosis antibody test and T-SPOT were negative.Diagnoses: Histologic examination revealed massive fibrinous exudation with organization within alveolar spaces and scattered neutrophilic infiltrates, which was consistent with AFOP.Interventions: This patient was treated with prednisolone therapy.Outcomes: Chest radiograph improvement and symptom improvement, including fever and respiratory symptoms, was observed after 2 week of oral prednisolone treatment. After 9-month of treatment, the patient was asymptomatic with stable disease and improved quality of life.Lessons: AFOP has unique pathologic manifestations; however, the condition is liable to be misdiagnosed as community-acquired pneumonia ortuberculosis. Antibiotics are ineffective, while some patients show good response to glucocorticoid therapy.Abbreviations: AFOP = acute fibrinous and organizing pneumonia, ALI = acute lung injury, DAD = diffuse alveolar damage, EP = eosinophil pneumonia, H&E = hematoxylin and eosin, NTMIs = nontuberculous mycobacterial infections, OP = organizing pneumonia. Abstract A dataset of 103 SARS-CoV isolates (101 human patients and 2 palm civets) was investigated on different aspects of genome polymorphism and isolate classification. The number and the distribution of single nucleotide variations (SNVs) and insertions and deletions, with respect to a "profile", were determined and discussed ("profile" being a sequence containing the most represented letter per position). Distribution of substitution categories per codon positions, as well as synonymous and non-synonymous substitutions in coding regions of annotated isolates, was determined, along with amino acid (a.a.) property changes. Similar analysis was performed for the spike (S) protein in all the isolates (55 of them being predicted for the first time). The ratio Ka/Ks confirmed that the S gene was subjected to the Darwinian selection during virus transmission from animals to humans. Isolates from the dataset were classified according to genome polymorphism and genotypes. Genome polymorphism yields to two groups, one with a small number of SNVs and another with a large number of SNVs, with up to four subgroups with respect to insertions and deletions. We identified three basic nine-locus genotypes: TTTT/TTCGG, CGCC/TTCAT, and TGCC/TTCGT, with four subgenotypes. Both classifications proposed are in accordance with the new insights into possible epidemiological spread, both in space and time. Abstract Background: Acute diarrhea is a common clinical presentation of dogs. The effect of specific anti-diarrheal probiotic pastes (ADPPs) in the management of acute, uncomplicated diarrhea in dogs is unknown.Hypothesis: Administration of an ADPP containing Enterococcus faecium 4b1707 will improve the clinical outcome of acute, uncomplicated diarrhea in dogs compared to placebo.Animals: One hundred forty-eight client-owned dogs with acute diarrhea as the main clinical sign.Methods: Double-blinded, placebo-controlled, randomized, blocked, multicenter clinical field study conducted at 14 primary care veterinary practices in the United Kingdom and Ireland.The ADPP was associated with better clinical outcome compared to placebo in dogs with acute, uncomplicated diarrhea. Dogs in the ADPP group had a significantly shorter duration of diarrhea (ADPP: median, 32 hours; 95% confidence interval [CI], 2-118; n = 51; Placebo: median, 47 hours; 95% CI, 4-167; n = 58; P = .008) and the rate of resolution of diarrhea was 1.60 times faster in the ADPP group than in the Placebo group (ratio, 1.60; 95% CI, 1.08-2.44; P = .02). Fewer dogs required additional medical intervention (AMI) for non-improvement or worsening in the ADPP group compared to the Placebo group (3.5% of dogs and 14.8% of dogs, respectively), with a relative risk of 0.88 (P = .04; AMI, ADPP, 3.5%, 2/57 dogs; Placebo, 14.8%, 9/61 dogs; relative risk, 0.88; 95% CI, 0.77-0.99).Conclusion and Clinical Importance: The ADPP may accelerate resolution of acute diarrhea in dogs and decrease the requirement for AMI.K E Y W O R D S Abstract In vitro integration of the polytopic, transmembrane lactose permease into membrane vesicles from Escherichia coli is demonstrated. To this end the enzyme was synthesized in a homologous, cell-free transcription-translation system. In this system, synthesis occurred in an essentially membrane-free environment leading to the formation of lactose permease aggregates, which were resistant to protease digestion and detergent solubilization. However, if inverted membrane vesicles from E. coli were included in the synthesis reaction, most de novo-synthesized lactose permease could be recovered from a membranecontaining subfraction (enriched in leader [signal] peptidase activity). This membrane association of lactose permease was Na2CO3 resistant, detergent sensitive, and yielded a distinct pattern of proteolytic cleavage peptides. Moreover, membrane vesicles when present cotranslationally during synthesis of lactose permease, acquired the capability to accumulate lactose, strongly suggesting a correct in vitro assembly of the enzyme.Because of the extensive aggregation of lactose permease synthesized in the absence of membranes, only low amounts originating from the soluble enzyme pool integrated posttranslationally into the membrane vesicles. Unlike the translocation of the outer membrane protein LamB into membrane vesicles, integration of lactose permease was found to be independent of the H+-motive force. Abstract Objective Outbreaks of transmissible respiratory infection are suspected to have significant effects on the health of pediatric and geriatric patients. The objective was to assess the impact of the Middle East respiratory syndrome (MERS) outbreak on the use of emergency resources.Methods An ecologic analysis of emergency department (ED) records between September and December 2015, was performed. Data was obtained from the National Emergency Department Information System database for Korea. All demographic and diagnostic data from patients presenting with febrile symptoms as a main complaint were collected. The data were compared to the equivalent period in the three years preceding the MERS outbreak in Korea.Following the MERS outbreak, there was an increase in overall ED visits by febrile patients and the proportion of visits by febrile patients, relative to total ED attendances. This effect was more prominent in the children under five years. The duration of the chief complaint before ED arrival and the length of ED stay were significantly increased among younger pediatric patients. Decreased body temperature on arrival was observed in younger pediatric patients.Conclusion MERS outbreak appears to have had a significant effects on ED use by febrile patients. The use of emergency care services by pediatric patients makes them more vulnerable to an outbreak of a transmissable disease. An effective strategy to control emergency center visits by non-urgent febrile patients and provide proper medical services is urgently needed. Abstract Sarcoidosis is an incurable, chronic granulomatous disease primarily involving the lungs and lymph nodes of unknown etiology, treated with non-specific antiinflammatory/immunosuppressive drugs. Persistently symptomatic patients worsen with a disabling, potentially fatal clinical course. To determine a possible infectious cause, we correlated in a case control study clinical information with the presence of bacterial DNA in sarcoidosis mediastinal lymph nodes compared to control lymph nodes resected during cancer surgery.We retrospectively studied formalin-fixed, paraffin-embedded, mediastinal lymph nodes from 30 sarcoidosis patients and 30 control lung cancer patients. Nucleic acids were extracted from nodes, were evaluated by rRNA PCR for bacterial 16S rDNA, and the result was sequenced and compared to a bacterial sequence library. Clinical information was correlated.: 11/30 (36.7%) of lymph nodes from sarcoidosis patients had detectable bacterial DNA, significantly more than control patient lymph nodes (2/30, 6.7%), p = 0.00516. At presentation, 19/30 (63.3%) sarcoidosis patients were symptomatic including all patients with detectable bacterial DNA. Radiographically, there were 18 Stage I and 12 Stage II patients. All Stage II patients were symptomatic and 75% had PCR-detectable bacteria. After a mean followup of 52.8 + 32.8 months, all patients with PCR-detectable bacteria were persistently symptomatic requiring treatment. DISCUSSION: 36.6% of sarcoidosis patients had detectable bacteria DNA on presentation, allwere quite symptomatic, and most were radiographically-advanced stage II patients. These findings suggest bacterial DNA-positive, symptomatic patients have more aggressive Abstract Cellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysin-rvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of isolated BAR domains in vitro, the current paradigm is that BAR domain-containing proteins polymerize into cylindrical scaffolds that stabilize lipid tubules. But in nature, proteins that contain BAR domains often also contain large intrinsically disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data suggest that the ability to concentrate disordered domains is a key driver of membrane remodeling and fission by BAR domain-containing proteins. Abstract Human coronavirus (HCoV), a member of Coronaviridae family, is the causative agent of upper respiratory tract infections and "atypical pneumonia". Despite severe epidemic outbreaks on several occasions and lack of antiviral drug, not much progress has been made with regard to an epitope-based vaccine designed for HCoV. In this study, a computational approach was adopted to identify a multiepitope vaccine candidate against this virus that could be suitable to trigger a significant immune response. Sequences of the spike proteins were collected from a protein database and analyzed with an in silico tool, to identify the most immunogenic protein.Both T cell immunity and B cell immunity were checked for the peptides to ensure that they had the capacity to induce both humoral and cell-mediated immunity. The peptide sequence from 88-94 amino acids and the sequence KSSTGFVYF were found as the most potential B cell and T cell epitopes, respectively. Furthermore, conservancy analysis was also done using in silico tools and showed a conservancy of 64.29% for all epitopes. The peptide sequence could interact with as many as 16 human leukocyte antigens (HLAs) and showed high cumulative population coverage, ranging from 75.68% to 90.73%. The epitope was further tested for binding against the HLA molecules, using in silico docking techniques, to verify the binding cleft epitope interaction. The allergenicity of the epitopes was also evaluated. This computational study of design of an epitope-based peptide vaccine against HCoVs allows us to determine novel peptide antigen targets in spike proteins on intuitive grounds, albeit the preliminary results thereof require validation by in vitro and in vivo experiments. Abstract Objective: Parasitic infestation is a major cause of losses in livestock production in tropical regions. A cross-sectional study was conducted to determine the prevalence of Gastro-intestinal (GI) parasites of dromedary camel (Camelus dromedarius) and fat-tailed sheep (dhumba), and the prevalence of hemoparasites in camel from Dhaka, Bangladesh. Materials and Methods: A total of 87 fecal samples (32 dhumba and 55 camel) and 55 camel blood samples were collected during September-October 2015. Fecal samples were examined by direct smear, sedimentation method, flotation technique, and McMaster technique for GI parasite. Giemsa stained blood smears were examined under microscope for hemoparasite detection. Results: 62% camel (n = 34; 95% confidence interval (CI): 47.7-74.6) were infected with at least one genus of parasite. 15% camel were harboring more than one genus of parasite. The prevalence of GI parasite and hemoparasite in camel were recorded as Trichuris spp. (n = 16; 29%; 95% CI: 17.6-42.9), Balantidium coli (n = 12; 22%; 95% CI: 11.8-35.0), Trichostrongylus spp. (n = 7; 13%; 95% CI: 5.3-24.5), Strongyloides spp. (n = 5; 9%; 95% CI: 3.0-20.0), Anaplasma spp. (n = 5; 9%; 95% CI: 3.02-20.0), Paragonimus spp. (n = 1; 2%; 95% CI: 0.05-9.7), Schistosoma spp. (n = 1; 2%; 95% CI: 0.05-9.7), Hymenolepis spp. (n = 1; 2%; 95% CI: 0.05-9.7), Moniezia spp. (n = 1; 2%; 95% CI: 0.05-9.7), and Babesia spp. (n = 1; 2%; 95% CI: 0.05-9.7). Mean EPG feces of camel was 291.76 ± 42.03 with a range of 0-1,400. Total 59.4% dhumba (n = 19; 95% CI: 41-76) were positive for GI parasite, including Trichostrongylus spp. (n = 10; 31.3%; 95% CI: 16.1-50), Strongyloides spp. (n = 9; 28%; 95% CI: 13.8-46.8), B. coli (n = 5; 15.6%; 95% CI: 5.3-32.8), and Trichuris spp. (n = 4; 12.5%; 95% CI: 3.5-28.9). Conclusions: High percentage of parasitic infestation in camel and dhumba in the present study refers to the necessity of use of anthelmintic for health and production improvement and to prevent zoonotic parasite transmission to animal handler and workers.ARTICLE HISTORYAccess article distributed under the terms of the Creative Commons Attribution 4.0 Licence (http://creativecommons.org/ licenses/by/4.0) Correspondence Ariful Islam arif@ecohealthalliance.org Program co-ordinator, USAID PREDICT-2 project, EcoHealth Alliance, Institute of Epidemiology, Disease control and Research (IEDCR), Mohakhali, Dhaka, Bangladesh How to cite: Islam A, Islam S, Ferdous J, Rahman MK, Uddin MH, Akter S, Rahman MH, Hassan MM. Diversity and prevalence of parasitic infestation with zoonotic potential in dromedary camel (Camelus dromedarius) and fat-tailed sheep (dhumba) in Bangladesh. J Adv Vet Anim Res 2019; 6(1):142-147. Abstract Determining the fitness of viral genotypes has become a standard practice in virology as it is essential to evaluate their evolutionary potential. Darwinian fitness, defined as the advantage of a given genotype with respect to a reference one, is a complex property that captures, in a single figure, differences in performance at every stage of viral infection. To what extent does viral fitness result from specific molecular interactions with host factors and regulatory networks during infection? Can we identify host genes in functional classes whose expression depends on viral fitness? Here, we compared the transcriptomes of tobacco plants infected with seven genotypes of tobacco etch potyvirus that differ in fitness. We found that the larger the fitness differences among genotypes, the more dissimilar the transcriptomic profiles are. Consistently, two different mutations, one in the viral RNA polymerase and another in the viral suppressor of RNA silencing, resulted in significantly similar gene expression profiles. Moreover, we identified host genes whose expression showed a significant correlation, positive or negative, with the virus' fitness. Differentially expressed genes which were positively correlated with viral fitness activate hormone-and RNA silencing-mediated pathways of plant defense. In contrast, those that were negatively correlated with fitness affect metabolism, reducing growth, and development. Overall, these results reveal the high information content of viral fitness and suggest its potential use to predict differences in genomic profiles of infected hosts. Abstract There is an increasing body of evidence suggesting that transmission of respiratory viruses occurs through the inhalation of virus-laden particles. Our study describes the use of an aerosol sampling system to monitor the prevalence of airborne viruses in a hospital setting. Using SKC AirCheck Touch pumps, with National Institute for Occupational Safety and Health (NIOSH) bioaerosol samplers and SKC filter cassette blanks, 28 aerosol samples were collected in a hospital ward in Singapore. Following DNA/RNA extraction, real-time RT-PCR/PCR was used for the detection of influenza A, B and D viruses, coronaviruses, enteroviruses, and adenoviruses. Airborne virus was detected in nine (32%) of 28 samples. Among the nine positive samples, eight were PCR-positive for adenovirus and one for influenza A virus. Our data suggest that bioaerosol sampling could be valuable in monitoring for airborne respiratory viruses in clinical environments to better understand the risk of infection during a hospital visit. Abstract Understanding transcription factor (TF) mediated control of gene expression remains a major challenge at the interface of computational and experimental biology. Computational techniques predicting TF-binding site specificity are frequently unreliable. On the other hand, comprehensive experimental validation is difficult and time consuming. We introduce a simple strategy that dramatically improves robustness and accuracy of computational binding site prediction. First, we evaluate the rate of recurrence of computational TFBS predictions by commonly used sampling procedures. We find that the vast majority of results are biologically meaningless. However clustering results based on nucleotide position improves predictive power. Additionally, we find that positional clustering increases robustness to long or imperfectly selected input sequences. Positional clustering can also be used as a mechanism to integrate results from multiple sampling approaches for improvements in accuracy over each one alone. Finally, we predict and validate regulatory sequences partially responsible for transcriptional control of the mammalian type A g-aminobutyric acid receptor (GABA A R) subunit genes. Positional clustering is useful for improving computational binding site predictions, with potential application to improving our understanding of mammalian gene expression. In particular, predicted regulatory mechanisms in the mammalian GABA A R subunit gene family may open new avenues of research towards understanding this pharmacologically important neurotransmitter receptor system. Abstract Background: Strategies are urgently needed to mitigate the risk of zoonotic disease emergence in southern China, where pathogens with zoonotic potential are known to circulate in wild animal populations. However, the risk factors leading to emergence are poorly understood, which presents a challenge in developing appropriate mitigation strategies for local communities.Methods: Residents in rural communities of Yunnan, Guangxi and Guangdong provinces were recruited and enrolled in this study. Data were collected through ethnographic interviews and field observations, and thematically coded and analysed to identify both risk and protective factors for zoonotic disease emergence at the individual, community and policy levels.Results: Eighty-eight ethnographic interviews and 55 field observations were conducted at nine selected sites. Frequent human-animal interactions and low levels of environmental biosecurity in local communities were identified as risks for zoonotic disease emergence. Policies and programmes existing in the communities provide opportunities for zoonotic risk mitigation.This study explored the relationship among zoonotic risk and human behaviour, environment and policies in rural communities in southern China. It identifies key behavioural risk factors that can be targeted for development of tailored risk-mitigation strategies to reduce the threat of novel zoonoses. Abstract Because many aspects of viral infection dynamics and inhibition are governed by stochastic processes, single-cell analysis should provide more information than approaches using population averaging. We have developed a microfluidic device composed of ~6000 wells, with each well containing a microstructure to capture single, infected cells replicating an enterovirus expressing a fluorescent reporter protein. We have used this system to characterize enterovirus inhibitors with distinct mechanisms of action. Single-cell analysis reveals that each class of inhibitor interferes with the viral infection cycle in a manner that can be distinguished by principal component analysis. Single-cell analysis of antiviral candidates not only reveals efficacy but also facilitates clustering of drugs with the same mechanism of action and provides some indication of the ease with which resistance will develop. Abstract Mycobacteriosis in swine is a common zoonosis found in abattoirs during meat inspections, and the veterinary authority is expected to inform the producer for corrective actions when an outbreak is detected. The expected value of the number of condemned carcasses due to mycobacteriosis therefore would be a useful threshold to detect an outbreak, and the present study aims to develop such an expected value through time series modeling. The model was developed using eight years of inspection data (2003 to 2010) obtained at 2 abattoirs of the Higashi-Mokoto Meat Inspection Center, Japan. The resulting model was validated by comparing the predicted timedependent values for the subsequent 2 years with the actual data for 2 years between 2011 and 2012. For the modeling, at first, periodicities were checked using Fast Fourier Transformation, and the ensemble average profiles for weekly periodicities were calculated. An Auto-Regressive Integrated Moving Average (ARIMA) model was fitted to the residual of the ensemble average on the basis of minimum Akaike's information criterion (AIC). The sum of the ARIMA model and the weekly ensemble average was regarded as the time-dependent expected value. During 2011 and 2012, the number of whole or partial condemned carcasses exceeded the 95% confidence interval of the predicted values 20 times. All of these events were associated with the slaughtering of pigs from three producers with the highest rate of condemnation due to mycobacteriosis. Abstract Chronic infection and cancer are associated with suppressed T cell responses in the presence of cognate antigen. Recent work identified memory-like CXCR5 + TCF1 + CD8 + T cells that sustain T cell responses during persistent infection and proliferate upon anti-PD1 treatment. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to CXCR5 + CD8 + T cell expansion in an IL-27-and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8 + T cells. We found that CD8 + T cell-intrinsic IL-27 signaling safeguards the ability of TCF1 hi cells to maintain proliferation and avoid terminal differentiation or programmed cell death. Mechanistically, IL-27 endowed rapidly dividing cells with IRF1, a transcription factor that was required for sustained division in a cell-intrinsic manner. These findings reveal that IL-27 opposes IFN-I to uncouple effector differentiation from cell division and suggest that IL-27 signaling could be exploited to augment self-renewing T cells in chronic infections and cancer. Abstract Mycoplasma pneumoniae infections mainly involve respiratory tract; however, also can manifestate other symptoms by site involved. Extrapulmonary manifestations of M. pneumoniae infection are rarely known to occur without pneumonia. Herein we report a case of a 9-year-old boy who presented with acute cholestatic hepatitis in the absence of pneumonia. Rhabdomyolysis, skin rash, and initial laboratory results suspicious of disseminated intravascular coagulopathy were also observed in this patient. M. pneumoniae infection was identified by a 4-fold increase in immunoglobulin G antibodies to M. pneumoniae between acute and convalescent sera by enzyme-linked immunosorbent assay. This is the first pediatric case in Korea of M. pneumoniae infection presenting with acute cholestatic hepatitis in the absence of pneumonia. Abstract The ability of a protein to fold into unique functional state or to stay intrinsically disordered is encoded in its amino acid sequence. Both ordered and intrinsically disordered proteins (IDPs) are natural polypeptides that use the same arsenal of 20 proteinogenic amino acid residues as their major building blocks. The exceptional structural plasticity of IDPs, their capability to exist as heterogeneous structural ensembles and their wide array of important disorder-based biological functions that complements functional repertoire of ordered proteins are all rooted within the peculiar differential usage of these building blocks by ordered proteins and IDPs. In fact, some residues (so-called disorder-promoting residues) are noticeably more common in IDPs than in sequences of ordered proteins, which, in their turn, are enriched in several orderpromoting residues. Furthermore, residues can be arranged according to their "disorder promoting potencies," which are evaluated based on the relative abundances of various amino acids in ordered and disordered proteins. This review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and concerns glutamic acid, which is the second most disorder-promoting residue. Abstract The ongoing Ebola virus (EBOV) disease (EVD) epidemic in Western Africa is the largest EVD outbreak recorded to date and requires the rapid development and deployment of antiviral measures. The viral glycoprotein (GP) facilitates host cell entry and, jointly with cellular interaction partners, constitutes a potential target for antiviral intervention. However, it is unknown whether the GPs of the currently and previously circulating EBOVs use the same mechanisms for cellular entry and are thus susceptible to inhibition by the same antivirals and cellular defenses. Here, we show that the GPs of the EBOVs circulating in 1976 and 2014 transduce the same spectrum of target cells, use the same cellular factors for host cell entry, and are comparably susceptible to blockade by antiviral interferon-induced transmembrane proteins and neutralizing antibody KZ52. Thus, the viruses responsible for the ongoing EVD epidemic should be fully susceptible to established antiviral strategies targeting GP and cellular entry factors. Abstract Complete Middle East respiratory syndrome coronavirus (MERS-CoV) genome sequences were obtained from nasal swabs of dromedary camels sampled in the Kingdom of Saudi Arabia through direct analysis of nucleic acid extracts or following virus isolation in cell culture. Consensus dromedary MERS-CoV genome sequences were the same with either template source and identical to published human MERS-CoV sequences. However, in contrast to individual human cases, where only clonal genomic sequences are reported, detailed population analyses revealed the presence of more than one genomic variant in individual dromedaries. If humans are truly infected only with clonal virus populations, we must entertain a model for interspecies transmission of MERS-CoV wherein only specific genotypes are capable of passing bottleneck selection.Citation Briese T, Mishra N, Jain K, Zalmout IS, Jabado OJ, Karesh WB, Daszak P, Mohammed OB, Alagaili AN, Lipkin WI. 2014. Middle East respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in Saudi Arabia. mBio 5(3): Abstract Diet-induced obesity is associated with systemic inflammation, which is considered to originate predominantly from the adipose tissue. Quercetin and resveratrol are two dietary polyphenols that exhibit anti-inflammatory properties and anti-insulin resistance when administered in isolation or combination (CQR). It remains unknown whether CQR reduces high fat diet (HFD)-induced obesity and inflammation in rats. In the current study, 46 male Wistar rats were divided into two groups, one of which was fed a normal diet (ND, 5.4% fat, w/w) and one of which was fed a HFD (45% fat, w/w) for 3 weeks. Following removal of the 12 most obesity-resistant rats from the HFD group, the remaining rats were divided into two sub-groups: A HFD group and a HFD+CQR group (administered 120 mg/kg/day resveratrol and 240 mg/kg/day quercetin). The results revealed that the HFD+CQR group had significantly lower body weights at 11 weeks compared with the HFD group and had significantly reduced visceral adipose tissue weights and adipocyte sizes. Serum lipid profiles were also significantly ameliorated in the HFD+CQR group. CQR attenuated the expression of systemic proinflammatory adipokines, including leptin, tumor necrosis factor-α, monocyte chemoattractant protein-1 and interleukin-6. It also reduced the recruitment of mast cells to the epididyotic adipose tissue (EAT). Furthermore, CQR reversed the HFD-induced suppression of 5'-adenosine monophosphate-activated protein kinase α1 (AMPKα1) phosphorylation and sirtuin 1 (SIRT1) expression in EAT. In conclusion, CQR may suppress obesity and associated inflammation via the AMPKα1/SIRT1 signaling pathway in rats fed a HFD. Abstract Bats are natural reservoirs of several important emerging viruses. Cross-species transmission appears to be quite common among bats, which may contribute to their unique reservoir potential. Therefore, understanding the importance of bats as reservoirs requires examining them in a community context rather than concentrating on individual species. Here, we use a network approach to identify ecological and biological correlates of cross-species virus transmission in bats and rodents, another important host group. We show that given our current knowledge the bat viral sharing network is more connected than the rodent network, suggesting viruses may pass more easily between bat species. We identify host traits associated with important reservoir species: gregarious bats are more likely to share more viruses and bats which migrate regionally are important for spreading viruses through the network. We identify multiple communities of viral sharing within bats and rodents and highlight potential species traits that can help guide studies of novel pathogen emergence. Abstract The induction of monocyte/macrophage procoagulant activity (PCA) has been implicated in the pathogenesis of murine hepatitis virus strain 3 (MHV-3) infection and disease. Previously, we have shown that induction of PCA by MHV-3 correlated with resistance/susceptibility to infection in different mouse strains. In this study, all BALB/cJ mice that were infected with 103 plaque-forming units of MHV-3 developed severe liver disease and died within 96-120 h. Examination of the livers of these animals showed marked hepatic necrosis, deposition of fibrin, and cellular expression of PCA by direct immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. Splenic mononuclear cells recovered from these mice expressed high concentrations of PCA with time after infection. Infusion into mice of a high-titered monoclonal antibody that neutralized PCA (3D4.3) attenuated the development of hepatic necrosis and enhanced survival in a dose-dependent manner. All of the animals receiving 100 #g, and 44% and 22% of the animals that received 50 and 25 #g per day, respectively, survived for 10 d and made a full recovery. Administration of the antibody resulted in a dose-dependent reduction in fibrin deposition, PCA expression as detected by direct immunofluorescence staining and by a functional assay. In animals treated with high concentrations of antibody, titers of antibody to PCA fell from 87 +_ 15/~g/ml to 100 +_ 7 ng/ml during the active phase of the disease, consistent with sequestration due to binding of the immunoglobulin to cells expressing PCA. Surviving animals, when rechallenged with MHV-3, had a 40% mortality, consistent with the known rates of metabolism of immunoglobulin. This further suggested that protection was by a passive mechanism. The results reported here demonstrate that a neutralizing antibody to PCA protects animals from fulminant hepatitis and death associated with MHV-3 infection, and supports the notion that PCA is a potent inflammatory mediator that plays a pivotal role in the pathogenesis of liver injury resulting from MHV-3 infection. Abstract Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) can be associated with various disorders. Recent investigation has involved clinical studies in collaboration with clinical investigators and pathologists on the pathogenetic mechanisms of ALI or ARDS caused by various disorders. This literature review includes a brief historical retrospective of ALI/ARDS, the neurogenic pulmonary edema due to head injury, the long-term experimental studies and clinical investigations from our laboratory, the detrimental role of NO, the risk factors, and the possible pathogenetic mechanisms as well as therapeutic regimen for ALI/ARDS. Abstract Hsueh P-R, Asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARSCoV-2): Facts and myths, Journal of Microbiology, Immunology and Infection, https://doi. Abstract Although the frequency of respiratory viral infection in patients with pulmonary acute respiratory distress syndrome (ARDS) is not uncommon, clinical significance of the condition remains to be further elucidated. The purpose of this study was to compare characteristics and outcomes of patients with pulmonary ARDS infected with influenza and other respiratory viruses. Methods: Clinical data of patients with pulmonary ARDS infected with respiratory viruses January 2014-June 2018 were reviewed. Respiratory viral infection was identified by multiplex reverse transcription-polymerase chain reaction (RT-PCR). Results: Among 126 patients who underwent multiplex RT-PCR, respiratory viral infection was identified in 46% (58/126): 28 patients with influenza and 30 patients with other respiratory viruses. There was no significant difference in baseline and clinical characteristics between patients with influenza and those with other respiratory viruses. The use of extracorporeal membrane oxygenation (ECMO) was more frequent in patients with influenza than in those with other respiratory viruses (32.1% vs 3.3%, p=0.006). Co-bacterial pathogens were more frequently isolated from respiratory samples of patients with pulmonary ARDS infected with influenza virus than those with other respiratory viruses. (53.6% vs 26.7%, p=0.036). There were no significant differences regarding clinical outcomes. In multivariate analysis, acute physiology and chronic health evaluation II was associated with 30-mortality (odds ratio, 1.158; 95% confidence interval, 1.022-1.312; p=0.022). Conclusion: Respiratory viral infection was not uncommon in patients with pulmonary ARDS. Influenza virus was most commonly identified and was associated with more co-bacterial infection and ECMO therapy. Abstract MINIREVIEWSComplementary examinations other than neuroimaging and neurosonology in acute stroke) is a peer-reviewed open access academic journal that aims to guide clinical practice and improve diagnostic and therapeutic skills of clinicians. The primary task of WJCC is to rapidly publish high- Abstract Acute respiratory tract infections (ARI) in infancy have been implicated in the development of chronic respiratory disease, but the complex interplay between viruses, bacteria and host is not completely understood. We aimed to prospectively determine whether nasal microbiota changes occur between the onset of the first symptomatic ARI in the first year of life and 3 weeks later, and to explore possible associations with the duration of respiratory symptoms, as well as with host, environmental and viral factors.Nasal microbiota of 167 infants were determined at both time-points by 16S ribosomal RNA-encoding gene PCR amplification and subsequent pyrosequencing. Infants were clustered based on their nasal microbiota using hierarchical clustering methods at both time-points.We identified five dominant infant clusters with distinct microbiota at the onset of ARI but only three clusters after 3 weeks. In these three clusters, symptom persistence was overrepresented in the Streptococcaceae-dominated cluster and underrepresented in the cluster dominated by "Others" ( p<0.001). Duration of symptoms was not associated with the type of respiratory virus.Infants with prolonged respiratory symptoms after their first ARI tend to exhibit distinct microbial compositions, indicating close microbiota-host interactions that seem to be of importance for symptom persistence and recovery.@ERSpublications Nasal microbiota in infants is associated with symptom persistence after acute symptomatic respiratory infections. Abstract Epidemics such as viral haemorrhagic fevers, severe acute respiratory syndrome, Middle East respiratory syndrome coronavirus or yet unknown ones have few chances of disappearing. Globalization, worldwide travel, climate change, social conflicts and wars, among others, are likely to favor the emergence of epidemics. Preparedness of hospitals to prevent the spread of these outbreaks is among the prioritized political programmes of many countries. The EuroNHID network has in the past drawn a map of features and equipment of hospitals across Europe to take care of highly contagious patients. We update the data regarding isolation capabilities and recommendations, with an emphasis on Mediterranean countries. Abstract In the wake of dynamic economic and political transitions worldwide, the Institute of Medicine recently released its report advocating investments in global health from the United States (US). The expert panel reinforces the 'transnational and interdisciplinary' nature of global health research and practice as an endeavor 'to improve health and achieve greater equity for all people worldwide.' This report was judiciously timed given the growing recognition of global health, and is also acknowledged for incorporating themes that are particularly pertinent to the twenty-first century. New paradigms are introduced, denouncing the dichotomous distinction between rich and poor countries with the rapidly transitioning countries emerging as global powers, and affirming the need for models of respectful partnership and wider translation of science into practice. Cultivating sustainable partnerships and investing in the understanding and combat of diseases worldwide will become increasingly important for the US to maintain its global competitiveness, and may offer lessons in innovation, efficiency, and organization of institutions and human resources. Abstract We provide the first genetic sequence data for a Dipylidium species from a wild carnivore plus an analysis of the effects of ecological, demographic, physiological and behavioural factors on Dipylidium sp. infection prevalence in a social carnivore, the spotted hyaena (Crocuta crocuta), in the Serengeti National Park, Tanzania. Our sequence data from a mitochondrial gene fragment (1176 base pair long) had a similarity of between 99% and 89% to Dipylidium caninum. We determined infection prevalence in 146 faecal samples from 124 known animals in three social groups (termed clans) using molecular screening and Dipylidium proglottid presence. Our analysis revealed significantly higher infection prevalence in juveniles (55%) than adults (15.8%), indicating that predominantly juveniles maintained infection in clans. The likelihood of infection in juveniles significantly: (1) increased as the number of adults and older juveniles (>6 months) at communal dens increased, implying a positive relationship between this factor and the size of the intermediate host (probably a flea species) population at communal dens; (2) decreased as the number of younger juveniles (<6 months) increased, suggesting that the chance of susceptible juveniles ingesting infected fleas during self-grooming declined as the number of infected fleas per younger juvenile declined; and (3) decreased during periods of low prey abundance in clan territories when an increased reliance on long-distances foraging excursions reduces the number of clan members visiting communal dens, possibly resulting in a decline in flea populations at dens. Long-distance foraging also increases the intervals (in days) between nursing visits by lactating females to their offspring. Lengthy intervals between milk intake by infected juveniles may reduce adult Dipylidium fecundity and hence decrease infection prevalence in the den flea population. Our study provides useful insights into Dipylidium epidemiology in a social carnivore population subject to large fluctuations in prey abundance. Abstract A number of virologic and Abstract Moraxella catarrhalis is a ubiquitous human-specific bacterium commonly associated with upper and lower respiratory tract infections, including otitis media, sinusitis and chronic obstructive pulmonary disease. The bacterium uses an autotransporter protein UspA1 to target an important human cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Using X-ray crystallography, we show that the CEACAM1 receptor-binding region of UspA1 unusually consists of an extended, rod-like left-handed trimeric coiled-coil. Mutagenesis and binding studies of UspA1 and the N-domain of CEACAM1 have been used to delineate the interacting surfaces between ligand and receptor and guide assembly of the complex. However, solution scattering, molecular modelling and electron microscopy analyses all indicate that significant bending of the UspA1 coiled-coil stalk also occurs. This explains how UspA1 can engage CEACAM1 at a site far distant from its head group, permitting closer proximity of the respective cell surfaces during infection. Abstract dence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well. Abstract The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rabl is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans-Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20°C or 43°C treatment, resulted in a significant shift of the distribution towards the trans-TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP3,S treatment of permeabilized cells, the receptor was detected in the/3-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL-containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions. p ROTEIN sorting/targeting along the exocytotic pathway is mediated by various types of targeting/sorting signals (Pelham, 1989; Hong and Tang, 1993) . The carboxyl-terminal Lys, Arp, Glu, Leu tetrapeptide (KDEL) ~ (HDEL in yeast) and related sequences have been shown to Abstract A 70-year-old woman was admitted to our hospital for dyspnea and a fever of 2 weeks duration. Chest imaging showed bilateral infiltration, and a rapid diagnostic test for influenza virus, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Legionella spp. was negative. She was intubated and mechanically ventilated and underwent bronchoalveolar lavage. Bronchoalveolar lavage fluid yielded no significant pathogens, and the multiplex polymerase chain reaction test was positive only for human bocavirus. Specific antibodies against significant pathogens were not increased in paired sera, so we diagnosed her with primary human bocavirus pneumonia. Abstract Background: Acquired myasthenia gravis (AMG) is increasingly recognized in cats, yet information regarding the natural history of the disease, treatment, and outcome including occurrence of immune and spontaneous remission remains limited.Objective: To determine the long-term outcome of cats with AMG without evidence of a cranial mediastinal mass (CMM).Animals: Eight cats diagnosed with AMG without evidence of a CMM.: Retrospective case series. The medical records of cats diagnosed with AMG between 2005 and 2018 from 2 veterinary referral hospitals were reviewed for inclusion. Inclusion criteria consisted of a diagnosis of AMG, thoracic imaging, serum biochemistry including measurement of creatine kinase, and a CBC. Exclusion criteria were the presence of an identifiable CMM, or administration of methimazole or carbimazole. Abstract This commentary presents a novel outlook for public health authorities in the affected countries to detect and respond quickly to the emerging public health threats such as Middle East respiratory syndrome coronavirus (MERS-CoV). Implementing an innovative electronic surveillance system called syndromic surveillance system is essential for global health security. Abstract Middle East respiratory syndrome (MERS) is a lethal respiratory disease -caused by MERScoronavirus (MERS-CoV) which was first identified in 2012. Especially, pregnant women can be expected as highly vulnerable candidates for this viral infection. In May 2015, this virus was spread in Korea and a pregnant woman was confirmed with positive result of MERS-CoV polymerase chain reaction (PCR). Her condition was improved only with conservative treatment. After a full recovery of MERS, the patient manifested abrupt vaginal bleeding with rupture of membrane. Under an impression of placenta abruption, an emergent cesarean section was performed. Our team performed many laboratory tests related to MERS-CoV and all results were negative. We report the first case of MERS-CoV infection during pregnancy occurred outside of the Middle East. Also, this case showed relatively benign maternal course which resulted in full recovery with subsequent healthy full-term delivery without MERS-CoV transmission. Abstract The mechanism by which yeast dipeptidyl aminopeptidase (DPAP) A, a type II integral mem- Abstract Ramsay Hunt syndrome (RHS) is characterized by varicella zoster virus (VZV) infection in the geniculate ganglion of the facial nerve. It typically presents with ipsilateral facial palsy and vesicles in the external auditory canal [1] . Recognizing atypical clinical manifestations of RHS, though not frequently reported, is of clinical importance for clinicians. For instance, RHS without vesicles, condition known as zoster sine herpete [2] , is difficult to distinguish itself from Bell's palsy. Although the vestibulocochlear nerve is frequently co-involved during the course of RHS, multiple lower cranial nerve involvement has rarely been described in the literature [5] [6] [7] [8] [9] [10] [11] [12] . Additionally, laryngitis due to VZV associated with RHS is not a well-recognized clinical entity. We herein report a case of Ramsay Hunt syndrome with zoster pharyngitis involving multiple lower cranial nerves.A 66-year-old woman with a history of hypertension was hospitalized because of progressive dizziness, right-side facial weakness and dysphagia. The patient was in her usual state of health until 6 days before admission, when sore throat and odynophagia developed, associated with subjective fevers and anorexia. Two days before admission, the patient reported dizziness, right-side facial weakness associated with pain radiating to the right ear, and dysphagia. The patient denied headache, photophobia, neck stiffness, nausea or vomiting. On the morning of admission, the patient developed right-side tinnitus, unsteady gait and inability to close the right eye.On physical examination, the patient appeared to be in mild distress. The temperature was 101.8 8F, blood pressure 176/93 mm Hg, pulse 102 beats per minute, respirations 20 breaths per minute and oxygen saturation 97% on room air. Her heart sounds were regular without murmurs and her lungs were clear to auscultation. On otologic examination, a few vesicles were observed in the external auditory canal of the right ear with a normal tympanic membrane. Neurological examination revealed peripheral facial nerve palsy (VII), with involvement of the left vestibulocochlear nerve (VIII), glossopharyngeal nerve (IX) and vagus nerve (X) ( Table 1) . Spinal accessory nerve (XI) and hypoglossal nerve (XII) were intact. Flexible laryngoscopic examination revealed erythema and erosive lesions on the right side of the epiglottis and arytenoid. There was an asymmetry of the soft palate. A biopsy of the erosive lesions was not performed. A non-contrast computed tomography of the head and temporal bone revealed no abnormalities.Ramsay Hunt syndrome with laryngitis due to varicella zoster virus was suspected and the patient was started on acyclovir and steroid therapy. After the completion of a 7-day course of the combination therapy, the patient's neurological symptoms improved except for the facial nerve palsy. Healed erosions on the IDCases 2 (2015) 47-48 A B S T R A C T Ramsay Hunt syndrome is characterized by varicella zoster virus infection affecting the geniculate ganglion of the facial nerve. It typically presents with vesicles in the external auditory canal associated with auricular pain and peripheral facial nerve paralysis. Although vestibulocochlear nerve is frequently co-involved during the course of Ramsay Hunt syndrome, multiple lower cranial nerve involvement has rarely been described in the literature. In addition, laryngitis due to varicella zoster virus is a diagnostic challenge due to its unfamiliarity among clinicians. We report a case of Ramsay Hunt syndrome with laryngitis involving multiple lower cranial nerves. Abstract Using a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4 + CD25 + FOXP3 + regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-b1 and TGF-b2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments. Abstract The accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b, 9b and ORF14), predicted unknown proteins (PUPs) encoded by the genes, are considered to be unique to the severe acute respiratory syndrome coronavirus (SARS-CoV) genome. These proteins play important roles in various biological processes mediated by interactions with their partners. However, very little is known about the interactions among these accessory proteins. Here, a EYFP (enhanced yellow fluorescent protein) bimolecular fluorescence complementation (BiFC) assay was used to detect the interactions among accessory proteins. 33 out of 81 interactions were identified by BiFC, much more than that identified by the yeast two-hybrid (Y2H) system. This is the first report describing direct visualization of interactions among accessory proteins of SARS-CoV. These findings attest to the general applicability of the BiFC system for the verification of protein-protein interactions. Abstract We previously demonstrated that transmissible gastroenteritis virus (TGEV) could induce apoptosis through caspase signaling. However, apoptosis was not completely prevented by caspases inhibitors, suggesting that there may be a caspase-independent pathway involved in TGEV-induced cell apoptosis. In this study, we investigated the regulation of apoptosis-inducing factor (AIF) on TGEV-induced apoptotic pathway. Results indicated that AIF translocated from the mitochondria to nucleus during TGEV infection, and the AIF inhibitor, N-phenylmaleimide (NP), significantly attenuated the apoptosis. In addition, the translocation of AIF was inhibited by Veliparib (ABT-888), an inhibitor of poly (ADP-ribose) polymerase (PARP). And the reactive oxygen species (ROS) scavenger, pyrrolidinedithiocarbamic (PDTC), redistributed AIF in the mitochondria and nucleus in TGEV-infected cells. Moreover, the protein levels in nucleus and the mRNA levels of AIF were inhibited in the presence of the p53 inhibitor, pifithrin-α (PFT-α) or in TGEV-infected p53−/−cells. Furthermore, TGEV-induced apoptosis was blocked by combination of three or more inhibitors, such as pan caspase inhibitor Z-VAD-FMK, NP, ABT-888, PDTC, PFT-α, to treat PK-15 cells. Taken together, these results suggest that the p53-and ROS-mediated AIF pathway and caspasedependent pathway were involved in TGEV-induced apoptosis. Abstract This guideline was developed as part of the 2016 Policy Research Servicing Project by the Korea Centers for Disease Control and Prevention. A multidisciplinary approach was taken to formulate this guideline to provide practical information about the diagnosis and treatment of adults with acute upper respiratory tract infection, with the ultimate aim to promote the appropriate use of antibiotics. The formulation of this guideline was based on a systematic literature review and analysis of the latest research findings to facilitate evidence-based practice, and focused on key questions to help clinicians obtain solutions to clinical questions that may arise during the care of a patient. This guideline mainly covers the subjects on the assessment of antibiotic indications and appropriate selection of antibiotics for adult patients with acute pharyngotonsillitis or acute sinusitis. Abstract This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.Background: Middle East respiratory syndrome coronavirus (MERS-CoV), which is not fully understood in regard to certain transmission routes and pathogenesis and lacks specific therapeutics and vaccines, poses a global threat to public health.To simulate the clinical aerosol transmission route, hDPP4 transgenic mice were infected with MERS-CoV by an animal nose-only exposure device and compared with instillation-inoculated mice. The challenged mice were observed for 14 consecutive days and necropsied on days 3, 5, 7, and 9 to analyze viral load, histopathology, viral antigen distribution, and cytokines in tissues.showed weight loss on days 7-11, obvious lung lesions on day 7, high viral loads in the lungs on days 3-9 and in the brain on days 7-9, and 60% survival. MERS-CoV instillation-inoculated mice exhibited clinical signs on day 1, obvious lung lesions on days 3-5, continuous weight loss, 0% survival by day 5, and high viral loads in the lungs and brain on days 3-5. Viral antigen and high levels of proinflammatory cytokines and chemokines were detected in the aerosol and instillation groups. Disease, lung lesion, and viral replication progressions were slower in the MERS-CoV aerosol-infected mice than in the MERS-CoV instillation-inoculated mice.Conclusion: hDPP4 transgenic mice were successfully infected with MERS-CoV aerosols via an animal nose-only exposure device, and aerosol-and instillation-infected mice simulated the clinical symptoms of moderate diffuse interstitial pneumonia.However, the transgenic mice exposed to aerosol MERS-CoV developed disease and lung pathology progressions that more closely resembled those observed in humans.animal nose-only exposure device, hDPP4 transgenic mice, intranasal instillation, MERS-CoV aerosol infection Abstract Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis of deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs. Abstract Infection and Drug Resistance Dovepress submit your manuscript | www.dovepress.com Dovepress 2321 R e v I e w open access to scientific and medical research Open Access Full Text Article Abstract: Health care-associated infections (HCAIs) are infections that occur while receiving health care, developed in a hospital or other health care facility that first appear 48 hours or more after hospital admission, or within 30 days after having received health care. Multiple studies indicate that the common types of adverse events affecting hospitalized patients are adverse drug events, HCAIs, and surgical complications. The US Center for Disease Control and Prevention identifies that nearly 1.7 million hospitalized patients annually acquire HCAIs while being treated for other health issues and that more than 98,000 patients (one in 17) die due to these. Several studies suggest that simple infection-control procedures such as cleaning hands with an alcohol-based hand rub can help prevent HCAIs and save lives, reduce morbidity, and minimize health care costs. Routine educational interventions for health care professionals can help change their hand-washing practices to prevent the spread of infection.In support of this, the WHO has produced guidelines to promote hand-washing practices among member countries. Abstract Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions. Abstract The purpose of this study was to investigate the genetic features of canine coronavirus (CCV) strains detected in Korea. M gene sequences obtained for isolates from 22 dogs with enteritis over a 5-year period were evaluated. Sequence comparison revealed that the 22 Korean CCV strains had an 87.2 to 100% nucleotide homology. Comparing to the typical reference CCV strains (type II), the nucleotide sequence of Korean strains had homology ranged from 86.3% to 98.3% (89.1% to 99.2% for the amino acid sequence) and 87.7% to 97.8% (92.4% to 100% for the amino acid sequence) when compared to FCoV-like CCV strains (type I). Three amino acid variations in the M gene were characteristic for the Korean CCV strains. Phylogenetic analysis demonstrated that the 22 Korean CCV strains belonged to four typical CCV clusters (i.e., a unique Korean CCV cluster, a type II and transmissible gastroenteritis virus cluster, an intermediate cluster between type I and II, and a type I cluster). This study was the first to identify genetic differences of the M gene from Korean CCV strains and provided a platform for molecular identification of different Korean CCV strains. Abstract A B S T R A C T Porcine Circovirus type 2 (PCV2) infections and associated diseases have been rarely studied in Africa. There is no report of PCV2 infection-associated morbidity and the level of awareness of stakeholders has never been investigated in Uganda. This cross sectional survey investigated the occurrence of Porcine Circovirus type 2systemic disease (PCV2-SD) among pigs and the associated level of awareness of stakeholders in Central Uganda. Data were collected using questionnaires, Focus Group Discussions (FGDs), key informant interviews and laboratory investigations. All respondents (n = 131) and farmers attending FGDs (n = 31) had never heard of PCV2-SD and only 16.7% (n = 2) of the interviewed animal health workers (n = 12) knew about the disease. Among the farms, 20 piglets presenting with a chronic wasting and a persistent diarrhea were detected and sampled for laboratory investigations. Severe lymphoid depletion with histiocytic and macrophage infiltration in lymphoid organs (n = 8), shortening of intestinal villi (n = 9), abscesses in various organs (n = 15) and granulomatous pneumonia (n = 2) were the major histopathological lesions described. Immunohistochemistry and PCR assays on organs with implicating lesions confirmed PCV2 infection in 25% (n = 5) of the 20 pigs. The study confirmed the occurrence of PCV2 infections among piglets with persistent diarrhea on pig farms in central Uganda and revealed a low level of associated knowledge among farmers and veterinary practitioners. The study arouses the need for systematic studies on prevalence of PCV2 infections and sensitization of stakeholders on occurrence of PCV2 infections in Uganda. Abstract cancer chemotherapy possesses high toxicity, particularly when a higher concentration of drugs is administered to patients. Therefore, searching for more effective compounds to reduce the toxicity of treatments, while still producing similar effects as current chemotherapy regimens, is required. currently, the search for potential anticancer agents involves a random, inaccurate process with strategic deficits and a lack of specific targets. For this reason, the initial in vitro high-throughput steps in the screening process should be reviewed for rapid identification of the compounds that may serve as anticancer agents. The present study aimed to investigate the potential use of the Pichia pastoris strain SMd1168H expressing dna topoisomerase i (SMd1168H-ToPoi) in a yeast-based assay for screening potential anticancer agents. The cell density that indicated the growth of the recombinant yeast without treatment was first measured by spectrophotometry. Subsequently, the effects of glutamate (agonist) and camptothecin (antagonist) on the recombinant yeast cell density were investigated using the same approach, and finally, the effect of camptothecin on various cell lines was determined and compared with its effect on recombinant yeast. The current study demonstrated that growth was enhanced in SMd1168H-ToPoi as compared with that in SMd1168H. Glutamate also enhanced the growth of the SMd1168H; however, the growth effect was not enhanced in SMd1168H-ToPoi treated with glutamate. By contrast, camptothecin caused only lower cell density and growth throughout the treatment of SMD1168H-TOPOI. The findings of the current study indicated that SMd1168H-ToPoi has similar characteristics to Mda-MB-231 cells; therefore, it can be used in a yeast-based assay to screen for more effective compounds that may inhibit the growth of highly metastatic breast cancer cells. Abstract Graphical Abstract Highlights d A Powassan virus LNP-mRNA vaccine induces potently neutralizing antibodies in mice d One dose of the mRNA vaccine protects against lethal Powassan virus challenge d The antibody response to the vaccine neutralizes other tickborne flaviviruses d The vaccine cross-protects against disease following challenge with Langat virus Abstract As the target organ for numerous pathogens, the lung epithelium exerts critical functions in health and disease. However, research in this area has been hampered by the quiescence of the alveolar epithelium under standard culture conditions. Here, we used human distal airway epithelial cells (DAECs) to generate alveolar epithelial cells. Long-term, robust growth of human DAECs was achieved using co-culture with feeder cells and supplementation with epidermal growth factor (EGF), Rho-associated protein kinase inhibitor Y27632, and the Notch pathway inhibitor dibenzazepine (DBZ). Removal of feeders and priming with DBZ and a cocktail of lung maturation factors prevented the spontaneous differentiation into airway club cells and instead induced differentiation to alveolar epithelial cells. We successfully transferred this approach to chicken distal airway cells, thus generating a zoonotic infection model that enables studies on influenza A virus replication. These cells are also amenable for gene knockdown using RNAi technology, indicating the suitability of the model for mechanistic studies into lung function and disease. Abstract Citation Fouchier RAM. 2015. Studies on influenza virus transmission between ferrets: the public health risks revisited. mBio 6(1):e02560-14. Abstract We have investigated the role of the somatostatin propeptide in mediating intracellular transport and sorting to the regulated secretory pathway. Using a retroviral expression vector, two fusion proteins were expressed in rat pituitary (GH3) cells: a control protein consisting of the/3-1actamase signal peptide fused to chimpanzee o~-globin (142 amino acids); and a chimera of the somatostatin signal peptide and proregion (82 amino acids) fused to ot-globin. Control globin was translocated into the endoplasmic reticulum as determined by accurate cleavage of its signal peptide; however, a-globin was not secreted but was rapidly and quantitatively degraded intracellularly with a hi2 of 4-5 min. Globin degradation was insensitive to chloroquine, a drug which inhibits lysosomal proteases, but was inhibited at 16°C suggesting proteolysis occurred during transport to the cis-Golgi apparatus. In contrast to the control globin, ,x,30% of the somatostatin propeptide-globin fusion protein was transported to the distal elements of the Golgi apparatus where it was endoproteolytically processed. Processing of the chimera occurred in an acidic intracellular compartment since cleavage was inhibited by 25 #M chloroquine. 60% of the transported chimera was cleaved at the Arg-Lys processing site in native prosomatostatin yielding "mature" a-globin. Most significantly, ",,50% of processed ot-globin was sorted to the regulated pathway and secreted in response to 8-Br-cAMP. We conclude that the somatostatin propeptide mediated transport of ot-globin from the endoplasmic reticulum to the trans-Golgi network by protecting molecules from degradation and in addition, facilitated packaging of c~-globin into vesicles whose secretion was stimulated by cAMP. Abstract Acute respiratory infections (ARIs) cause large disease burden each year. The codetection of viral and bacterial pathogens is quite common; however, the significance for clinical severity remains controversial. We aimed to identify viruses and bacteria in hospitalized children with ARI and the impact of mixed detections.Hospitalized children with ARI aged 16 were recruited from 2009 to 2013 at the Children's . Nasopharyngeal aspirates (NPAs) were collected for detection of common respiratory viruses by reverse transcription polymerase chain reaction (RT-PCR) or PCR. Bacteria were isolated from NPAs by routine culture methods. Detection and codetection frequencies and clinical features and severity were compared.Of the 3181 hospitalized children, 2375 (74.7%) were detected with !1 virus and 707 (22.2%) with !1 bacteria, 901 (28.3%) with !2 viruses, 57 (1.8%) with !2 bacteria, and 542 (17.0%) with both virus and bacteria. The most frequently detected were Streptococcus pneumoniae, respiratory syncytial virus, parainfluenza virus, and influenza virus. Clinical characteristics were similar among different pathogen infections for older group (!6 years old), with some significant difference for the younger. Cases with any codetection were more likely to present with fever; those with !2 virus detections had higher prevalence of cough; cases with virus and bacteria codetection were more likely to have cough and sputum. No significant difference in the risk of pneumonia, severe pneumonia, and intensive care unit admission were found for any codetection than monodetection.There was a high codetection rate of common respiratory pathogens among hospitalized pediatric ARI cases, with fever as a significant predictor. Cases with codetection showed no significant difference in severity than those with single pathogens. (Medicine 94(16):e742) Abbreviations: AdV = adenovirus, ARI = acute respiratory infection, BoV = bocavirus, CoV = coronavirus, ICU = intensive care unit, IFVA = influenza A virus, IFVB = influenza B virus, MPV = metapneumovirus, PIV = parainfluenza virus, RSV = respiratory syncytial virus, RV = rhinovirus. Abstract Balkan endemic nephropathy (BEN), originally described in 1956, is a unique familial, chronic renal disease encountered with a high-prevalence rate in Serbia, Bulgaria, Romania, Croatia and Bosnia and Herzegovina. The most prominent features of the disease are its endemic nature, longincubation period, familial clustering of the disease and an unusually high incidence of associated upper urothelial cancer (UUC). There are no clear-cut data on BEN incidence and prevalence, since the studies carried out in different endemic areas yielded contradictory information. In spite of intermittent variations, the incidence of new cases has remained stable over time. It has been estimated that almost 100 000 people are at risk of BEN, whereas 25 000 have the disease. The clinical signs and symptoms of BEN are non-specific and often remain unrecognized for years. There are no pathognomonic diagnostic features of BEN, but the set of epidemiological, clinical and biochemical data along with the pattern of pathologic injury in the absence of any other renal diseases are highly suggestive of this entity. Although the aetiology has been extensively studied, fostering the publication of various hypotheses, only one of them has provided conclusive evidence related to the aetiology of BEN. Studies conducted over the past decade have provided particularly strong arguments that BEN and UUC are caused by chronic poisoning with aristolochic acids (AAs). In light of these later studies, one can raise the question whether AAs could be responsible for previously and currently widespread unrecognized global renal disease and UUC. Abstract Aberrant expression of DNA polymerase b, a key enzyme involved in base excision repair, leads to genetic instability and carcinogenesis. Pol b expression has been previously shown to be regulated at the level of transcription, but there is also evidence of post-transcriptional regulation, since rat transcripts undergo alternative polyadenylation, and the resulting 3 0 UTR contain at least one regulatory element. Data presented here indicate that RNA of the short 3 0 UTR folds to form a strong secondary structure (hairpin). Its regulatory role was established utilizing a luciferase-based reporter system. Further studies led to the identification of a protein factor, which binds to this element-the antiapoptotic, cytoskeleton-related protein Hax-1. The results of in vitro binding analysis indicate that the formation of the RNA-protein complex is significantly impaired by disruption of the hairpin motif. We demonstrate that Hax-1 binds to Pol b mRNA exclusively in the form of a dimer. Biochemical analysis revealed the presence of Hax-1 in mitochondria, but also in the nuclear matrix, which, along with its transcript-binding properties, suggests that Hax-1 plays a role in post-transcriptional regulation of expression of Pol b. Abstract Cyclosporin A (CsA) ~ is a potent reversible immunosuppressive agent which is able to suppress allograft rejection and acute allogeneic graft-versus-host disease (GVHD) (1-7). Although CsA dramatically improves survival after allogeneic marrow transplantation, rats treated with a short course of CsA develop delayed onset GVHD after CsA withdrawal. This paper presents new evidence which indicates that GVHD may develop upon CsA withdrawal following not only allogeneic but also syngeneic and even autologous bone marrow reconstitution.Rats. Lewis female RT1 ~ rats, 6-8 wk old, were purchased from Harlan Sprague Dawley, Inc., Indianapolis, IN.Radiation. Lewis rats were irradiated on day -1 (1,020 rad) at 120 rad/min from a dual source Cs ~3~ small animal irradiator (Atomic Energy of Canada Ltd., Ottawa, Ontario). Leg-shielded rats were then anesthetized with chloral hydrate, their right tibias shielded with a lead doughnut 1.5 cm thick and 2.5 cm long, and irradiated with 1,020 rad.Marrow Transplantation. Donor marrow from tibias, femurs, and humeri was sus-7 pended at 6 × 10 nucleated cells/ml in Hanks' solution supplemented with 50 U/ml penicillin and 50 ng/ml streptomycin. On day 0 rats received 1 ml via the tail vein.Antibiotics. Rats received medicated drinking water supplemented with bactrim, neomycin, and polymyxin B and were given 2.5 mg/kg gentamycin per day subcutaneously for 20 d. Rats that developed overt clinical infections were routinely sacrificed and excluded from the study.Cyclosporin A. CsA was the generous gift of Sandoz, Inc., Hanover, NJ. Emulphor EL-620 was obtained from GAF Corp., New York. Powdered CsA was dissolved in absolute ethanol, added to emulphor, and mixed with water to yield a final emulphor concentration of 5%. Rats were weighed daily and received 1 ml/100 g body weight per day subcutaneously from day 0 to 40 or as indicated.Assessment of GVHD. Rats were examined daily for signs of clinical GVHD such as red ears, dermatitis, or diarrhea. Skin biopsies were taken at frequent intervals. When rats developed severe clinical GVHD characterized by weight loss, hunched appearance, and extensive dermatitis they were sacrificed and autopsied. Previously described criteria were Abstract Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV), a phlebovirus in the family Bunyaviridae, is an emerging tick-borne infectious disease that impacts humans. This disease manifests as a decreased blood cell count and multi-organ failure, with a case-fatality rate of more than 12% in China. Because vaccines or antiviral drugs for the treatment of this disease are not available, monitoring the SFTS circulation in animals and controlling the tick-mammal cycle are important for preventing SFTS. Monoclonal antibodies against the recombinant nucleoprotein of SFTSV were generated to develop a competitive enzyme-linked immunosorbent assay (cELISA) for the detection of antibodies against SFTSV infection in cattle. The specificity and sensitivity of cELISA was assessed by comparing the results of this assay to those of an immunofluorescence assay (IFA). The results of the cELISA using 416 field bovine serum samples and laboratory-immunized positive sera showed 98.1% consistency with those of the IFA. The cELISA used in this study did not show cross-reactivity with antisera against other viral cattle diseases. The cELISA presented in this study can be applied to detect antibodies against SFTSV in cattle. Abstract The 7-year medical education program in Taiwan has been established since 1949. More than 60 years later, many medical professionals have observed and voiced its deficiencies following the outbreak of severe acute respiratory syndrome. The deficiencies are three-fold: (1) specialties are excessively institutionalized, (2) students engage in passive learning and memorization, and (3) passing one written national examination serves as the means of granting permanent physician qualification. The situation has aroused concerns and discussions among medical professionals and educators for a new medical education program. Authorized by the Conference of Deans of Medical Schools in Taiwan, Prof. Chyi-Her Lin assembled a team for planning medical curricular reform. Subsequently, Prof. Shan-Chwen Chang organized a task force team which has been monitoring the new 6-year program since 2013. The aims of medical reform by Prof. Lin are (1) to eliminate the specialty training part, (2) to use innovative teaching methods to motivate students to learn proactively, and (3) to implement competency-based medical education. Now, the first class of physicians will enter the workplace in 2019, subject to various clinical challenges. Abstract Introduction respiratory pathogens associated with childhood pneumonia are often detected in the upper respiratory tract of healthy children, making their contribution to pneumonia difficult to determine. We aimed to determine the contribution of common pathogens to pneumonia adjusting for rates of asymptomatic detection to inform future diagnosis, treatment and preventive strategies. Methods a case-control study was conducted among children <18 years in Perth, Western australia. cases were children hospitalised with radiologically confirmed pneumonia; controls were healthy children identified from outpatient and local immunisation clinics. nasopharyngeal swabs were collected and tested for 14 respiratory viruses and 6 bacterial species by Polymerase chain reaction (Pcr). For each pathogen, adjusted odds ratio (aOr; 95% ci) was calculated using multivariate logistic regression and population-attributable fraction (95% ci) for pneumonia was estimated. Results From May 2015 to October 2017, 230 cases and 230 controls were enrolled. at least one respiratory virus was identified in 57% of cases and 29% of controls (aOr: 4.7; 95% ci: 2.8 to 7.8). at least one bacterial species was detected in 72% of cases and 80% of controls (aOr: 0.7; 95% ci: 0.4 to 1.2). respiratory syncytial virus (rSV) detection was most strongly associated with pneumonia (aOr: 58.4; 95% ci: 15.6 to 217.5). Mycoplasma pneumoniae was the only bacteria associated with pneumonia (aOr: 14.5; 95% ci: 2.2 to 94.8). We estimated that rSV, human metapneumovirus (HMPV), influenza, adenovirus and Mycoplasma pneumoniae were responsible for 20.2% (95% ci: 14.6 to 25.5), 9.8% (5.6% to 13.7%), 6.2% (2.5% to 9.7%), 4% (1.1% to 7.1%) and 7.2% (3.5% to 10.8%) of hospitalisations for childhood pneumonia, respectively. Conclusions respiratory viruses, particularly rSV and HMPV, are major contributors to pneumonia in australian children. Abstract In the present study we have dissected the transport pathways between the ER and the Golgi complex using a recently introduced (Kuismanen, E., J. Jiintti, V. Miikiranta, and M. Sariola. 1992. J. Cell Sci. 102:505-513) inhibition of transport by caffeine at 20~ Recovery of the Golgi complex from brefeldin A (BFA) treatment was inhibited by caffeine at reduced temperature (20~ suggesting that caffeine inhibits the membrane traffic between the ER and the Golgi complex. Caffeine at 20~ did not inhibit the BFA-induced retrograde movement of the Golgi membranes. Further, incubation of the cells in 10 mM caffeine at 20~ had profound effects on the distribution and the organization of the pre-Golgi and the Golgi stack membranes. Caffeine treatment at 20~ resulted in a selective and reversible translocation of the pre-and cis-Golgi marker protein (p58) to the periphery of the cell. This caffeine-induced effect on the Golgi complex was different from that induced by BFA, since mannosidase II, a Golgi stack marker, remained perinucleady located and the Golgi stack coat protein, B-COP, was not detached from Golgi membranes in the presence of 10 mM caffeine at 20~ Electron microscopic analysis showed that, in the presence of caffeine at 20~ the morphology of the Golgi stack was altered and accumulation of numerous small vesicles in the Golgi region was observed. The results in the present study suggest that caffeine at reduced temperature (20~ reveals a functional interface between the pre-Golgi and the Golgi stack. Abstract Porcine epidemic diarrhea virus (PEDV) is a contagious coronavirus infecting pigs that leads to significant economic losses in the swine industry. Given that PEDV infection occurs in gut epithelial cells mainly via the fecal-oral route, induction of PEDV-specific immune responses in the mucosal compartment is required for protective immunity against viral infection. However, an effective mucosal vaccine against the currently prevalent PEDV strain is not available. In this study, we demonstrated that the N-terminal domain (NTD) of the spike (S) protein of PEDV represents a new vaccine candidate molecule to be applied via the mucosal route. We first established an Escherichia coli expression system producing the partial NTD (NTD 231-501 ) of the PEDV S protein. Orally administered NTD 231-501 protein specifically interacted with the apical area of M cells in the follicle-associated epithelium of Peyer's patch. Additionally, the NTD protein induced antigen-specific immune responses in both the systemic and mucosal immune compartments when administered orally. Collectively, we propose the NTD of the PEDV S protein to be a candidate mucosal vaccine molecule. Abstract Multiple choice question Objective structured clinical examination Postgraduate training Standardized patient a b s t r a c t Background: Postgraduate year training programs play an important role in the development of a comprehensive medical education. The goal of these training programs is to inculcate in physicians the expected level of skill in patient care. After the initiation of such programs in the USA, Europe, and Japan, studies were conducted in Taiwan to investigate relevant training methods, and a training system was established in 2003. Beginning with 3-month programs, followed by 6-month programs, the programs were constantly modified and enhanced by the establishment of the 1-year training program in 2011. This year was the transition period from the 6-month programs to the 1-year programs. Methods: We used a 50-item multiple choice question (MCQ) test and six 10-min stations for objective structured clinical examination (OSCE), which was composed of four stations relating to standardized patients and two stations concerning the clinical skill evaluation, to evaluate the learning results of the trainees. The trainees were divided into four groups according to the training program. Results: There was no significant difference between the performance of the 6 months and 1-year groups. The p values were 0.424 in the MCQ test and 0.082 in the OSCE evaluation. Conclusion: A well-designed postgraduate training program should develop trainees' competencies. The results of this study may provide useful insight for ways to improve the design of training programs. Further investigation to better understand the impact of different programs is warranted. Abstract Background: Neurological diseases have become an obvious challenge due to insufficient therapeutic intervention. Therefore, novel drugs for various neurological disorders are in desperate need. Recently, compelling evidence has demonstrated that chemokine receptor CXCR3, which is a G protein-coupled receptor in the CXC chemokine receptor family, may play a pivotal role in the development of neurological diseases. The aim of this review is to provide evidence for the potential of CXCR3 as a therapeutic target for neurological diseases.English journal articles that focused on the invovlement of CXCR3 in neurological diseases were searched via PubMed up to May 2017. Moreover, reference lists from identified articles were included for overviews.The expression level of CXCR3 in T cells was significantly elevated in several neurological diseases, including multiple sclerosis (MS), glioma, Alzheimer's disease (AD), chronic pain, human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and bipolar disorder. CXCR3 antagonists showed therapeutic effects in these neurological diseases.These studies provided hard evidence that CXCR3 plays a vital role in the pathogenesis of MS, glioma, AD, chronic pain, HAM/TSP and bipolar disorder. CXCR3 is a crucial molecule in neuroinflammatory and neurodegenerative diseases. It regulates the activation of infiltrating cells and resident immune cells. However, the exact functions of CXCR3 in neurological diseases are inconclusive. Thus, it is important to understand the topic of chemokines and the scope of their activity in neurological diseases. Abstract Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the two primary causes of upper respiratory tract disease in cats. The aim of this study was to demonstrate the distribution of FCV and FHV-1 among the feline population of several counties in Rio Grande do Sul State, Brazil. To this end, conjunctival and nasal swabs were collected from 302 cats from different locations, including households, breeding catteries, veterinary clinics, animal hospitals and experimental research facilities. The samples were collected between July 2006 to June 2009. The virus isolation was performed in CRFK cells and, subsequently, the identification was confirmed by PCR. FCV, FHV-1, or both were isolated from 55 cats from 28 different locations. FCV alone was isolated from 52.7% (29/55) of the animals that tested positively, FHV-1 alone was isolated from 38.2% (21/55) of the animals that tested positively, and coinfection were detected in 9.1% (5/55) of the animals that tested positively. Virus detection was more prevalent in cats that were less than 1 year old, among animals that shared a living space with other cats, and females. FCV and FHV-1 were isolated from vaccinated cats. In addition, both viruses were isolated from cats that showed no signs of disease. The results suggest that a carrier state is common for both viruses in the evaluated population. A search for other causes of respiratory disease in that population is necessary; and further studies relating to the molecular characterization of viruses and vaccine efficacy are also necessary. Abstract Pulmonary infection is a common complication after lung transplantation, and early detection is crucial for outcome. However, the condition can be clinically difficult to diagnose and to distinguish from rejection. The aim of this prospective study was to evaluate heparin-binding protein (HBP), lysozyme, and the cytokines interleukin (IL)-1β, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF) in bronchoalveolar lavage fluid (BALF) as potential biomarkers for pulmonary infection in lung-transplanted patients. One hundred thirteen BALF samples from 29 lung transplant recipients were collected at routine scheduled bronchoscopies at 3 and 6 months, or on clinical indication. Samples were classified into no, possible, probable, or definite infection at the time of sampling. Rejection was defined by biopsy results. HBP, lysozyme, and cytokines were analyzed in BALF and correlated to likelihood of infection and rejection. All biomarkers were significantly increased in BALF during infection, whereas patients with rejection presented low levels that were comparable to noninfection samples. HBP, IL-1β, and IL-8 were the best diagnostic markers of infection with area under the receiver-operating characteristic curve values of 0.88, 0.91, and 0.90, respectively. In conclusion, HBP, IL-1β, and IL-8 could be useful diagnostic markers of pulmonary infection in lung-transplanted patients. K E Y W O R D S basic (laboratory) research/science, bronchoalveolar lavage (BAL), clinical research/practice, cytokines/cytokine receptors, infectious disease, lung disease: infectious, lung transplantation/ pulmonology, rejection Abstract The vision of legendary criminologist Cesare Lombroso to use scientific theories of individual causes of crime as a basis for screening and prevention programmes targeting individuals at risk for future criminal behaviour has resurfaced, following advances in genetics, neuroscience and psychiatric epidemiology. This article analyses this idea and maps its ethical implications from a public health ethical standpoint. Twenty-seven variants of the new Lombrosian vision of forensic screening and prevention are distinguished, and some scientific and technical limitations are noted. Some lures, biases and structural factors, making the application of the Lombrosian idea likely in spite of weak evidence are pointed out and noted as a specific type of ethical aspect. Many classic and complex ethical challenges for health screening programmes are shown to apply to the identified variants and the choice between them, albeit with peculiar and often provoking variations. These variations are shown to actualize an underlying theoretical conundrum in need of further study, pertaining to the relationship between public health ethics and the ethics and values of criminal law policy. Abstract The aim of the present study was to screen the anti-dengue potential of crude leaf extracts of two plants from Pavetta tomentosa and Tarenna asiatica. For larvicidal assay, the acetone extract of both plants showed maximum effects, with the least LC 50 and LC 90 values (P. tomentosa (5.968 and 7.493 μg/ml) and T. asiatica (1.288 and 1.992 μg/ml)) and the same extract of both plants exhibited better pupicidal potency. The adulticidal activity of both plants (0-60 min interval periods) recorded best results in acetone extracts and the LC 50 and LC 90 values were recorded as P. tomentosa (32.105 and 41.001 μg/ml) and T. asiatica (09.012 and 11.854 μg/ml). Among the two plants P. tomentosa acetone leaf extract have good antiviral property against Dengue viral cell line. In addition, the phytochemical nature of the plant reveals the presence of saponins, flavonoids and alkaloids in all the tested extracts of both plants. GC-MS analysis revealed Hexanedioic acid, Bis(2-Ethylhexyl) Ester (22.54) and 2, 6,10,14,18, 2,6,10, 15, 19,15,19,23-Hexamethyl-(ALL-E)-(25.33) identified as two major phytoconstitutents in P. tomentosa and Tetracontane (23.580) is a major compound identified from T. asiatica acetone extracts. The functional groups of chemical compounds (aromatis, alkanes, alkyls and carboxylic acids) from P. tomentosa and T. asiatica were analyzed by FT-IR spectrum. Abstract The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe acute respiratory tract infection with a high fatality rate in humans. Coronaviruses are capable of infecting multiple species and can evolve rapidly through recombination events. Here, we report the complete genomic sequence analysis of a MERS-CoV strain imported to China from South Korea. The imported virus, provisionally named ChinaGD01, belongs to group 3 in clade B in the whole-genome phylogenetic tree and also has a similar tree topology structure in the open reading frame 1a and -b (ORF1ab) gene segment but clusters with group 5 of clade B in the tree constructed using the S gene. Genetic recombination analysis and lineage-specific singlenucleotide polymorphism (SNP) comparison suggest that the imported virus is a recombinant comprising group 3 and group 5 elements. The time-resolved phylogenetic estimation indicates that the recombination event likely occurred in the second half of 2014. Genetic recombination events between group 3 and group 5 of clade B may have implications for the transmissibility of the virus. IMPORTANCE The recent outbreak of MERS-CoV in South Korea has attracted global media attention due to the speed of spread and onward transmission. Here, we present the complete genome of the first imported MERS-CoV case in China and demonstrate genetic recombination events between group 3 and group 5 of clade B that may have implications for the transmissibility of MERS-CoV. Tan W. 2015. Origin and possible genetic recombination of the Middle East respiratory syndrome coronavirus from the first imported case in China: phylogenetics and coalescence analysis. mBio 6(5):e01280-15. Abstract Intracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SMmediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Abstract . Use of highly pathogenic avian influenza A(H5N1) gain-of-function studies for molecular-based surveillance and pandemic preparedness. mBio 5(6):e02431-14. Abstract Since the 2006 Pandemic Influenza Preparedness and Response Plan according to the World Health Organization's recommendation, the Republic of Korea has prepared and periodically evaluated the plan to respond to various public health crises including pandemic influenza. Korea has stockpiled 13,000,000 doses of antiviral drugs covering 26% of the Korean population and runs 519 isolated beds in 16 medical institutions. The division of public health crisis response in Korea Centers for Disease Control and Prevention are in charge of responding to public health crises caused by emerging infectious diseases including severe acute respiratory syndrome, avian influenza human infection, and pandemic influenza. Its job description includes preparing for emerging infectious diseases, securing medical resources during a crisis, activating the emergency response during the crisis, and fortification of capabilities of public health personnel. It could evolve into a comprehensive national agency to deal with public health crisis based on the experience of previous national emerging infectious diseases. Abstract Invariant (Ii) chain is a membrane-spanning protein that is found associated intracellularly with class II histocompatibility antigens.In the endoplasmic reticulum Ii chain spans the membrane and exposes the NH2 terminus on the cytoplasmic and the C O O H terminus on the lumenal side. This orientation across the membrane is demonstrated directly with the monoclonal antibody In-l, which exclusively recognizes the NH2 terminal cytoplasmically exposed part of Ii chain.Membrane insertion of Ii chain requires signal recognition particle and docking protein. When tested in a wheat germ cell free system, signal recognition particle arrests translation of Ii chain. No signal sequence is cleaved from Ii chain upon membrane insertion. Abstract V920, rVSVDG-ZEBOV-GP, is a recombinant vesicular stomatitis-Zaire ebolavirus vaccine which has shown an acceptable safety profile and provides a protective immune response against Ebola virus disease (EVD) induced by Zaire ebolavirus in humans. The purpose of this study was to determine whether the V920 vaccine is capable of replicating in arthropod cell cultures of relevant vector species and of replicating in live mosquitoes. While the V920 vaccine replicated well in Vero cells, no replication was observed in Anopheles or Aedes mosquito, Culicoides biting midge, or Lutzomyia sand fly cells, nor in live Culex or Aedes mosquitoes following exposure through intrathoracic inoculation or feeding on a high-titer infectious blood meal. The insect taxa selected for use in this study represent actual and potential epidemic vectors of VSV. V920 vaccine inoculated into Cx. quinquefasciatus and Ae. aegypti mosquitoes demonstrated persistence of replication-competent virus following inoculation, consistent with the recognized biological stability of the vaccine, but no evidence for active virus replication in live mosquitoes was observed. Following administration of an infectious blood meal to Ae. aegypti and Cx. quinquefasciatus mosquitoes at a titer several log 10 PFU more concentrated than would be observed in vaccinated individuals, no infection or dissemination of V920 was observed in either mosquito species. In vitro and in vivo data gathered during this study support minimal risk of the vector-borne potential of the V920 vaccine. Abstract Abbreviations used in this paper: BiP, immunoglobin-binding protein; FRT, ferritinlike protein recombination target; hIre1, human Ire1-; MEF, mouse embryonic fibroblast; qPCR, quantitative real-time PCR; RIDD, regulated Ire1-dependent decay; Tm, tunicamycin; UPR, unfolded protein response.Note added in proof. Ire1 but not Ire1-I642G activated by 1NM-PP1 was also recently found to degrade ER-localized mRNAs and promote apoptosis in INS-1 cells (Han et al. 2009. Cell. Abstract Objective: This study aimed to evaluate the prevalence and related factors of post-traumatic stress disorder (PTSD) symptoms among doctors and nurses who were exposed to H7N9 patients during the H7N9 influenza epidemic. To provide scientific basis for promoting the physical and psychological health of these staff members. Method: The 102 medical staff workers who were exposed to H7N9 patients were recruited through convenient sampling between January 2015 and May 2016. We used a self-reported questionnaire, the PTSD Checklist-Civilian Version (PCL-C), to evaluate the PTSD symptoms among doctors and nurses from an intensive care unit (n ¼ 61), a respiratory department (n ¼ 20), and an emergency department (n ¼ 21). We then analyzed the related factors. Results: Around 20.59% of the tested doctors and nurses showed PTSD symptoms. The sample had a mean PCL-C score of 30.00 ± 9.95. The differences in the scores of doctors and nurses with different genders, ages, professional titles, contact frequencies, trainings, and experiences were statistically significant (P < 0.05, P < 0.01). Moreover, t-tests and one-way analysis of variance showed that nurses received higher scores than doctors, female participants received higher scores than male participants, and the participants with low professional title and high contact frequency, aged between 20 years and 30 years, with less than five years of work experience, having not received related training and with no related experience obtained higher PCL-C scores than the others (P < 0.05, P < 0.01). Conclusion: The PTSD level of doctors and nurses after their exposure to H7N9 patients was high, which warrant further research. Health and medical institutions should pay attention to the physical and psychological health of these staff members. Abstract Although generally safe and effective, severe perioperative complications, including cardiac arrest, may occur during general anesthesia in infants. With the emergence of evidence that specific anesthetic agents may affect future neurocognitive outcomes, there has been an increased focus on alternatives to general anesthesia, including spinal anesthesia. We present a case of cardiac arrest during general anesthesia in an infant who required urologic surgery. During the subsequent anesthetic care, spinal anesthesia was offered as an alternative to general anesthesia. The risks of severe perioperative complications during general anesthesia are reviewed, etiologic factors for such events are presented, and the use of spinal anesthesia as an alternative to general anesthesia is discussed. Abstract Introduction: Considerable advocacy, funding, training, and technical support have been provided to South Asian countries to strengthen One Health (OH) collaborative approaches for controlling diseases with global human pandemic potential since the early 2000s. It is essential that the OH approach continues to be strengthened given South Asia is a hot spot for emerging and endemic zoonotic diseases. The objectives of this article are to describe OH research and training and capacity building activities and the important developments in government support for OH in these countries to identify current achievements and gaps. Materials and methods: A landscape analysis of OH research, training, and government support in South Asia was generated by searching peer-reviewed and grey literature for OH research publications and reports, a questionnaire survey of people potentially engaged in OH research in South Asia and the authors' professional networks. Results: Only a small proportion of zoonotic disease research conducted in South Asia can be described as truly OH, with a significant lack of OH policy-relevant research. A small number of multisectoral OH research and OH capacity building programmes were conducted in the region. The governments of Bangladesh and Bhutan have established operational OH strategies, with variable progress institutionalising OH in other countries. Identified gaps were a lack of useful scientific information and of a collaborative culture for formulating and implementing integrated zoonotic disease control policies and the need for ongoing support for transdisciplinary OH research and policy-relevant capacity building programmes. Discussion: Overall we found a very small number of truly OH research and capacity building programmes in South Asia. Even though significant progress has been made in institutionalising OH in some South Asian countries, further behavioural, attitudinal, and institutional changes are required to strengthen OH research and training and implementation of sustainably effective integrated zoonotic disease control policies. C ollaborative One Health (OH) approaches that integrate the formulation, funding, implementation, and governance of control policies involving human, animal, and wildlife populations are required to more effectively control endemic zoonotic diseases and to detect and manage emerging zoonotic diseases (1). This is especially important in South Asia, which is a hot spot for endemic and emerging infectious zoonotic diseases (2, 3). Abstract . Observation of vimentin rearrangement in HFF cells infected withT. gondii. HFF cells were infected with T. gondii RH strain for 18 h or uninfected (controls), and then fixed with paraformaldehyde. An indirect immunofluorescence assay (IFA) was performed. The results of IFA demonstrate that host cell vimentin was rearranged and accumulated around the T. gondii parasitophorous vacuoles (arrowheads). This phenomenon was not observed in uninfected cells. Figure S2. Host cell vimentin had no obvious effect on the proliferation of T. gondii.A. qRT-PCR was performed to verify the knockdown of vimentin in HFF cells treated with siRNA (t test, ***p≤0.001). B. Knockdown of vimentin was also demonstrated by Western Blot. C. Number of tachyzoites per parasitophorous vacuole (PV) in untreated, ctrl siRNA-treated, and vimentin siRNA treated cells.The number of vacuoles containing one, two, four, or eight parasites was visualized under a fluorescence microscope (100×). Means ± SD combined from three independent experiments, each performed in triplicate, were analyzed by two-way ANOVA. No significant difference was found among these three groups, hence these data clearly demonstrated that vimentin expression levels did not affect the proliferation of T. gondii. Abstract & $ %The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their 'fresh' host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its 'fresh' host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA. Abstract The recent emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) underscore the urgent need for the development of novel, safe, and effective vaccines against the prevailing strain. In this study, we generated a cold-adapted live attenuated vaccine candidate (Aram-P29-CA) by short-term passage of a virulent PEDV isolate at successively lower temperatures in Vero cells. Whole genome sequencing identified 12 amino acid changes in the cold-adapted strain with no insertions and deletions throughout the genome. Animal inoculation experiments confirmed the attenuated phenotype of Aram-P29-CA virus in the natural host. Pregnant sows were orally administered P29-CA live vaccines two doses at 2-week intervals prior to parturition, and the newborn piglets were challenged with the parental virus. The oral homologous prime-boost vaccination of P29-CA significantly improved the survival rate of the piglets and notably mitigated the severity of diarrhea and PEDV fecal shedding after the challenge. Furthermore, strong antibody responses to PEDV were detected in the sera and colostrum of immunized sows and in the sera of their offspring. These results demonstrated that the cold-adapted attenuated virus can be used as a live vaccine in maternal vaccination strategies to provide durable lactogenic immunity and confer passive protection to litters against PEDV. Abstract Background. Administration of convalescent plasma, serum, or hyperimmune immunoglobulin may be of clinical benefit for treatment of severe acute respiratory infections (SARIs) of viral etiology. We conducted a systematic review and exploratory meta-analysis to assess the overall evidence.Methods. Healthcare databases and sources of grey literature were searched in July 2013. All records were screened against the protocol eligibility criteria, using a 3-stage process. Data extraction and risk of bias assessments were undertaken.Results. We identified 32 studies of SARS coronavirus infection and severe influenza. Narrative analyses revealed consistent evidence for a reduction in mortality, especially when convalescent plasma is administered early after symptom onset. Exploratory post hoc meta-analysis showed a statistically significant reduction in the pooled odds of mortality following treatment, compared with placebo or no therapy (odds ratio, 0.25; 95% confidence interval, .14-.45; I 2 = 0%). Studies were commonly of low or very low quality, lacked control groups, and at moderate or high risk of bias. Sources of clinical and methodological heterogeneity were identified.Conclusions. Convalescent plasma may reduce mortality and appears safe. This therapy should be studied within the context of a well-designed clinical trial or other formal evaluation, including for treatment of Middle East respiratory syndrome coronavirus CoV infection. Abstract Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it attractive candidate vector to prevent infectious diseases. Attenuated MV have acquired the ability to use the complement regulator CD46 as a major receptor to mediate virus entry and intercellular fusion. Therefore, attenuated MV strains preferentially infect and destroy a wide variety of cancer cells making them also attractive oncolytic vectors. The use of recombinant MV vector has to comply with various regulatory requirements, particularly relating to the assessment of potential risks for human health and the environment. The present article highlights the main characteristics of MV and recombinant MV vectors used for vaccination and virotherapy and discusses these features from a biosafety point of view. Abstract One quarter of all deaths worldwide each year result from infectious diseases caused by microbial pathogens. Pathogens infect and cause disease by producing virulence factors that target host cell molecules. Studying how virulence factors target host cells has revealed fundamental principles of cell biology. These include important advances in our understanding of the cytoskeleton, organelles and membrane-trafficking intermediates, signal transduction pathways, cell cycle regulators, the organelle/protein recycling machinery, and cell-death pathways. Such studies have also revealed cellular pathways crucial for the immune response. Discoveries from basic research on the cell biology of pathogenesis are actively being translated into the development of host-targeted therapies to treat infectious diseases. Thus there are many reasons for cell biologists to incorporate the study of microbial pathogens into their research programs. Abstract Titanium dioxide nanoparticles (TiO 2 NPs) induce lung inflammation in experimental animals. In this study, we conducted a comprehensive toxicogenomic analysis of lung responses in mice exposed to six individual TiO 2 NPs exhibiting different sizes (8, 20 and 300 nm), crystalline structure (anatase, rutile or anatase/rutile) and surface modifications (hydrophobic or hydrophilic) to investigate whether the mechanisms leading to TiO 2 NP-induced lung inflammation are property specific. A detailed histopathological analysis was conducted to investigate the long-term disease implications of acute exposure to TiO 2 NPs. C57BL/6 mice were exposed to 18, 54, 162 or 486 µg of TiO 2 NPs/mouse via single intratracheal instillation. Controls were exposed to dispersion medium only. Bronchoalveolar lavage fluid (BALF) and lung tissue were sampled on 1, 28 and 90 days post-exposure. Although all TiO 2 NPs induced lung inflammation as measured by the neutrophil influx in BALF, rutile-type TiO 2 NPs induced higher inflammation with the hydrophilic rutile TiO 2 NP showing the maximum increase. Accordingly, the rutile TiO 2 NPs induced higher number of differentially expressed genes. Histopathological analysis of lung sections on Day 90 post-exposure showed increased collagen staining and fibrosislike changes following exposure to the rutile TiO 2 NPs at the highest dose tested. Among the anatase, the smallest TiO 2 NP of 8 nm showed the maximum response. The anatase TiO 2 NP of 300 nm was the least responsive of all. The results suggest that the severity of lung inflammation is property specific; however, the underlying mechanisms (genes and pathways perturbed) leading to inflammation were the same for all particle types. While the particle size clearly influenced the overall acute lung responses, a combination of small size, crystalline structure and hydrophilic surface contributed to the long-term pathological effects observed at the highest dose (486 µg/mouse). Although the dose at which the pathological changes were observed is considered physiologically high, the study highlights the disease potential of certain TiO 2 NPs of specific properties. Abstract Background: Chronic Hepatitis B (CHB) remains a major problem for global public health. Viral persistence and immune defects are the two major reasons for CHB, and it was hypothesized that based on a transient clearance of serum viral DNA and HBsAg "window stage", active immunization against hepatitis B virus (HBV) might initiate effective host immune responses versus HBV to achieve functional cure of CHB. Methods: Two experimental mouse models that mice hydrodynamic injected HBV DNA or infected with recombinant AAV/HBV were used. The "sandwich" therapeutic effect by using a potent human anti-HBsAg neutralizing monoclonal antibody (G12) in combination with antiviral drug tenofovir disoproxil fumarate (TDF), followed by active immunization with HBsAg-HBsAb (mYIC) was evaluated. Findings: A single G12 injection rapidly cleared serum HBsAg in HDI-HBV carrier mice, with a synergistic effect in decreasing viral DNA load when TDF was given orally. When both serum viral DNA and HBsAg load became low or undetectable, mYIC was administered. A more effective clearance of viral DNA and HBsAg was observed and serum HBsAb was developed only in these "sandwich"-treated mice. Efficient intrahepatic anti-HBV immune responses were also observed in these mice, including the formation of aggregates of myeloid cells with CD8 + T cells and increased TNF-α, granzyme B production.The "sandwich" combination therapy not only efficiently decreased HBsAg and HBV DNA levels but also induced effective cellular and humoral immunity, which may result in functional cure of CHB.Research in context sectionPersistence of HBV cccDNA and various host immune defects were shown as the two main obstacles for HBV clearance. Blocking a single target will not be sufficient to achieve functional cure of CHB. A very small percentage of CHB patients could attain "functional cure" by antiviral treatment fol- * Corresponding authors. .cn (Z. Yuan). lowed by interferon therapy. Therapeutic vaccination for CHB showed substantial efficacy. A humanized HBsAb G12 could efficiently block HBV infection and decrease HBsAg level in mice model.This research showed that when both serum viral DNA and HB-sAg load became low or undetectable by antiviral and HBsAb treatment, therapeutic vaccine (mYIC) add on induced effective, host humoral and intrahepatic cellular anti-HBV immune responses, with more rapid and efficient clearance of HBV and HBsAg.Based on optimized antiviral treatment with potent human HB-sAb to rapidly decrease serum HBsAg, followed by boosting host with active immunization provides a novel strategy for functional cure of CHB. Abstract Fibrillarin, one of the major proteins of the nucleolus, plays several essential roles in ribosome biogenesis including pre-rRNA processing and 2 0 -O-ribose methylation of rRNA and snRNAs. Recently, it has been shown that fibrillarin plays a role in virus infections and is associated with viral RNPs. Here, we demonstrate the ability of recombinant fibrillarin 2 from Arabidopsis thaliana (AtFib2) to interact with RNAs of different lengths and types including rRNA, snoRNA, snRNA, siRNA and viral RNAs in vitro. Our data also indicate that AtFib2 possesses two RNA-binding sites in the central (138-179 amino acids) and C-terminal (225-281 amino acids) parts of the protein, respectively. The conserved GCVYAVEF octamer does not bind RNA directly as suggested earlier, but may assist with the proper folding of the central RNA-binding site. Abstract Background and Purpose Middle East respiratory syndrome (MERS) has a high mortality rate and pandemic potential. However, the neurological manifestations of MERS have rarely been reported since it first emerged in 2012.We evaluated four patients with laboratory-confirmed MERS coronavirus (CoV) infections who showed neurological complications during MERS treatment. These 4 patients were from a cohort of 23 patients who were treated at a single designated hospital during the 2015 outbreak in the Republic of Korea. The clinical presentations, laboratory findings, and prognoses are described. Abstract Objective: In this study we execute a rational screen to identify Chinese medical herbs that are commonly used in treating viral respiratory infections and also contain compounds that might directly inhibit 2019 novel coronavirus (2019-nCoV), an ongoing novel coronavirus that causes pneumonia. Methods: There were two main steps in the screening process. In the first step we conducted a literature search for natural compounds that had been biologically confirmed as against sever acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus. Resulting compounds were cross-checked for listing in the Traditional Chinese Medicine Systems Pharmacology Database. Compounds meeting both requirements were subjected to absorption, distribution, metabolism and excretion (ADME) evaluation to verify that oral administration would be effective. Next, a docking analysis was used to test whether the compound had the potential for direct 2019-nCoV protein interaction. In the second step we searched Chinese herbal databases to identify plants containing the selected compounds. Plants containing 2 or more of the compounds identified in our screen were then checked against the catalogue for classic herbal usage. Finally, network pharmacology analysis was used to predict the general in vivo effects of each selected herb. Results: Of the natural compounds screened, 13 that exist in traditional Chinese medicines were also found to have potential anti-2019-nCoV activity. Further, 125 Chinese herbs were found to contain 2 or more of these 13 compounds. Of these 125 herbs, 26 are classically catalogued as treating viral respiratory infections. Network pharmacology analysis predicted that the general in vivo roles of these 26 herbal plants were related to regulating viral infection, immune/inflammation reactions and hypoxia response. Conclusion: Chinese herbal treatments classically used for treating viral respiratory infection might contain direct anti-2019-nCoV compounds. Abstract Astroviruses and kobuviruses are frequently found in mammalian feces, including that of humans. The present study examined fecal samples from 91 Korean dogs suffering from diarrhea. Canine astroviruses (CAstVs) and canine kobuviruses (CKoVs) were identified in 2 (2.1%) and 46 (50.6%) dogs, respectively. Nucleotide sequence analysis coupled with phylogenetic analysis using the neighbor-joining method showed that CAstVs clustered into four genetically diverse groups. Two Korean CAstVs belonged to group 2 alongside strains isolated in Italy and France. Twelve of the Korean CKoVs belonged to a single clade, along with strain UK003 identified in the UK and six CKoVs identified in the USA. Thus, the results suggest that the Korean strain of CAstV is closely related to strains isolated in Europe. Surely, CKoV in South Korea could identify the circulation among dogs population. Abstract Word Counts: 299 words (abstract); 2658 words (text);Objective: This study investigated the associations between syphilis prevalence and age among blood donors, and described the distribution of serological titres among syphilis-infected donors, aiming at confirming the syphilis epidemic characteristics and promoting effective interventions for older adults.Methods: Data were obtained from the Shenzhen Program for Syphilis Prevention and Control in 2014-2017. Blood samples were screened using the enzyme-linked immunosorbent assay (ELISA), and confirmed using the Treponema pallidum particle agglutination assay (TPPA) and toluidine red unheated serum test (TRUST).: Among 394 792 blood donors, 733 were TPPA and TRUST positive, and 728 were only TPPA positive. The overall prevalence of syphilis infection was 370.1 per 100 000 [95% confidence interval (CI), 351.1-389.0 per 100 000]; the prevalence of active infection was 185.7 per 100 000 (95% CI, 172.2-199.1 per 100 000). People aged ≥45 years displayed a prevalence of 621.8 per 100 000 in syphilis infection and 280.5 per 100 000 in active infection, which were 3.8 times and 2.4 times higher than that for people aged <25 years. The prevalence of syphilis infection (χ 2 trend = 311.9, p trend < 0.001) and active infection (χ 2 trend = 72.1, p trend < 0.001) increased significantly with age. After stratification of gender and year of donation , the increasing trend of prevalence with age remained(p trend < 0.05), except for the prevalence of active infection in males (χ 2 trend = 0.923, p trend = 0.337) and females (χ 2 trend = 0.224, p trend = 0.636) in 2014. About 16.3% of patients aged ≥45 years had a TRUST titre of ≥1∶8, lower than that of patients aged <25 years (51.3%) and 25-34 years (34.1%).The findings confirm the high prevalence of syphilis among older adults, and suggest the need to increase awareness among healthcare providers and deliver more-targeted prevention interventions for older adults to promote early testing. Abstract We report the complete genome sequences of a buffalo coronavirus (BufCoV HKU26) detected from the faecal samples of two domestic water buffaloes (Bubalus bubalis) in Bangladesh. They possessed 98-99% nucleotide identities to bovine coronavirus (BCoV) genomes, supporting BufCoV HKU26 as a member of Betacoronavirus 1. Nevertheless, BufCoV HKU26 possessed distinct accessory proteins between spike and envelope compared to BCoV. Sugar-binding residues in the N-terminal domain of S protein in BCoV are conserved in BufCoV HKU26.New Microbes and New Infections Abstract To describe radiologic findings of adenovirus pneumonia and to understand clinico-radiological features associated with progression to acute respiratory distress syndrome (ARDS) in patients with adenovirus pneumonia. Materials and Methods: This study included 19 patients diagnosed with adenovirus pneumonia at a tertiary referral center, in the period between March 2003 and April 2015. Clinical findings were reviewed, and two radiologists assessed imaging findings by consensus. Chi-square, Fisher's exact, and Student's t tests were used for comparing patients with and without subsequent development of ARDS. Results: Of 19 patients, nine were immunocompromised, and 10 were immunocompetent. Twelve patients (63%) progressed to ARDS, six of whom (32%) eventually died from the disease. The average time for progression to ARDS from symptom onset was 9.6 days. Initial chest radiographic findings were normal (n = 2), focal opacity (n = 9), or multifocal or diffuse opacity (n = 8). Computed tomography (CT) findings included bilateral (n = 17) or unilateral (n = 2) ground-glass opacity with consolidation (n = 14) or pleural effusion (n = 11). Patients having subsequent ARDS had a higher probability of pleural effusion and a higher total CT extent compared with the non-ARDS group (p = 0.010 and 0.007, respectively). However, there were no significant differences in clinical variables such as patient age and premorbid condition. Conclusion: Adenovirus pneumonia demonstrates high rates of ARDS and mortality, regardless of patient age and premorbid conditions, in the tertiary care setting. Large disease extent and presence of pleural effusion on CT are factors suggestive of progression to ARDS. Abstract Cellular sensing of virus-derived nucleic acids is essential for early defenses against virus infections. In recent years, the discovery of DNA sensing proteins, including cyclic GMP-AMP synthase (cGAS) and gamma-interferoninducible protein (IFI16), has led to understanding of how cells evoke strong innate immune responses against incoming pathogens carrying DNA genomes. The signaling stimulated by DNA sensors depends on the adaptor protein STING (stimulator of interferon genes), to enable expression of antiviral proteins, including type I interferon.To facilitate efficient infections, viruses have evolved a wide range of evasion strategies, targeting host DNA sensors, adaptor proteins and transcription factors. In this review, the current literature on virus-induced activation of the STING pathway is presented and we discuss recently identified viral evasion mechanisms targeting different steps in this antiviral pathway. Abstract Background Severe Acute Respiratory Syndrome (SARS) was first reported in November 2002 in China, and spreads to about 30 countries over the next few months. While the characteristics of epidemic transmission are individually assessed, there are also important implicit associations between them.Methods A novel methodological framework was developed to overcome barriers among separate epidemic statistics and identify distinctive SARS features. Individual statistics were pair-wise linked in terms of their common features, and an integrative epidemic network was formulated.The study of associations between important SARS characteristics considerably enhanced the mainstream epidemic analysis and improved the understanding of the relationships between the observed epidemic determinants. The response of SARS transmission to various epidemic control factors was simulated, target areas were detected, critical time and relevant factors were determined.Conclusion It was shown that by properly accounting for links between different SARS statistics, a data-based analysis can efficiently reveal systematic associations between epidemic determinants. The analysis can predict the temporal trend of the epidemic given its spatial pattern, to estimate spatial exposure given temporal evolution, and to infer the driving forces of SARS transmission given the spatial exposure distribution. Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/)NOTEVirologyABSTRACT. To trace the prevalence of canine distemper virus (CDV) in diarrhoetic dogs, a total of 201 stool samples were collected in the Heilongjiang province of northeastern China from May 2014 to April 2015. The 201 fecal samples were subjected to the detection of CDV by using RT-PCR targeting the partial N gene, phylogenetic analysis based on the complete H gene, and coinfection analysis. Results indicated that 24.88% (50/201) of the samples were positive for CDV. The fifty CDV samples exhibited an overall co-infection rate of 94% (47/50) with four enteric viruses (82%, 41/50) and five bacteria (72%, 36/50). The positivity rate of CDV exhibited differences among regions, seasons, ages and immunization status. Phylogenetic analysis of the complete H genes (n=6) revealed that the CDV strains identified in our study belonged to the Asia-1 group, and showed genetic diversities. These data provide evidence that there are a number of genetically diverse CDV Asia-1 strains circulating in diarrhoetic dogs in northeastern China; the CDV-affected animals exhibit the high co-infection with other enteric viruses and bacteria. Abstract We have studied the role of restrictions to lateral mobility in the segregation of proteins to apical and basolateral domains of MDCK epithelial cells. Radioimmunoassay and semiquantitative video analysis of immunofluorescence on frozen sections showed that one apical and three basolateral glycoproteins, defined by monoclonal antibodies and binding of beta-2microglobulin, were incompletely extracted with 0.5 % Triton X-100 in a buffer that preserves the cortical cytoskeleton (Fey, E. G., K. Abstract With the advent of deep sequencing, genomic surveillance has become a popular method for detection of infectious disease, supplementing information gathered by classic clinical or serological techniques to identify host-determinant markers and trace the origin of transmission. However, two main factors complicate genomic surveillance. First, pathogens exhibiting high genetic diversity demand higher levels of scrutiny to obtain an accurate representation of the entire population. Second, current systems of detection are nonuniform, with significant gaps in certain geographic locations and animal reservoirs. Despite past unforeseen pandemics like the 2009 swine-origin H1N1 influenza virus, there is no standardized way of evaluating surveillance. A more complete surveillance system should capture a greater proportion of pathogen diversity. Here we present a novel quantitative method of assessing the completeness of genomic surveillance that incorporates the time of sequence collection, as well as the pathogen's evolutionary rate. We propose the q2 coefficient, which measures the proportion of sequenced isolates whose closest neighbor in the past is within a genetic distance equivalent to 2 years of evolution, roughly the median time of changing strain selection for influenza A vaccines. Easily interpretable and significantly faster than other methods, the q2 coefficient requires no full phylogenetic characterization or use of arbitrary clade definitions. Application of the q2 coefficient to influenza A virus confirmed poor sampling of swine and avian populations and identified regions with deficient surveillance. We demonstrate that the q2 coefficient can not only be applied to other pathogens, including dengue and West Nile viruses, but also used to describe surveillance dynamics, particularly the effects of different public health policies. IMPORTANCE Surveillance programs have become key assets in determining the emergence or prevalence of pathogens circulating in human and animal populations. Genomic surveillance, in particular, provides comprehensive information on the history of isolates and potential molecular markers for infectivity and pathogenicity. Current techniques for evaluating genomic surveillance are inaccurate, ignoring the pathogen's evolutionary rate and biodiversity, as well as the timing of sequence collection. Using sequence data, we propose the q2 coefficient as a quantitative measure of surveillance completeness that combines elements of time and evolution without defining arbitrary criteria for clades or species. Through several case studies of influenza A, dengue, and West Nile viruses, we employed the q2 coefficient to identify sampling deficiencies in different host species and locations, as well as examine the effects of different public health policies through historical records of the q2 coefficient. These results can guide public health agencies to focus resource allocation and virus collection to bolster specific problems in surveillance.Citation Chan JM, Rabadan R. 2013. Quantifying pathogen surveillance using temporal genomic data. mBio 4(1):e00524-12. Abstract Previously developed Asn-Gly-Arg (NGR) peptide-modified multifunctional poly(ethyleneimine)-poly(ethylene glycol) (PEI-PEG)-based nanoparticles (TPIC) have been considered to be promising carriers for the co-delivery of DNA and doxorubicin (DOX). As a continued effort, the aim of the present study was to further evaluate the interaction between TPIC and human umbilical vein endothelial cells (HUVEC) to better understand the cellular entry mechanism. In the present investigation, experiments relevant to co-localization, endocytosis inhibitors and factors influencing the internalization were performed. Without any treatment, there was no co-localization between aminopeptidase N/CD13 (APN/CD13) and caveolin 1 (CAV1). However, co-localization between CD13 and CAV1 was observed when cells were incubated with an anti-CD13 antibody or TPIC. As compared with antibody treatment, TPIC accelerated the speed and enhanced the degree of co-localization. TPIC entered HUVEC not only together with CD13 but also together with CAV1. However, this internalization was not dependent on the enzyme activity of CD13 but could be inhibited by methyl-β-eyclodextfin (MβCD), further identifying the involvement of caveolae-mediated endocytosis (CvME). This conclusion was also verified by endocytosis inhibitor experiments. Abstract Transmissible gastroenteritis (TGE), caused by transmissible gastroenteritis virus (TGEV), is a highly infectious disease in pigs. Vaccination is an effective approach to prevent TGEV infection. Here, we evaluated the potential of TGEV S1 as a DNA vaccine and porcine interleukin (pIL)-12 as an adjuvant in a mouse model. A DNA vaccine was constructed with the TGEV S1 gene to induce immune response in an experimental mouse model; pIL-12 was chosen as the immunological adjuvant within this DNA vaccine. The pVAX1-(TGEV-S1) and pVAX1-(pIL-12) vectors were transfected into BHK-21 cells and expressed in vitro. Experimental mice were separately immunized with each of the recombinant plasmids and controls through the intramuscular route. The lymphocytes isolated from the blood and spleen were analyzed for proliferation, cytotoxic activities, and populations of CD4 + and CD8 + cells. The titers of TGEV S1 in an enzyme-linked immunosorbent assay (ELISA) and TGEV neutralizing antibodies and the concentrations of interferon (IFN)-γ and IL-4 were also analyzed in the serum. The plasmids pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could be expressed in BHK-21 cells, and the combination of pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could induce a significant increase in all markers. pIL-12 could act as an immunological adjuvant in the DNA vaccine for TGEV-S1. Furthermore, the DNA vaccine prepared using TGEV-S1 and porcine IL-12 could induce excellent humoral and cellular immune responses.Transmissible gastroenteritis is a highly infectious and acute digestive disease caused by transmissible gastroenteritis virus (TGEV) infection. The classical symptoms of this disease include severe diarrhea, vomiting, and dehydration. Swine of different ages and breeds are susceptible to TGEV infection. TGEV is often isolated in cases of poly-infections involving other pathogens and mainly infects piglets. The mortality rate can be as high as 100% and may decline as the animal gets older; however, the decrease in the feed utilization of infected animals may have huge economic consequences [11, 16] .TGEV is a coronavirus and contains a positive-sense single-stranded RNA genome [28] , which encodes four structural proteins and three non-structural proteins. Spike (S) protein is the major component of the virion surface and possesses four antigen sites, Abstract Defensins are antimicrobial peptides that participate in the innate immunity of hosts. Humans constitutively and/or inducibly express α-and β-defensins, which are known for their antiviral and antibacterial activities. This review describes the application of human defensins. We discuss the extant experimental results, limited though they are, to consider the potential applicability of human defensins as antiviral agents. Given their antiviral effects, we propose that basic research be conducted on human defensins that focuses on RNA viruses, such as human immunodeficiency virus (HIV), influenza A virus (IAV), respiratory syncytial virus (RSV), and dengue virus (DENV), which are considered serious human pathogens but have posed huge challenges for vaccine development for different reasons. Concerning the prophylactic and therapeutic applications of defensins, we then discuss the applicability of human defensins as antivirals that has been demonstrated in reports using animal models. Finally, we discuss the potential adjuvant-like activity of human defensins and propose an exploration of the 'defensin vaccine' concept to prime the body with a controlled supply of human defensins. In sum, we suggest a conceptual framework to achieve the practical application of human defensins to combat viral infections.Abstract Review Abstract These authors contributed equally to this work. Abstract Microglia are prominent immune cells in the central nervous system (CNS) and are critical players in both neurological development and homeostasis, and in neurological diseases when dysfunctional. Our previous understanding of the phenotypes and functions of microglia has been greatly extended by a dearth of recent investigations. Distinct genetically defined subsets of microglia are now recognized to perform their own independent functions in specific conditions. The molecular profiling of single microglial cells indicates extensively heterogeneous reactions in different neurological disorders, resulting in multiple potentials for crosstalk with other kinds of CNS cells such as astrocytes and neurons. In settings of neurological diseases it could thus be prudent to establish effective cell-based therapies by targeting entire microglial networks. Notably, activated microglial depletion through genetic targeting or pharmacological therapies within a suitable time window can stimulate replenishment of the CNS niche with new microglia. Additionally, enforced repopulation through provision of replacement cells also represents a potential means of exchanging dysfunctional with functional microglia. In each setting the newly repopulated microglia might have the potential to resolve ongoing neuroinflammation. In this review, we aim to summarize the most recent knowledge of microglia and to highlight microglial depletion and subsequent repopulation as a promising cell replacement therapy. Although glial cell replacement therapy is still in its infancy and future translational studies are still required, the approach is scientifically sound and provides new optimism for managing the neurotoxicity and neuroinflammation induced by activated microglia. K E Y W O R D S cell replacement therapy, depletion, microglia, neuroinflammation Abstract With the increase in international human and material exchanges, contagious and infectious epidemics are occurring. One of the effective methods of epidemic inhibition is the rapid development and supply of vaccines. Considering the safety of the brain during vaccine development is very important. However, manuals for brain safety assays for new vaccines are not uniform or effective globally. Therefore, the aim of this study is to establish a positive-control protocol for an effective brain safety test to enhance rapid vaccine development. The blood-brain barrier's tight junctions provide selective defense of the brain; however, it is possible to destroy these important microstructures by administering lipopolysaccharides (LPSs), thereby artificially increasing the permeability of brain parenchyma. In this study, test conditions are established so that the degree of brain penetration or brain destruction of newly developed vaccines can be quantitatively identified. The most effective conditions were suggested by measuring time-dependent expressions of tight junction biomarkers (zonula occludens-1 [ZO-1] and occludin) in two types of mice (C57BL/6 and ICR) following exposure to two types of LPS (Salmonella and Escherichia). In the future, we hope that use of the developed positive-control protocol will help speed up the determination of brain safety of novel vaccines. Abstract MBP-tagged TRIM25 protein was expressed in E.coli Rosetta 2 (DE3) TM cells (Millipore).Transformed cells were grown in LB broth containing 0.2% glucose at 37 °C. Then, 0.2 mM IPTG was added and the cells were incubated under shaking conditions for 18 h at 25 °C, after which the cells were harvested by centrifugation. The pellet was resuspended in T1 buffer (20 mM Tris/Cl(pH 7.4), 1 mM EDTA, 1 mM sodium azide, and 1 mM DTT) and sonicated. After centrifugation, the supernatant was loaded onto a DEAE ion-exchange column (GE healthcare) and the flow throw was collected. Next, the pooled sample was applied to amylose resin (NEB). The column was washed with T2 buffer (T1 buffer + 200 mM NaCl) and eluted with T3 buffer (T2 buffer + 10 mM maltose). The purity of the proteins was analyzed by 10% SDS-PAGE. The final yield of MBP-TRIM25 was determined by the Bradford assay with BSA as the standard and the samples were stored at �80 °C.His-tagged RIG-I was expressed in E. coli BL21(DE3) cells. The transformed cells were grown in LB broth at 37 °C and then further incubated at 4 °C for 3 h. Subsequently, 0.25 mM IPTG was added and the cells were incubated under shaking conditions for 18 h at 25 °C, after which the cells were harvested by centrifugation. The pellet was resuspended in R1 buffer (50 mM HEPES; pH 7.4, 10% glycerol, 500 mM NaCl, 0.2% NP-40, 10 mM imidazole, 1 mM PMSF, 0.5 mg/ml lysozyme, and protease inhibitor cocktail) and sonicated. After centrifugation, the supernatant was applied to a Ni-NTA chelating agarose (Incospham). The column was washed with R2 buffer (50 mM HEPES; pH 7.4, 10% glycerol, 500 mM NaCl, 0.2% NP-40, and 50 mM imidazole) and the His-tagged RIG-I was eluted by increasing the imidazole concentration. The purest fractions were applied to a heparin-Sepharose column (GE healthcare), and the resin was washed with R3 buffer (50 mM Tris/Cl(pH 7.4), 10% glycerol, 0.2% NP-40, and 20 mM NaCl) and eluted with increasing concentrations of NaCl (up to 500 mM). The final yield of His-RIG-I was determined by the Bradford assay (Bio-Rad) with BSA as the standard.RNA Aptamers Specific to NS1 Protein of Influenza Virus Hye-Min Woo et al. Abstract of NPEC1-Bmi1 cells, an immortalized nasopharyngeal epithelial cell line. Furthermore, efficient infection by this mechanism involved the activation of the PI3K/AKT signaling pathway. Thus, our study identified "in-cell infection" as a novel mechanism for EBV infection. Given the diversity of virus-infected cells and the prevalence of cell-in-cell structures during chronic infection, we speculate that "in-cell infection" is likely a general mechanism for EBV and other viruses to infect non-susceptible ECs. Abstract In vivo serial passage of non-pathogenic viruses has been shown to lead to increased viral virulence, and although the precise mechanism(s) are not clear, it is known that both host and viral factors are associated with increased pathogenicity. Under-or overnutrition leads to a decreased or dysregulated immune response and can increase viral mutant spectrum diversity and virulence. The objective of this study was to identify the role of viral mutant spectra dynamics and host immunocompetence in the development of pathogenicity during in vivo passage. Because the nutritional status of the host has been shown to affect the development of viral virulence, the diet of animal model reflected two extremes of diets which exist in the global population, malnutrition and obesity. Sendai virus was serially passaged in groups of mice with differing nutritional status followed by transmission of the passaged virus to a second host species, guinea pigs. Viral population dynamics were characterized using deep sequence analysis and computational modeling. Histopathology, viral titer and cytokine assays were used to characterize viral virulence. Viral virulence increased with passage and the virulent phenotype persisted upon passage to a second host species. Additionally, nutritional status of mice during passage influenced the phenotype. Sequencing revealed the presence of several non-synonymous changes in the consensus sequence associated with passage, a majority of which occurred in the hemagglutinin-neuraminidase and polymerase genes, as well as the presence of persistent high frequency variants in the viral population. In particular, an N1124D change in the consensus sequences of the polymerase gene was detected by passage 10 in a majority of the animals. In vivo comparison of an 1124D plaque isolate to a clone with 1124N genotype indicated that 1124D was associated with increased virulence. Abstract Background: The largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection outside Middle East Asia in 2015 has necessitated the rapid expansion of laboratories that conduct MERS-CoV molecular testing in Korea, together with external quality assessment (EQA) to evaluate the assays used.Methods: The EQA program consisted of two phases; self-validation and blind assessment. For the first EQA phase, in vitro transcribed upstream region of the envelope gene (upE) and the open reading frame (ORF)1a RNAs were used at a concentration of 1,000 copies/μL. The test panel for the second EQA phase consisted of RNA extracts from three samples, which were obtained from two MERS-CoV positive patients and one MERS-CoV negative patient.Results: The first EQA phase results for 46 participants showed a linear relationship between the threshold cycle (CT) values of RNA materials and the logarithmic concentrations for both upE and ORF1a gene targets (R 2 = 0.73 and 0.75, respectively). The mean CT value for each concentration was different depending on which commercial kit was used for the assay. Among the three commonly used kits, PowerChek MERS Real-Time PCR kit (KogeneBiotech, Korea) showed the lowest CT values at all concentrations of upE and most concentrations of ORF1a. The second EQA phase results for 47 participants were 100% correct for all tested samples.Conclusions: This EQA survey demonstrates that the MERS-CoV molecular testing performed in Korea during the 2015 outbreak is of robust capability. However, careful establishment and validation of a cut-off value are recommended to ensure good analytical sensitivity. Abstract We investigated the clinical presentations, diagnostic and therapeutic modalities, and prognosis from follow-up of infants with apparent life-threatening events (ALTE). In addition, the relationship between the clinical characteristics of patients and significant intervention scores was analyzed. We enrolled patients younger than 12 months who were diagnosed with ALTE from January 2005 to December 2012. There were 29 ALTE infants with a peak incidence of age younger than 1 month (48.3%). The most common symptoms for ALTE diagnosis were apnea (69.0%) and color change (58.6%). Eleven patients appeared normal upon arrival at hospital but 2 patients required cardiopulmonary resuscitation during the initial ALTE. The most common ALTE cause was respiratory disease, including respiratory infection and upper airway anomalies (44.8%). There were 20 cases of repeat ALTE and 2 cases of death during hospitalization. Four patients (15.4%) experienced recurrence of ALTE after discharge and 4 patients (15.4%) showed developmental abnormalities during the follow-up period. The patients with ALTE during sleep had lower significant intervention scores (P = 0.015) compared to patients with ALTE during wakefulness and patients with previous respiratory symptoms had higher significant intervention scores (P = 0.013) than those without previous respiratory symptoms.Although not statistically significant, there was a weak positive correlation between the patient's total ALTE criteria and total significant intervention score (Fig. 2 , r = 0.330, P = 0.080). We recommend that all ALTE infants undergo inpatient observation and evaluations with at least 24 hr of cardiorespiratory monitoring, and should follow up at least within a month after discharge. Abstract Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many densegranule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts.IMPORTANCE Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammalsincluding humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies.Blazejewski T, Nursimulu N, Pszenny V, Dangoudoubiyam S, Namasivayam S, Chiasson MA, Chessman, Tonkin M, Swapna LS, Hung SS, Bridgers J, Ricklefs SM, Boulanger MJ, Dubey JP, Porcella SF, Kissinger JC, Howe DK, Grigg ME, Parkinson J. 2015. Systems-based analysis of the Sarcocystis neurona genome identifies pathways that contribute to a heteroxenous life cycle. mBio 6(1):e02445-14. Abstract Frameshifting is an essential process that regulates protein synthesis in many viruses. The ribosome may slip backward when encountering a frameshift motif on the messenger RNA, which usually contains a pseudoknot structure involving tertiary base pair interactions. Due to the lack of detailed molecular explanations, previous studies investigating which features of the pseudoknot are important to stimulate frameshifting have presented diverse conclusions. Here we constructed a bimolecular pseudoknot to dissect the interior tertiary base pairs and used single-molecule approaches to assess the structure targeted by ribosomes. We found that the first ribosome target stem was resistant to unwinding when the neighboring loop was confined along the stem; such constrained conformation was dependent on the presence of consecutive adenosines in this loop. Mutations that disrupted the distal base triples abolished all remaining tertiary base pairs. Changes in frameshifting efficiency correlated with the stem unwinding resistance. Our results demonstrate that various tertiary base pairs are coordinated inside a highly efficient frameshift-stimulating RNA pseudoknot and suggest a mechanism by which mechanical resistance of the pseudoknot may persistently act on translocating ribosomes. Abstract The ongoing new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) outbreak is spreading in China, but it has not yet reached its peak. Five million people emigrated from Wuhan before lockdown, potentially representing a source of virus infection. Determining case distribution and its correlation with population emigration from Wuhan in the early stage of the epidemic is of great importance for early warning and for the prevention of future outbreaks.The official case report on the COVID-19 epidemic was collected as of January 30, 2020. Time and location information on COVID-19 cases was extracted and analyzed using ArcGIS and WinBUGS software. Data on population migration from Wuhan city and Hubei province were extracted from Baidu Qianxi, and their correlation with the number of cases was analyzed.The COVID-19 confirmed and death cases in Hubei province accounted for 59.91% (5806/9692) and 95.77% (204/213) of the total cases in China respectively. Hot spot provinces included Sichuan and Yunnan, which are adjacent to Hubei. The time risk of Hubei province on the following day was 1.960 times that on the previous day. The number of cases in some cities was relatively low, but the time risk appeared to be continuously rising. The correlation coefficient between the provincial number of cases and emigration from Wuhan was up to 0.943. The lockdown of 17 cities in Hubei province and the implementation of nationwide control measures efficiently prevented an exponential growth in the number of cases.The population that emigrated from Wuhan was the main infection source in other cities and provinces. Some cities with a low number of cases showed a rapid increase in case load. Owing to the upcoming Spring Festival return wave, understanding the risk trends in different regions is crucial to ensure preparedness at both the individual and organization levels and to prevent new outbreaks. Abstract Cross-linking of surface receptors results in 1. Abbreviations used in this paper: 2-IT, 2-iminothiolane; ct2-M , a2-macroglobulin; I3-VLDL, 13-very low density lipoprotein; Au-F-Tfl0, fluorescein-labeled-multivalent transferrin-colloidal gold conjugate; Cy3-Tf, Cy3-1abeled-transferrin; DAB, diaminobenzadine; DiI-LDL, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled-low density lipoprotein; F-Tfl0, fluorescein-labeled-multivalent transferrin; LDL, low density lipoprotein; sulfo-MBS, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; Tf, transferrin; TfR, transferrin receptor; Tfl0, multivalent transferrin. Abstract Public health officials are increasingly recognizing the need to develop disease-forecasting systems to respond to epidemic and pandemic outbreaks. For instance, simple epidemic models relying on a small number of parameters can play an important role in characterizing epidemic growth and generating short-term epidemic forecasts. In the absence of reliable information about transmission mechanisms of emerging infectious diseases, phenomenological models are useful to characterize epidemic growth patterns without the need to explicitly model transmission mechanisms and the natural history of the disease. In this article, our goal is to discuss and illustrate the role of regularization methods for estimating parameters and generating disease forecasts using the generalized Richards model in the context of the 2014e15 Ebola epidemic in West Africa. Abstract The preceding communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell Biol. 101:1578-1590 described the lipid composition of a series of mouse fibroblast cell lines which vary in susceptibility to the fusogenic effects of polyethylene glycol (PEG). Two alterations in lipid content were found to be directly correlated with resistance to PEG-induced cell fusion: increases in fatty acyl chain saturation, and the elevation of neutral glycerides, including an unusual ether-linked compound. In this study, we have probed the association between lipid composition and cell fusion through the use of fatty acid supplements to the cellular growth medium, and show that the fusibility of cells can be controlled by altering their acyl chain composition. The parental Clone 1D cells contain moderately unsaturated fatty acids with a ratio of saturates to polyunsaturates (S/P) ,~ 1, and fuse virtually to completion following a standard PEG treatment. By contrast, the lipids of a highly fusion-resistant mutant cell line, F40, are highly saturated (S/P = 4). When the S/P ratio of Clone 1D cells was increased to approximate that normally found in F40 cells by growth in the presence of high concentrations of saturated fatty acids, they became highly resistant to PEG. Reduction of the S/P ratio of F40 cells by growth in cis-polyunsaturated fatty acids rendered them susceptible to fusion. Cell lines Fa, F16, etc., which are normally intermediate between Clone 1D and F40 in both lipid composition and fusion response, can be altered in either direction (towards either increased or decreased susceptibility to fusion) by the addition of appropriate fatty acids to the growth medium. Although trans-unsaturated fatty acids have phase-transition temperatures roughly similar to saturated compounds, and might therefore be expected to affect membrane fluidity in a similar manner, trans-unsaturated fatty acids exerted the same effect as cisunsaturates on the control of PEG-induced cell fusion. This observation suggests that the control of cell fusion by alteration of fatty acid content is not due to changes in membrane fluidity, and thus that the fatty acids are involved in some other way in the modulation of cell fusion. Abstract The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe respiratory infection with a high mortality rate (~35%). MERS-CoV has been a global threat due to continuous outbreaks in the Arabian peninsula and international spread by infected travelers since 2012. From May to July 2015, a large outbreak initiated by an infected traveler from the Arabian peninsula swept South Korea and resulted in 186 confirmed cases with 38 deaths (case fatality rate, 20.4%).Here, we show the rapid emergence and spread of a mutant MERS-CoV with reduced affinity to the human CD26 receptor during the South Korean outbreak. We isolated 13 new viral genomes from 14 infected patients treated at a hospital and found that 12 of these genomes possess a point mutation in the receptor-binding domain (RBD) of viral spike (S) protein. Specifically, 11 of these genomes have an I529T mutation in RBD, and 1 has a D510G mutation. Strikingly, both mutations result in reduced affinity of RBD to human CD26 compared to wild-type RBD, as measured by surface plasmon resonance analysis and cellular binding assay. Additionally, pseudotyped virus bearing an I529T mutation in S protein showed reduced entry into host cells compared to virus with wild-type S protein. These unexpected findings suggest that MERS-CoV adaptation during human-to-human spread may be driven by host immunological pressure such as neutralizing antibodies, resulting in reduced affinity to host receptor, and thereby impairs viral fitness and virulence, rather than positive selection for a better affinity to CD26.and resulted in 186 confirmed cases with 38 deaths. This is the largest outbreak outside the Middle East, and it raised strong concerns about the possible emergence of MERS-CoV mutations. Here, we isolated 13 new viral genomes and found that 12 of them possess a point mutation in the receptor-binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, CD26, compared to the wild-type virus. These unexpected findings suggest that MERS-CoV adaptation in humans may be driven by host immunological pressure. N-H. 2016. Spread of mutant Middle East respiratory syndrome coronavirus with reduced affinity to human CD26 during the South Korean outbreak. mBio 7(2):e00019-16. Abstract Mesenchymal stem cells have been widely studied to promote local bone regeneration of osteonecrosis of the femoral head (ONFH). Previous studies observed that dimethyloxaloylglycine (DMOG) enhanced the angiogenic and osteogenic activity of mesenchymal stem cells by activating the expression of hypoxia inducible factor-1α (HIF-1α), thereby improving the bone repair capacity of mesenchymal stem cells. In the present study, it was investigated whether DMOG could increase the bone repair capacity of adipose-derived stem cells (ASCs) in the treatment of ONFH. Western blot analysis was performed to detect HIF-1α protein expression in ASCs treated with different concentrations of DMOG. The results showed DMOG enhanced HIF-1α expression in ASCs in a dose-dependent manner at least for 7 days. Furthermore, DMOG-treated ASCs were transplanted into the necrotic area of a rabbit model of ONFH to treat the disease. Four weeks later, micro-computed tomography (CT) quantitative analysis showed that 58.8±7.4% of the necrotic area was regenerated in the DMOG-treated ASCs transplantation group, 45.5±3.4% in normal ASCs transplantation group, 25.2±2.8% in only core decompression group and 10.6±2.6% in the untreated group. Histological analysis showed that transplantation of DMOG-treated ASCs clearly improved the bone regeneration of the necrotic area compared with the other three groups. Micro-CT and immunohistochemical analysis demonstrated the revasculation of the necrotic area were also increased significantly in the DMOG-treated ASC group compared with the control groups. Thus, it is hypothesized that DMOG could increase the bone repair capacity of ASCs through enhancing HIF-1α expression in the treatment of ONFH. Abstract We have previously reported intra-segmental crossovers in Brome mosaic virus (BMV) RNAs. In this work, we studied the homologous recombination of BMV RNA in three different hosts: barley (Hordeum vulgare), Chenopodium quinoa, and Nicotiana benthamiana that were co-infected with two strains of BMV: Russian (R) and Fescue (F). Our work aimed at (1) establishing the frequency of recombination, (2) mapping the recombination hot spots, and (3) addressing host effects. The F and R nucleotide sequences differ from each other at many translationally silent nucleotide substitutions. We exploited this natural variability to track the crossover sites. Sequencing of a large number of cDNA clones revealed multiple homologous crossovers in each BMV RNA segment, in both the whole plants and protoplasts. Some recombination hot spots mapped at similar locations in different hosts, suggesting a role for viral factors, but other sites depended on the host. Our results demonstrate the chimeric ('mosaic') nature of the BMV RNA genome. Abstract Graphical Abstract Highlights d ER stress pathways can mediate immune recognition of zoonotic viruses d Glycosylation status of viral proteins regulates activation of ER stress d Acute lung injury from pandemic influenza viruses is dependent on this activation d Adaptation through glycan addition mediates immune escape of seasonal IAV Abstract Extracorporeal membrane oxygenation (ECMO) has developed as a critical tool permitting lung protection in severe respiratory failure. Its use was largely confined to acute respiratory distress syndrome [1]; however, as technology has advanced, it is now used in a range of respiratory diseases, including asthma. In the context of near-fatal asthma exacerbations, ECMO provides a management strategy for difficult-to-ventilate patients who would otherwise be unlikely to survive. Importantly, in asthma, traditional mechanical ventilation strategies can be associated with volutrauma and barotrauma due to the high pressures required in the presence of severe bronchospasm [2] . To date, there is a paucity of data for ECMO use in acute asthma and it is unknown whether specific clinical or inflammatory characteristics are associated with the need for ECMO.We performed a retrospective review of all adult asthmatics requiring mechanical ventilatior and/or ECMO for near-fatal asthma admitted to our single large tertiary hospital between 2011 and 2016. Clinical and demographic data including ventilator parameters, biochemical and immunological indices, and microbiology isolates were recorded. We compared patients requiring ECMO with patients requiring conventional mechanical ventilation only, to identify any factors that were significantly associated with the need for ECMO.All data, including demographic, physiological and laboratory data, utilised in this study were collected as part of routine acute asthma care in our intensive care unit (ICU) and tertiary severe asthma centre. No additional ethical approval was required. 76 patients (46% female, mean±SD age 39±16 years) with a primary diagnosis of acute asthma associated with the clinical features of a near-fatal exacerbation requiring ECMO or mechanical ventilation were included in this analysis. 22 patients (29%) required ECMO and 54 (71%) required conventional mechanical ventilation only. Compared to patients requiring mechanical ventilation, those requiring ECMO were more likely to be female (72% versus 35%, p=0.003) and younger (mean age 30±14 versus 43±15 years, p=0.002). Prior to intervention, patients requiring ECMO had a higher total white cell count (15.5±6.4 versus 12.2±4.4×10 9 L −1 , p=0.013), were more acidotic (mean pH 7.12±0.17 versus 7.27±0.09, p<0.001) and hypercapnic (mean carbon dioxide tension 12.8±4.1 versus 7.26±2.1 kPa, p<0.001). No statistically significant differences in oxygenation (oxygen tension), C-reactive protein (CRP), total serum immunoglobulin E or blood eosinophil counts at the time of admission to the ICU were observed between groups. Patients requiring ECMO were more likely to have a positive fungal isolate from bronchoalveolar lavage (BAL) fluid than those requiring mechanical ventilation (36% versus 10%, p=0.026). Rhinovirus was also identified in a greater proportion of respiratory isolates in the ECMO cohort compared to mechanical ventilation (27.2% versus 6.9%, p=0.048) with a strong trend towards a greater likelihood of any respiratory virus isolation in the ECMO group (54.5% versus 27.6%, p=0.053). There was no difference in the @ERSpublications Patients with near-fatal asthma requiring ECMO are more likely to be younger and female and are also likely to have positive viral and fungal isolates on bronchoalveolar lavage when compared to those receiving conventional mechanical ventilation http://bit.ly/2S38SaCCite this article as: Patel S, Shah NM, Malhotra AM, et al. Inflammatory and microbiological associations with near-fatal asthma requiring extracorporeal membrane oxygenation. ERJ Open Res Abstract Plant-based biomanufacturing of therapeutic proteins is a relatively new platform with a small number of commercial-scale facilities, but offers advantages of linear scalability, reduced upstream complexity, reduced time to market, and potentially lower capital and operating costs. In this study we present a detailed process simulation model for a large-scale new "greenfield" biomanufacturing facility that uses transient agroinfiltration of Nicotiana benthamiana plants grown hydroponically indoors under lightemitting diode lighting for the production of a monoclonal antibody. The model was used to evaluate the total capital investment, annual operating cost, and cost of goods sold as a function of mAb expression level in the plant (g mAb/kg fresh weight of the plant) and production capacity (kg mAb/year). For the Base Case design scenario (300 kg mAb/year, 1 g mAb/kg fresh weight, and 65% recovery in downstream processing), the model predicts a total capital investment of $122 million dollars and cost of goods sold of $121/g including depreciation. Compared with traditional biomanufacturing platforms that use mammalian cells grown in bioreactors, the model predicts significant reductions in capital investment and >50% reduction in cost of goods compared with published values at similar production scales. The simulation model can be modified or adapted by others to assess the profitability of alternative designs, implement different process assumptions, and help guide process development and optimization. Abstract Objective: The purpose of this study was to quantify lesions on chest radiographs in patients with severe acute respiratory syndrome (SARS) and analyze the severity of the lesions with clinical parameters.Two experienced radiologists reviewed chest radiographs of 28 patients with SARS. Each lung was divided into upper, middle, and lower zones. A SARS-related lesion in each zone was scored using a four-point scale: zero to three. The mean and maximal radiographic scores were analyzed statistically to determine if the scorings were related to the laboratory data and clinical course.Results: Forward stepwise multiple linear regression showed that the mean radiographic score correlated most significantly with the number of hospitalized days (p < 0.001). The second most significant factor was the absolute lymphocyte count (p < 0.001) and the third most significant factor was the number of days of intubation (p = 0.025). The maximal radiographic score correlated best with the percentage of lymphocytes in a leukocyte count (p < 0.001), while the second most significant factor was the number of hospitalized days (p < 0.001) and the third most significant factor was the absolute lymphocyte count (p = 0.013). The mean radiographic scores of the patients who died, with comorbidities and without a comorbidity were 11.1, 6.3 and 2.9, respectively (p = 0.032). The corresponding value for maximal radiographic scores were 17.7, 9.7 and 6.0, respectively (p = 0.033).The severity of abnormalities quantified on chest radiographs in patients with SARS correlates with the clinical parameters. Abstract Ebolaviruses are highly pathogenic in humans and nonhuman primates and pose a severe threat to public health. The interferon-induced transmembrane (IFITM) proteins can restrict entry of ebolaviruses, influenza A viruses, and other enveloped viruses. However, the breadth and mechanism of the antiviral activity of IFITM proteins are incompletely understood. Here, we employed ebolavirus glycoprotein-pseudotyped vectors and ebolavirus-like particles to address this question. We show that IFITM proteins inhibit the cellular entry of diverse ebolaviruses and demonstrate that type I interferon induces IFITM protein expression in macrophages, major viral targets. Moreover, we show that IFITM proteins block entry of influenza A viruses and ebolaviruses by different mechanisms and provide evidence that antibodies and IFITM proteins can synergistically inhibit cellular entry of ebolaviruses. These results provide insights into the role of IFITM proteins in infection by ebolaviruses and suggest a mechanism by which antibodies, though poorly neutralizing in vitro, might contribute to viral control in vivo. Abstract Waddlia chondrophila, an obligate intracellular bacterium of the Chlamydiales order, is considered as an agent of bovine abortion and a likely cause of miscarriage in humans. Its role in respiratory diseases was questioned after the detection of its DNA in clinical samples taken from patients suffering from pneumonia or bronchiolitis. To better define the role of Waddlia in both miscarriage and pneumonia, a tool allowing large-scale serological investigations of Waddlia seropositivity is needed. Therefore, enriched outer membrane proteins of W.chondrophila were used as antigens to develop a specific ELISA.After thorough analytical optimization, the ELISA was validated by comparison with micro-immunofluorescence and it showed a sensitivity above 85% with 100% specificity. The ELISA was subsequently applied to human sera to specify the role of W. chondrophila in pneumonia. Overall, 3.6% of children showed antibody reactivity against W. chondrophila but no significant difference was observed between children with and without pneumonia. Proteomic analyses were then performed using mass spectrometry, highlighting members of the outer membrane protein family as the dominant proteins. The major Waddlia putative immunogenic proteins were identified by immunoblot using positive and negative human sera. The new ELISA represents an efficient tool with high throughput applications. Although no association with pneumonia and Waddlia seropositivity was observed, this ELISA could be used to specify the role of W. chondrophila in miscarriage and in other diseases. Abstract Graphical Abstract Highlights d PI4KB activity expedites the formation of coxsackievirus replication organelles (ROs) d PI4KB inhibition impairs polyprotein processing, which is rescued by a 3A mutation d Upon PI4KB inhibition, this mutant replicates at the Golgi in the absence of ROs d Innate immune responses are not enhanced when RO biogenesis is delayed Correspondence m.barcena@lumc.nl (M.B.), f.j.m.vankuppeveld@uu.nl (F.Enteroviruses reorganize cellular endomembranes into replication organelles (ROs) for genome replication. Although enterovirus replication depends on phosphatidylinositol 4-kinase type IIIb (PI4KB), its role, and that of its product, phosphatidylinositol 4-phosphate (PI4P), is only partially understood. Exploiting a mutant coxsackievirus resistant to PI4KB inhibition, we show that PI4KB activity has distinct functions both in proteolytic processing of the viral polyprotein and in RO biogenesis. The escape mutation rectifies a proteolytic processing defect imposed by PI4KB inhibition, pointing to a possible escape mechanism. Remarkably, under PI4KB inhibition, the mutant virus could replicate its genome in the absence of ROs, using instead the Golgi apparatus. This impaired RO biogenesis provided an opportunity to investigate the proposed role of ROs in shielding enteroviral RNA from cellular sensors. Neither accelerated sensing of viral RNA nor enhanced innate immune responses was observed. Together, our findings challenge the notion that ROs are indispensable for enterovirus genome replication and immune evasion. Abstract Background: The genus Orthobornavirus comprises non-segmented, negative-stranded RNA viruses able to infect humans, mammals, reptiles and various birds. Parrot bornavirus 1 to 8 (PaBV-1 to 8) causes neurological and/or gastrointestinal syndromes and death on psittacines. We aimed to identify and to produce epidemiologic knowledge about the etiologic agent associated with a death of two female Psittacus erithacus (grey parrot). Methods and Results: Both parrots were submitted for a complete standardised necropsy. Tissue samples were analysed by PCR. The findings in necropsy were compatible with bornavirus infection. Analysis revealed PaBV-4 related with genotypes detected in captive and in wild birds. The N and X proteins of PaBV-4 were more related to avian bornaviruses, while phosphoprotein was more related to variegated squirrel bornavirus 1 (VSBV-1). Within the P gene/phosphoprotein a highly conserved region between and within bornavirus species was found. Conclusions: Portugal is on the routes of the intensive world trade of psittacines. Broad screening studies are required to help understanding the role of wild birds in the emergence and spread of pathogenic bornaviruses. PaBV-4 phosphoprotein is closer to VSBV-1 associated with lethal encephalitis in humans than with some of the avian bornaviruses. The highly conserved P gene/ phosphoprotein region is a good target for molecular diagnostics screenings.ARTICLE HISTORY Abstract Background: Disseminated or fatal Zika virus (ZIKV) infections were reported in immunosuppressed patients. Existing interferon-signaling/receptor-deficient mouse models may not be suitable for evaluating treatment effects of recombinant interferons. Methods: We developed a novel mouse model for ZIKV infection by immunosuppressing BALB/c mice with dexamethasone. Results: Dexamethasone-immunosuppressed male mice (6-8 weeks) developed disseminated infection as evidenced by the detection of ZIKV-NS1 protein expression and high viral loads in multiple organs. They had ≥10% weight loss and high clinical scores soon after dexamethasone withdrawal (10 dpi), which warranted euthanasia at 12 dpi. Viral loads in blood and most tissues at 5 dpi were significantly higher than those at 12 dpi (P b 0.05). Histological examination revealed prominent inflammatory infiltrates in multiple organs, and CD45+ and CD8+ inflammatory cells were seen in the testis. These findings suggested that clinical deterioration occurred during viral clearance by host immune response. Type I interferon treatments improved clinical outcome of mice (100% vs 0% survival). Conclusions: Besides virus dissemination, inflammation of various tissues, especially orchitis, may be potential complications of ZIKV infection with significant implications on disease transmission and male fertility. Interferon treatment should be considered in patients at high risks for ZIKV-associated complications when the potential benefits outweigh the side effects of treatment. Abstract Background: BCoV is identified in both healthy and diarrheic calves, complicating its assessment as a primary pathogen. Objectives: To investigate the detection rates of bovine coronavirus (BCoV) in feces of healthy and diarrheic calves and to describe the usefulness of a pancoronavirus reverse transcriptase (RT) PCR (PanCoV-RT-PCR) assay to identify BCoV in samples of diarrheic calves.Animals: Two hundred and eighty-six calves <21 days. Calves with liquid or semiliquid feces, temperature >39.5°C, and inappetence were considered as cases, and those that had pasty or firm feces and normal physical examination were designated as controls.Methods: Prospective case-control study. A specific BCoV-RT-PCR assay was used to detect BCoV in fecal samples. Association between BCoV and health status was evaluated by exact and random effect logistic regression. Fecal (n = 28) and nasal (n = 8) samples from diarrheic calves were tested for the presence of BCoV by both the PanCoV-RT-PCR and a specific BCoV-RT-PCR assays. A Kappa coefficient test was used to assess the level of agreement of both assays.Results: BCoV was detected in 55% (157/286) of calves; 46% (66/143), and 64% (91/143) of healthy and diarrheic calves, respectively. Diarrheic calves had higher odds of BCoV presence than healthy calves (OR: 2.16, 95% CI: 1.26 to 3.83, P = 0.004). A good agreement between PanCoV-RT-PCR and BCoV-RT-PCR to detect BCoV was identified (j = 0.68, 95% CI: 0.392 to 0.967; P < 0.001).Conclusions and Clinical Importance: BCoV was more likely to be detected in diarrheic than healthy calves. The PanCoV-RT-PCR assay can be a useful tool to detect CoV samples from diarrheic calves. Abstract Single-stranded RNAs (ssRNAs) are ubiquitous RNA elements that serve diverse functional roles. Much of our understanding of ssRNA conformational behavior is limited to structures in which ssRNA directly engages in tertiary interactions or is recognized by proteins. Little is known about the structural and dynamic behavior of free ssRNAs at atomic resolution. Here, we report the collaborative application of nuclear magnetic resonance (NMR) and replica exchange molecular dynamics (REMD) simulations to characterize the 12 nt ssRNA tail derived from the prequeuosine riboswitch. NMR carbon spin relaxation data and residual dipolar coupling measurements reveal a flexible yet stacked core adopting an A-form-like conformation, with the level of order decreasing toward the terminal ends. An A-to-C mutation within the polyadenine tract alters the observed dynamics consistent with the introduction of a dynamic kink. Pre-ordering of the tail may increase the efficacy of ligand binding above that achieved by a random-coil ssRNA. The REMD simulations recapitulate important trends in the NMR data, but suggest more internal motions than inferred from the NMR analysis. Our study unmasks a previously unappreciated level of complexity in ssRNA, which we believe will also serve as an excellent model system for testing and developing computational force fields. Abstract AbstracL Expression of hepatitis B surface antigen (HBsAg), the major envelope protein of the virus, in the absence of other viral proteins leads to its secretion as oligomers in the form of disk-like or tubular lipoprotein particles. The observation that these lipoprotein particles are heavily disulphide crosslinked is paradoxical since HBsAg assembly is classically believed to occur in the ER, and hence in the presence of high levels of protein disulphide isomerase (PDI) which should resolve these higher intermolecular crosslinks. Indeed, incubation of mature, highly disulphide crosslinked HBsAg with recombinant PDI causes the disassembly of HBsAg to dimers. We have used antibodies against resident ER proteins in double immunofluorescence studies to study the stages of the conversion of the HBsAg from individual protein subunits to the secreted, crosslinked, oligomer. We show that HBsAg is rapidly sorted to a post-ER, pre-Golgi compartment which excludes PDI and other major soluble resident ER proteins although it overlaps with the distribution of rab2, an established marker of an intermediate compartment. Kinetic studies showed that disulphide-linked HBsAg dimers began to form during a short (2 rain) pulse, increased in concentration to peak at 60 rain, and then decreased as the dimers were crosslinked to form higher oligomers. These higher oligomers are the latest identifiable intracellular form of HBsAg before its secretion (h/2 = 2 h). Brefeldin A treatment does not alter the localization of HBsAg in this PDI excluding compartment, however, it blocks the formation of new oligomers causing the accumulation of dimeric HBsAg. Hence this oligomerization must occur in a pre-Golgi compartment. These data support a model in which rapid dimer formation, catalyzed by PDI, occurs in the ER, and is followed by transport of dimers to a pre-Golgi compartment where the absence of PDI and a different lumenal environment allow the assembly process to be completed.NVELOPED animal viruses have been major tools in the characterization of the secretory pathway and in advancing our understanding of the mechanisms of protein sorting. Frequently, particular features of the pathways of protein traffic are exploited in the maturation pathways of different viruses so that a study of virus assembly leads to unexpected rewards in terms of an understanding of intracellular transport. This paper presents a study of the assembly of the lipoprotein particle formed by the transmembrane glycoprotein of hepatitis B virus. This particle has the unusual feature of being stabilized by extensive intermolecutax crosslinks and here we show that this reflects its assembly in an intermediate compartment between the ER and the Golgi.Hepatitis B virus (HBV) ~ is a small (440/~) DNA virus 1. Abbt~*viations used in this paper: BfA, Brefeldin A; BiP, Ig heavy chain binding protein; HBsAg, hepatitis B surface antigen protein; HBV, hepatitis B virus; PDI, protein disulphide isornerase; St9, a clonal isolate of Spodopterafrugiperda IPLB-Sf21-AE cells; anti-KXsKDEL a polyclonal antibody developed in rabbits against a mixture of peptides with the sequences KXXXXXKDEL where X indicates a position filled by a mixture of alanine, aspartic acid, histidine, glutamine, leucine, tyrosine, and lysine.which is the type virus of the hepadnaviridae (hepatotropic DNA viruses). HBV consists of a nucleoprotein core containing a relatively small DNA genome (3,200 bp) surrounded by a protein capsid composed of the core protein, hepatitis B core antigen. The nucleocapsid is surrounded by an envelope in which the major protein component is the 226 amino acid hepatitis B surface antigen (HBsAg) which exists in nonglycosylated 24-kD (P24) and in N-glycosylated 27-kD (GP27) forms. In addition to being an envelope protein of the infectious virion, HBsAg is assembled into coreless, lipoprotein particles that are secreted from HBV infected cells and form the bulk of HBsAg in the circulation. These noninfectious lipoprotein particles occur in two morphological forms: 22-nm diameter particles which appear as spherical when negatively stained for EM and filamentous particles of the same diameter and variable length (Bayer et al., 1968; Robinson and Lutwick, 1976; Dane et al., 1970) . The "spherical" forms appear disk-like when visualized by cryoelectron microscopy and could represent fragments of the tubular forms (Huovila, A.-P. J., and S. D. Fuller, manuscript in preparation). The round 22-rim I-IBsAg particles isolated from the sera 9 The Rockefeller University Press, Abstract Background: Hepatitis C virus (HCV) is major cause of liver cirrhosis in humans. HCV capsid (core) protein (HCVcp) is a highly demanded antigen for various diagnostic, immunization and pathogenesis studies. Plants are considered as an expression system for producing safe and inexpensive biopharmaceutical proteins. Although invention of transgenic (stable) tobacco plants expressing HCVcp with proper antigenic properties was recently reported, no data for "transient-expression" that is currently the method of choice for rapid, simple and lower-priced protein expression in plants is available for HCVcp. Objectives: The purpose of this study was to design a highly codon-optimized HCVcp gene for construction of an efficient transient-plant expression system for production of HCVcp with proper antigenic properties in a regional tobacco plant (Iranian Jafarabadi-cultivar) by evaluation of different classes of vectors and suppression of gene-silencing in tobacco.A codon-optimized gene encoding the Kozak sequence, 6xHis-tag, HCVcp (1-122) and KDEL peptide in tandem (from N-to C-terminal) was designed and inserted into potato virus-X (PVX) and classic pBI121 binary vectors in separate cloning reactions. The resulted recombinant plasmids were transferred into Agrobacterium tumefaciens and vacuum infiltrated into tobacco leaves. The effect of gene silencing suppressor P19 protein derived from tomato bushy stunt virus on the expression yield of HCVcp by each construct was also evaluated by co-infiltration in separate groups. The expressed HCVcp was evaluated by dot and western blotting and ELISA assays. Results: The codon-optimized gene had an increased adaptation index value (from 0.65 to 0.85) and reduced GC content (from 62.62 to 51.05) in tobacco and removed the possible deleterious effect of "GGTAAG" splice site in native HCVcp. Blotting assays via specific antibodies confirmed the expression of the 15 kDa HCVcp. The expression level of HCVcp was enhanced by 4-5 times in P19 co-agroinfiltrated plants with better outcomes for PVX, compared to pBI121 vector (0.022% versus 0.019% of the total soluble protein). The plant-derived HCVcp (pHCVcp) could properly identify the HCVcp antibody in HCV-infected human sera compared to Escherichia coli-derived HCVcp (eHCVcp), indicating its potential for diagnostic/immunization applications. Conclusions: By employment of gene optimization strategies, use of viral-based vectors and suppression of plant-derived gene silencing effect, efficient transient expression of HCVcp in tobacco with proper antigenic properties could be possible. Abstract Upon viral infection, the 2', 5'-oligoadenylate synthetase (OAS)-ribonuclease L (RNaseL) system works to cleave viral RNA, thereby blocking viral replication. However, it is unclear whether OAS proteins have a role in regulating gene expression. Here, we show that OAS1 and OAS3 act as negative regulators of the expression of chemokines and interferonresponsive genes in human macrophages. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology was used to engineer human myeloid cell lines in which the OAS1 or OAS3 gene was deleted. Neither OAS1 nor OAS3 was exclusively responsible for the degradation of rRNA in macrophages stimulated with poly(I:C), a synthetic surrogate for viral double-stranded (ds)RNA. An mRNA sequencing analysis revealed that genes related to type I interferon signaling and chemokine activity were increased in OAS1 −/− and OAS3 −/−TLR4-mediated induction of those genes was modulated by OAS1 in macrophages. However, stimulation of these cells with type I interferons had no effect on OAS1-or OAS3mediated chemokine secretion. These data suggest that OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages. [BMB Reports 2019; 52(2): 133-138] BMB Rep. 2019; 52(2): 133-138 www.bmbreports.org Abstract Objectives Feline leukaemia virus (FeLV), a gamma retrovirus, causes diseases of the feline haematopoietic system that are invariably fatal. Rapid and accurate testing at the point-of-need (PON) supports prevention of virus spread and management of clinical disease. This study evaluated the performance of an insulated isothermal PCR (iiPCR) that detects proviral DNA, and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA, for FeLV detection at the PON. Methods Mycoplasma haemofelis, feline coronavirus, feline herpesvirus, feline calicivirus and feline immunodeficiency virus were used to test analytical specificity. In vitro transcribed RNA, artificial plasmid, FeLV strain American Type Culture Collection VR-719 and a clinical FeLV isolate were used in the analytical sensitivity assays. A retrospective study including 116 clinical plasma and serum samples that had been tested with virus isolation, real-time PCR and ELISA, and a prospective study including 150 clinical plasma and serum samples were implemented to evaluate the clinical performances of the iiPCR-based methods for FeLV detection. Results Ninety-five percent assay limit of detection was calculated to be 16 RNA and five DNA copies for the RT-iiPCR, and six DNA copies for the iiPCR. Both reactions had analytical sensitivity comparable to a reference realtime PCR (qPCR) and did not detect five non-target feline pathogens. The clinical performance of the RT-iiPCR and iiPCR had 98.82% agreement (kappa[κ] = 0.97) and 100% agreement (κ = 1.0), respectively, with the qPCR (n = 85). The agreement between an automatic nucleic extraction/RT-iiPCR system and virus isolation to detect FeLV in plasma or serum was 95.69% (κ = 0.95) and 98.67% (κ = 0.85) in a retrospective (n = 116) and a prospective (n = 150) study, respectively. Conclusions and relevance These results suggested that both RT-iiPCR and iiPCR assays can serve as reliable tools for PON FeLV detection. Abstract Background: Human infections with zoonotic coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, have raised great public health concern globally. Here, we report a novel batorigin CoV causing severe and fatal pneumonia in humans. Methods: We collected clinical data and bronchoalveolar lavage (BAL) specimens from five patients with severe pneumonia from Jin Yin-tan Hospital of Wuhan, Hubei province, China. Nucleic acids of the BAL were extracted and subjected to next-generation sequencing. Virus isolation was carried out, and maximum-likelihood phylogenetic trees were constructed. Results: Five patients hospitalized from December 18 to December 29, 2019 presented with fever, cough, and dyspnea accompanied by complications of acute respiratory distress syndrome. Chest radiography revealed diffuse opacities and consolidation. One of these patients died. Sequence results revealed the presence of a previously unknown b-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. These isolates showed 79.0% nucleotide identity with the sequence of SARS-CoV (GenBank NC_004718) and 51.8% identity with the sequence of MERS-CoV (GenBank NC_019843). The virus is phylogenetically 1 closest to a bat SARS-like CoV (SL-ZC45, GenBank MG772933) with 87.6% to 87.7% nucleotide identity, but is in a separate clade. Moreover, these viruses have a single intact open reading frame gene 8, as a further indicator of bat-origin CoVs. However, the amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating that these viruses might use the same receptor. Conclusion: A novel bat-borne CoV was identified that is associated with severe and fatal respiratory disease in humans. Abstract To determine the prevalence and characteristics of birth defects in perinatal infants in Hubei Province during 200l-2008. Methods: The prevalence of birth defects in perinatal infants delivered after 28 weeks or more was analyzed in Hubei surveillance hospitals during 200l-2008. Results: The incidence of birth defects in perinatal infants from 200l to 2008 was 120.0 per 10,000 births, and was increased by about 41% from 81. 1 in 2001 to 138.5 per 10,000 births in 2008. The incidence in the first 4 years (2005)(2006)(2007)(2008) was much higher than the latter four (2001-2004) (χ 2 =77.64 , P <0.05). The difference in prevalence between urban and rural was of no significance in 2008 (χ 2 =0.03，P >0.05), but that between male and female was significant (χ 2 =5.24 , P <0.05), as the former prevalence was much higher. The prevalence of birth defects was slightly higher among mothers over 35 years old than those under 35 years old, but with no significance (χ 2 =1.98, P >0.05). The two leading birth defects were cleft lip and/or palate and polydactyly, followed by congenital heart disease, hydrocephaly, external ear malformation and neural tube defects. The prevalence of congenital heart disease was rising. Conclusions: Eight years' birth defects data indicate that the birth defect rate was on the rise and the birth defects prevalence in Hubei province should be valued. Abstract Some proteins are expressed as a result of a ribosome frameshifting event that is facilitated by a slippery site and downstream secondary structure elements in the mRNA. This review summarizes recent progress in understanding mechanisms of -1 frameshifting in several viral genes, including IBV 1a/ 1b, HIV-1 gag-pol, and SFV 6K, and in Escherichia coli dnaX. The exact frameshifting route depends on the availability of aminoacyl-tRNAs: the ribosome normally slips into the -1-frame during tRNA translocation, but can also frameshift during decoding at condition when aminoacyl-tRNA is in limited supply. Different frameshifting routes and additional slippery sites allow viruses to maintain a constant production of their key proteins. The emerging idea that tRNA pools are important for frameshifting provides new direction for developing antiviral therapies. Abstract The activation of somatic mutations conferring sensitivity to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors has been widely used in the development of advanced or metastatic primary lung cancer therapy. Therefore, identification of EGFR mutations is essential. In the present study, a loop-mediated isothermal amplification (LAMP) method was used to identify EGFR mutations, and its efficiency was compared with the Therascreen quantitative PCR assay. Using LAMP and Therascreen to analyze surgically resected tissue samples from patients with pulmonary adenocarcinoma, EGFR mutations were observed in 32/59 tumor samples (LAMP) and 33/59 tumor samples (Therascreen). Notably, the LAMP assay identified one tumor as wild-type, which had previously been identified as a deletion mutation in exon 19 via the Therascreen assay (Case X). However, the direct sequencing to confirm the EGFR status of the Case X adhered to the results of the LAMP assay. Further experiments using Case X DNA identified this exon 19 deletion mutation using both methods. In addition, a novel deletion mutation in exon 19 of the EGFR was identified. Overall, the present study shows that the LAMP method may serve as a valuable alternative for the identification oncogene mutations. Abstract Porcine epidemic diarrhoea virus (PEDV) causes acute and severe watery diarrhoea and dehydration, as well as 50-100 per cent mortality in piglets. For the PEDV diagnosis, a rapid test kit that is specific and sensitive to PEDV is critical to monitor this disease at pig farms. The present study aimed to develop an immunochromatographic assay (ICA) strip test for detecting PEDV in faecal swabs. The newly developed diagnostic test showed a detection limit of 10 4.0 TCID 50 /ml of PEDV. Using faecal swab samples, the relative sensitivity and specificity of the ICA kit were 95.0 per cent and 98.6 per cent, respectively, compared with those of real-time RT-PCR. In samples from piglets experimentally infected with PEDV, the results showed 100 per cent agreement with those found by real-time RT-PCR. Our developed test strip will be useful for rapid diagnosis and can be used for epidemiological surveillance of PEDV infection. Abstract To assess the role of spatial proximity, defined as patients sharing bays, in the spread of norovirus during outbreaks in hospitals.Enhanced surveillance of norovirus outbreaks between November 2009 and November 2011.Data were gathered during 149 outbreaks of norovirus in hospital wards from five hospitals in two major cities in England serving a population of 2 million. We used the time between the first two cases of each outbreak to estimate the serial interval for norovirus in this setting.This distribution and dates of illness onset were used to calculate epidemic trees for each outbreak. We then used a permutation test to assess whether proximity, for all outbreaks, was more extreme than would be expected by chance under the null hypothesis that proximity was not associated with transmission risk.Sixty-five outbreaks contained complete data on both onset dates and ward position. We estimated the serial interval to be 1.86 days (95% confidence intervals 1.6-2.2 days), and with this value found strong evidence to reject the null hypothesis that proximity was unimportant (p < 0.001). Sensitivity analysis using different values of the serial interval showed that there was evidence to reject the null hypothesis provided the assumed serial interval was less than 2.5 days.Our results provide evidence that patients occupying the same bay as patients with symptomatic norovirus infection are at an increased risk of becoming infected by these patients compared with patients elsewhere in the same ward. Abstract Combination antiretroviral therapy fails in complete suppression of HIV-1 due to drug resistance and persistent latency. Novel therapeutic intervention requires knowledge of intracellular pathways responsible for viral replication, specifically those untargeted by antiretroviral drugs. An understudied phenomenon is the nucleolar localization of Rev phosphoprotein, which completes nucleocytoplasmic transport of unspliced/partially spliced HIV mRNA through multimerization with intronic cis-acting targets-the Rev-response element (RRE). Rev contains a nucleolar localization signal (NoLS) comprising the COOH terminus of the arginine-rich motif for accumulation within nucleoli-speculated as the interaction ground for Rev with cellular proteins mediating mRNA-independent nuclear export and splicing. Functionality of Rev nucleolar access during HIV-1 production and infection was investigated in the context of deletion and single-point mutations within Rev-NoLS. Mutations induced upon Rev-NoLS are hypothesized to inactivate the HIV-1 infectious cycle. HIV-1 HXB2 replication ceased with Rev mutations lacking nucleolar access due to loss or replacement of multiple arginine residues. Rev mutations missing single arginine residues remained strictly nucleolar in pattern and participated in proviral production, however, with reduced efficiency. Viral RNA packaging also decreased in efficiency after expression of nucleolar-localizing mutations. These results were observed during propagation of variant HIV-1 NL4-3 containing nucleolar-localizing mutations within the viral backbone (M4, M5, and M6). Lentiviral particles produced with Rev single-point mutations were transducible at extremely low frequency. Similarly, HIV-1 NL4-3 Rev-NoLS variants lost infectivity, unlike virulent WT (wild type) HIV-1 NL4-3 . HIV-1 NL4-3 variants were capable of CD4 + host entry and reverse transcription as WT HIV-1 NL4-3 , but lacked ability to complete a full infectious cycle. We currently reveal that viral integration is deregulated in the presence of Rev-NoLS mutations. Abstract The purpose of this study was to evaluate the viral frequency, seasonality, and clinical and demographic features of patients hospitalized with acute bronchiolitis. A cross-sectional, descriptive study was performed in 316 infants younger than 2 years of age who were hospitalized for acute viral bronchiolitis. Respiratory tract infection agents were investigated with polymerase chain reaction (PCR). A total of 316 infants were included in this study. Of the 316 infants, at least one respiratory tract pathogen was detected in 75% (237/316). Respiratory syncytial virus (RSV) was the most common virus identified in 127 infants (40.1%) followed by rhinovirus (n = 78, 24.6%). In this study, where viral agents were determined via PCR in patients who were followed-up due to the diagnosis of acute bronchiolitis, RSV was detected as the most common agent, as in other studies. In almost half of the RSV-positive patients, RSV was accompanied by a second or third agent. Abstract In humans, infection with the coronavirus, especially the severe acute respiratory syndrome coronavirus (SARS-CoV) and the emerging Middle East respiratory syndrome coronavirus (MERS-CoV), induces acute respiratory failure, resulting in high mortality. Irregular coronavirus related epidemics indicate that the evolutionary origins of these two pathogens need to be identified urgently and there are still questions related to suitable laboratory animal models. Thus, in this review we aim to highlight key discoveries concerning the animal origin of the virus and summarize and compare current animal models.animal models, coronaviruses, reservoirs, the Middle East respiratory syndrome (canonical form), the severe acute respiratory syndromeThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Abstract Background/purpose: The licensed dentists in Taiwan should join the postgraduate year training program for dentists (PGYD) since 2010. This study aimed to analyze the project types and the geographical distribution of the PGYD training institutions in Taiwan from 2010 to 2018. Materials and methods: From 2010 to 2018, 735 hospitals and clinics participated in four types of PGYD project including hospital as the single training institution (project A), clinic as the single training institution (project B), hospital as the main training institution in the joint training group (project C), and clinic as the main training institution in the joint training group (project D). The project types and the geographical distribution of the training institutions were analyzed. Abstract Background: Middle East Respiratory Syndrome (MERS) is a respiratory disease caused by a novel coronavirus that was identified in 2012 in Saudi Arabia. It is associated with significant mortality and morbidity. We identified factors associated with the Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) infection among suspected cases presented with sign and symptoms of upper respiratory infection or exposure to the virus. We also looked at the impact of medication history on virus transmission. Method: We included subjects with suspected MERS-CoV infection and confirmed cases of MERS infection. Subjects were excluded if there were any missing data that prevent the final analysis. Descriptive statistics were used to report demographic data. Percentages and frequencies were used to summarize the categorical variables, while means and standard deviations were calculated for continuous variables. Logistic regression was used to assess the risk of MERS-CoV infection among the suspected cases. A value of p < 0.05 was considered statistically significant. Results: A total of 16,189 suspected cases were identified, complete data were analyzed for 3154 to assess factors that are independently associated with MERS-CoV infection. MERS-CoV infection was associated with age (adjusted odds ratio [AOR] = 1.06; 95% CI [1.02-1.098], P-value = 0.004), male gender (AOR = 1.617; 95% CI [1.365-1.77], P-value < 0.001) and diabetes (AOR = 1.68; 95% CI [1.346-1.848], P-value = 0.002. There was no significant association with the other comorbidities. Medication history was not associated with an increase or decrease the likelihood of the infection. Conclusions: MERS-Cov infection is more common in male, advanced age and diabetes. No medications were associated with an increase or decrease the likelihood of the infection. This is important to focus on screening and detection to this patient population. Abstract Background: Despite the introduction of a polymerase chain reaction (PCR) test for the diagnosis of respiratory viral infection (RVI), guidance on the application of this test and the management of RVI in immunocompromised children is lacking. This study evaluated the clinical characteristics of RVI and established strategies for the PCR test in children and adolescents with hematological malignancies.The medical records of the enrolled patients were retrospectively reviewed. Results: A total of 93 respiratory illnesses were included. Group I included 46 (49.5%) cases of RVI, including 31 (67.4%) upper and 15 (32.6%) lower respiratory infections. Rhinovirus (37.0%) was the most common viral pathogen. Significantly more patients in Group I had community-acquired respiratory illnesses (p=0.003) and complained of rhinorrhea (p<0.001) and sputum (p=0.008) than those in Group II. In Group I, significantly more patients with lower respiratory infections had uncontrolled underlying malignancies (p=0.038) and received reinduction or palliative chemotherapy (p=0.006) than those with upper respiratory infections. Conclusions: A multiplex PCR test should be considered for RVI diagnosis in immunocompromised children and adolescents with respiratory symptoms, especially in those with rhinorrhea or sputum prominent over a cough. The early application of the PCR test in patients with uncontrolled underlying malignancies may improve outcomes. Abstract Taiwan's National Laboratory System is one of the action packages of the Global Health Security Agenda, which was launched by the World Health Organization (WHO) to promote health security as an international priority and to encourage progress toward full implementation of the WHO International Health Regulations (IHR) 2005. The mission of each national laboratory system is to conduct real-time biosurveillance and effective laboratory-based diagnostics, as measured by a nationwide laboratory system able to reliably conduct diagnoses on specimens transported properly to designated laboratories from at least 80% of the regions in the country. In Taiwan, the national laboratory system for public health is well-established and coordinated by the Taiwan Centers for Disease Control (CDC), which is the government authority in charge of infectious disease prevention and intervention. Through the national laboratory system, Taiwan CDC effectively detects and characterizes pathogens that cause communicable diseases across the entire country, including both known and novel threats, and also conducts epidemiologic analyses of infectious diseases. In this article, we describe the national laboratory system for public health in Taiwan. We provide additional information on the national influenza laboratory surveillance network to demonstrate how our national laboratory systems work in practice, including descriptions of long-term seasonal influenza characterization and successful experiences identifying novel H7N9 and H6N1 influenza viruses. Abstract Infectious bronchitis (IB) is a highly contagious disease involving mostly upper respiratory tract in chickens, leading to significant economic losses in the poultry industry worldwide. One of the major concerns regarding to IB is the emergence of new types of infectious bronchitis viruses (IBVs). The purpose of this study was to identify the IBVs isolated from Iranian broiler chickens with respiratory symptoms. Twenty-five broiler flocks around Ahwaz (southwest of Iran) were examined for IBV. The specimens including trachea, lung, liver, kidney, and ceacal tonsil, were collected from diseased birds and inoculated into chicken embryonated eggs. Harvested allantoic fluids were subjected to reverse transcription polymerase chain reaction (RT-PCR) using primers in order to amplify spike 1 (S1) gene of IBV. The RT-PCR products of four IBV isolates were sequenced. The results showed that from 25 examined flocks with respiratory disease, 12 flocks (48.00%) were positive for IBV. In phylogenetic analysis, our isolates were closely related to the QX-like viruses such as PCRLab/06/2012 (Iran), QX, HC9, HC10, CK/CH/GX/NN11-1, CK/CH/JS/YC11-1, CK/CH/JS/2010/13, CK/CH/JS/2011/2 (China), QX/SGK-21, QX/SGK-11 (Iraq) with nucleotide homology up to 99.00%. This study indicates the role of IBVs in the respiratory disorders of broiler flocks located in southwest Iran, and also the existence of a variant of IBV, which is distinguishable from the other Iranian variants. Abstract The burden of stroke is comparatively greater in Asian countries than in the Western world. While there has been a documented recent decline in the incidence of stroke in several Western nations due to better risk factor management, much less is known about the nature and trajectory of stroke in Asia over the last decade. The objective of this study was to explore risk factors, medication use, incidence, and one-year recurrence of stroke in Taiwan. Methods We conducted a nationwide cohort study by reviewing all hospitalized patients ( ≥ 18 years) with a primary diagnosis of ischemic stroke between 2001 and 2011 from Taiwan National Health Insurance Research Database. Results A total of 291,381 first-ever ischemic stroke patients were enrolled between 2000 and 2011. The average age was about 70 years and approximately 58.6% of them were men. While prevalence of diabetes mellitus and hyperlipidemia, as well as use of statins, antiplatelet agents, and oral anticoagulant agents for atrial fibrillation significantly increased; incidence (142.3 vs. 129.5 per 100,000 in 2000 and 2011, respectively) and one-year recurrence (9.6% vs. 7.8% in 2000 and 2011, respectively) of stroke declined during this period of time.Conclusions Over the last decade in Taiwan, rates of primary ischemic stroke and one-year recurrent stroke decreased by 9% and 18% respectively. Abstract An outbreak of COVID-19 developed aboard the Princess Cruises Ship during January eFebruary 2020. Using mathematical modeling and time-series incidence data describing the trajectory of the outbreak among passengers and crew members, we characterize how the transmission potential varied over the course of the outbreak. Our estimate of the mean reproduction number in the confined setting reached values as high as~11, which is higher than mean estimates reported from community-level transmission dynamics in China and Singapore (approximate range: 1.1e7). Our findings suggest that R t decreased substantially compared to values during the early phase after the Japanese government implemented an enhanced quarantine control. Most recent estimates of R t reached values largely below the epidemic threshold, indicating that a secondary outbreak of the novel coronavirus was unlikely to occur aboard the Diamond Princess Ship. Abstract a b s t r a c t A 3.5 year-old cat was admitted to the University of Melbourne Veterinary Teaching Hospital for chronic vomiting. Abdominal ultrasonography revealed a focal, circumferential thickening of the wall of the duodenum extending from the pylorus aborally for 3 cm, and an enlarged gastric lymph node. Cytology of fine-needle aspirates of the intestinal mass and lymph node revealed an eosinophilic inflammatory infiltrate and numerous extracellular septate acute angle branching fungal-type hyphae. Occasional hyphae had globose terminal ends, as well as round to oval blastospores and germ tubes. Candida albicans was cultured from a surgical biopsy of the duodenal mass. No underlying host immunodeficiencies were identified. Passage of an abrasive intestinal foreign body was suspected to have caused intestinal mucosal damage resulting in focal intestinal candidiasis. The cat was treated with a short course of oral itraconazole and all clinical signs resolved. Abstract Small RNAs play a critical role in host-pathogen interaction. Indeed, small RNA-mediated silencing or RNA interference (RNAi) is one of the earliest forms of antiviral immunity. Although it represents the main defense system against viruses in many organisms, the antiviral role of RNAi has not been clearly proven in higher vertebrates. However, it is well established that their response to viral infection relies on the recognition of viral RNAs by host pattern recognition receptors (PRRs) to trigger activation of the interferon pathway. In the present work, we report the existence of a novel small noncoding RNA population produced in mammalian cells upon RNA virus infection. Using Sindbis virus (SINV) as a prototypic arbovirus model, we profiled the small RNA population of infected cells in both human and African green monkey cell lines. Here, we provide evidence for the presence of discrete small RNAs of viral origin that are not associated with the RNA-induced silencing complex (RISC), that are highly expressed and detected by Northern blot analysis, and that accumulate as 21-to 28-nucleotide (nt) species during infection. We report that the cellular antiviral endoribonuclease RNase L cleaves the viral genome, producing in turn the small RNAs. Surprisingly, we uncovered the presence of a modification on the 3=-end nucleotide of SINV-derived viral small RNAs (SvsRNAs) that might be at the origin of their stability. Altogether, our findings show that stable modified small viral RNAs could represent a novel way to modulate host-virus interaction upon SINV infection.Citation Girardi E, Chane-Woon-Ming B, Messmer M, Kaukinen P, Pfeffer S. 2013. Identification of RNase L-dependent, 3=-end-modified, viral small RNAs in Sindbis virusinfected mammalian cells. mBio 4(6):e00698-13. Abstract Organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. Stress response can either repair the damage or activate one of the programmed cell death (PCD) mechanisms, for example apoptosis, and finally end in cell death. One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. This process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rRNA is less susceptible to degradation in respiring cells with functional defence against oxidative stress. In addition, RNA fragmentation is independent of two yeast apoptotic factors, metacaspase Yca1 and apoptosis-inducing factor Aif1, but it relies on the apoptotic chromatin condensation induced by histone H2B modifications. These data describe a novel phenotype for certain stress-and ageingrelated PCD pathways in yeast. Abstract A 2-to 4-year-old uncastrated male Siberian tiger (Panthera tigris altica) bred in a local wild animal park presented with generalized clinical signs including abdominal pain, fever, lethargy, and anorexia, along with subcutaneous nodules along the trunk. The patient subsequently died of chronic, progressive dyspnea despite 45 days of antibiotic treatment. At necropsy, mesenteric fat inflammation and multiple subcutaneous, peritoneal, and intraabdominal nodules were observed. The lungs demonstrated congestion and heavy coagulation, and necrotic foci were observed on the cut surface. Histopathologically, the nodules were identified as granulomatous fatty tissue with numerous lymphocytes, infiltration with lipid-laden macrophages, and fibrosis. These changes were also noted in the lung. The etiology of this condition remains undetermined. Abstract The complement system, an important part of innate immunity, plays a critical role in pathogen clearance. Unregulated complement activation is likely to play a crucial role in the pathogenesis of acute lung injury (ALI) induced by highly pathogenic virus including influenza A viruses H5N1, H7N9, and severe acute respiratory syndrome (SARS) coronavirus. In highly pathogenic virus-induced acute lung diseases, high levels of chemotactic and anaphylatoxic C5a were produced as a result of excessive complement activaiton. Overproduced C5a displays powerful biological activities in activation of phagocytic cells, generation of oxidants, and inflammatory sequelae named ''cytokine storm'', and so on. Blockade of C5a signaling have been implicated in the treatment of ALI induced by highly pathogenic virus. Herein, we review the literature that links C5a and ALI, and review our understanding of the mechanisms by which C5a affects ALI during highly pathogenic viral infection. In particular, we discuss the potential of the blockade of C5a signaling to treat ALI induced by highly pathogenic viruses. Abstract Two of the earliest Middle East respiratory syndrome (MERS) cases were men who had visited the Doha central animal market and adjoining slaughterhouse in Qatar. We show that a high proportion of camels presenting for slaughter in Qatar show evidence for nasal MERS-CoV shedding (62/105). Sequence analysis showed the circulation of at least five different virus strains at these premises, suggesting that this location is a driver of MERS-CoV circulation and a high-risk area for human exposure. No correlation between RNA loads and levels of neutralizing antibodies was observed, suggesting limited immune protection and potential for reinfection despite previous exposure. Abstract The most common cause of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is respiratory infection. Most studies of bacterial or viral cause in AECOPD have been conducted in Western countries. We investigated bacterial and viral identification rates in AECOPD in Korea.We reviewed and analyzed medical records of 736 cases of AECOPD at the Korea University Guro Hospital. We analyzed bacterial and viral identification rates and classified infections according to epidemiological factors, such as Global Initiative for Chronic Obstructive Lung Disease stage, mortality, and seasonal variation. Results: The numbers of AECOPD events involving only bacterial identification, only viral identification, bacterial-viral co-identification, and no identification were 200 (27.2%), 159 (21.6%), 107 (14.5%), and 270 (36.7%), respectively. The most common infectious bacteria identified were Pseudomonas aeruginosa (13.0%), Streptococcus pneumoniae (11.4%), and Haemophilus influenzae (5.3%); the most common viruses identified were influenza virus (12.4%), rhinovirus (9.4%), parainfluenza virus (5.2%), and metapneumovirus (4.9%). The bacterial identification rate tended to be higher at more advanced stages of chronic obstructive pulmonary disease (p=0.020 overall, p=0.011 for P. aeruginosa, p=0.048 for S. pneumoniae). Staphylococcus aureus and Klebsiella pneumoniae were identified more in mortality group (p=0.003 for S. aureus, p=0.009 for K. pneumoniae). All viruses were seasonal (i.e., greater prevalence in a particular season; p<0.050). Influenza virus and rhinovirus were mainly identified in the winter, parainfluenza virus in the summer, and metapneumovirus in the spring. Conclusion: This information on the epidemiology of respiratory infections in AECOPD will improve the management of AECO-PD using antibiotics and other treatments in Korea. Abstract Houttuynia cordata Thunb. is a traditional herb used for clearing heat and eliminating toxins, and has also been used for the treatment of severe acute respiratory syndrome (SARS). In vitro, the crude H. cordata polysaccharides (CHCP) exhibited potent anti-complementary activity through both the classical and alternative pathways by acting on components C3 and C4 of the complement system without interfering with the coagulation system. This study was to investigate the preventive effects of CHCP on acute lung injury (ALI) induced by hemorrhagic shock plus lipopolysaccharide (LPS) instillation (two-hit) and LPS-induced fever in rats. CHCP significantly attenuated pulmonary injury in the "two-hit" ALI model by reducing pulmonary edema and protein exudation in bronchoalveolar lavage fluid (BALF). In addition, it reduced the deposit of complement activation products in the lung and improved oxidantantioxidant imbalance. Moreover, CHCP administration inhibited fever in rats, reduced the number of leukocytes and restored serum complement levels. The inhibition on the inappropriate activation of complement system by CHCP may play an important role in its beneficial effects on inflammatory diseases. The anti-complementary polysaccharides are likely to be among the key substances for the heatclearing function of H. cordata. Abstract We have recently reported the isolation of a novel virus, provisionally designated C/swine/Oklahoma/1334/2011 (C/ OK), with 50% overall homology to human influenza C viruses (ICV), from a pig in Oklahoma. Deep RNA sequencing of C/OK virus found a matrix 1 (M1) protein expression strategy that differed from that of ICV. The novelty of C/OK virus prompted us to investigate whether C/OK virus could exist in a nonswine species. Significantly, we found that C/OK virus was widespread in U.S. bovine herds, as demonstrated by reverse transcription (RT)-PCR and serological assays. Genome sequencing of three bovine viruses isolated from two herds in different states further confirmed these findings. To determine whether swine/bovine C/OK viruses can undergo reassortment with human ICV, and to clarify the taxonomic status of C/OK, in vitro reassortment and serological typing by agar gel immunodiffusion (AGID) were conducted. In vitro reassortment using two human ICV and two swine and bovine C/OK viruses demonstrated that human ICV and C/OK viruses were unable to reassort and produce viable progeny. Antigenically, no cross-recognition of detergent split virions was observed in AGID between human and nonhuman viruses by using polyclonal antibodies that were reactive to cognate antigens. Taken together, these results demonstrate thatC/OK virus is genetically and antigenically distinct from ICV. The classification of the new virus in a separate genus of the Orthomyxoviridae family is proposed. The finding of C/OK virus in swine and bovine indicates that this new virus may spread and establish infection in other mammals, including humans.Here, we show molecular and serological data demonstrating widespread circulation of similar viruses in bovines. Deep RNA sequencing, phylogenetic analysis, and in vitro reassortment experiments demonstrate that animal ICV-like viruses are genetically distinct from human ICV. Antigenically, we show that ICV-like viruses are not recognized by ICV antibodies. En masse, these results suggest that bovine influenza virus warrants classification as a new genus of influenza virus. The finding of this novel virus that can infect multiple mammalian species warrants further research into its role in human health. . 2014. Characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the Orthomyxoviridae family. mBio 5(2):e00031-14. Abstract Synthetic biology, application of synthetic chemistry to biology, is a broad term that covers the engineering of biological systems with structures and functions not found in nature to process information, manipulate chemicals, produce energy, maintain cell environment and enhance human health. Synthetic biology devices contribute not only to improve our understanding of disease mechanisms, but also provide novel diagnostic tools. Methods based on synthetic biology enable the design of novel strategies for the treatment of cancer, immune diseases metabolic disorders and infectious diseases as well as the production of cheap drugs. The potential of synthetic genome, using an expanded genetic code that is designed for specific drug synthesis as well as delivery and activation of the drug in vivo by a pathological signal, was already pointed out during a lecture delivered at Kuwait University in 2005. Of two approaches to synthetic biology, top-down and bottom-up, the latter is more relevant to the development of personalized medicines as it provides more flexibility in constructing a partially synthetic cell from basic building blocks for a desired task. Abstract An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-qTOF-MS/MS) method was developed and validated for the simultaneous determination of glycyrrhizin and glycyrrhetic acid. These analytes were separated on a reverse phase C18 column using a mobile phase of acetonitrile:2% acetic acid in water (75:25, v/v) with a flow rate of 200 mL/min. The qTOF-MS was operated under multiple reaction monitoring (MRM) mode using the electrospray ionization (ESI) technique with positive ion polarity. A comparison of three different extraction techniques i.e. accelerated solvent extraction (ASE), extraction under ultrasonic waves (USW) and the classical extraction by percolation (CE) method was done and quantification of these extracts was also carried out by the proposed method. Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) has caused human respiratory infections with a high case fatality rate since 2012. However, the mode of virus transmission is not well understood. The findings of epidemiological and virological studies prompted us to hypothesize that the human gastrointestinal tract could serve as an alternative route to acquire MERS-CoV infection. We demonstrated that human primary intestinal epithelial cells, small intestine explants, and intestinal organoids were highly susceptible to MERS-CoV and can sustain robust viral replication. We also identified the evidence of enteric MERS-CoV infection in the stool specimen of a clinical patient. MERS-CoV was considerably resistant to fed-state gastrointestinal fluids but less tolerant to highly acidic fasted-state gastric fluid.In polarized Caco-2 cells cultured in Transwell inserts, apical MERS-CoV inoculation was more effective in establishing infection than basolateral inoculation. Notably, direct intragastric inoculation of MERS-CoV caused a lethal infection in human DPP4 transgenic mice. Histological examination revealed MERS-CoV enteric infection in all inoculated mice, as shown by the presence of virus-positive cells, progressive inflammation, and epithelial degeneration in small intestines, which were exaggerated in the mice pretreated with the proton pump inhibitor pantoprazole. With the progression of the enteric infection, inflammation, virus-positive cells, and live viruses emerged in the lung tissues, indicating the development of sequential respiratory infection. Taken together, these data suggest that the human intestinal tract may serve as an alternative infection route for MERS-CoV. Abstract Recurring epidemics of kiwifruit (Actinidia spp.) bleeding canker disease are caused by Pseudomonas syringae pv. actinidiae (Psa). In order to strengthen understanding of population structure, phylogeography, and evolutionary dynamics, we isolated Pseudomonas from cultivated and wild kiwifruit across six provinces in China. Based on the analysis of 80 sequenced Psa genomes, we show that China is the origin of the pandemic lineage but that strain diversity in China is confined to just a single clade. In contrast, Korea and Japan harbor strains from multiple clades. Distinct independent transmission events marked introduction of the pandemic lineage into New Zealand, Chile, Europe, Korea, and Japan. Despite high similarity within the core genome and minimal impact of within-clade recombination, we observed extensive variation even within the single clade from which the global pandemic arose. Abstract DNA microarrays have emerged as a viable platform for detection of pathogenic organisms in clinical and environmental samples. These microbial detection arrays occupy a middle ground between low cost, narrowly focused assays such as multiplex PCR and more expensive, broad-spectrum technologies like high-throughput sequencing. While pathogen detection arrays have been used primarily in a research context, several groups are aggressively working to develop arrays for clinical diagnostics, food safety testing, environmental monitoring and biodefense. Statistical algorithms that can analyze data from microbial detection arrays and provide easily interpretable results are absolutely required in order for these efforts to succeed. In this article, we will review the most promising array designs and analysis algorithms that have been developed to date, comparing their strengths and weaknesses for pathogen detection and discovery. Abstract Objectives: The outbreak of Middle Eastern respiratory syndrome coronavirus (MERS-CoV) was one of the major events in South Korea in 2015. In particular, this study pays attention to formulating a mathematical model for MERS transmission dynamics and estimating transmission rates. Methods: Incidence data of MERS-CoV from the government authority was analyzed for the first aim and a mathematical model was built and analyzed for the second aim of the study. A mathematical model for MERS-CoV transmission dynamics is used to estimate the transmission rates in two periods due to the implementation of intensive interventions. Results: Using the estimates of the transmission rates, the basic reproduction number was estimated in two periods. Due to the superspreader, the basic reproduction number was very large in the first period; however, the basic reproduction number of the second period has reduced significantly after intensive interventions. Conclusion: It turned out to be the intensive isolation and quarantine interventions that were the most critical factors that prevented the spread of the MERS outbreak. The results are expected to be useful to devise more efficient intervention strategies in the future. Abstract Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110-124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression. Abstract A 62-year-old man presented to our hospital for the further evaluation and treatment of his back pain, general fatigue, and dyspnea, which had developed 4 days after the 29th administration of nivolumab to treat his lung cancer. Based on his clinical history, elevated serum cardiac enzyme values, and cardiac magnetic resonance (CMR) imaging and myocardial biopsy findings, he was diagnosed with myocarditis induced by nivolumab. Corticosteroid therapy improved his condition, and CMR performed on hospital day 11 also showed remarkable improvement. Although nivolumab-induced myocarditis is rare, cardiologists should consider it when encountering patients treated with such a drug for malignant disease. Abstract The 2015 Middle East Respiratory Syndrome Coronavirus (MERS-CoV) outbreak in Korea caused major economic and social problems. The control intervention was conducted during the MERS-CoV outbreak in Korea immediately after the confirmation of the index case. This study investigates whether the early risk communication with the general public and mass media is an effective preventive strategy. Methods: The SEIR (Susceptible, Exposed, Infectious, Recovered) model with estimated parameters for the time series data of the daily MERS-CoV incidence in Korea was considered from May to December 2015. For 10,000 stochastic simulations, the SEIR model was computed using the Gillespie algorithm. Depending on the time of control intervention on the 20th, 40th, and 60th days after the identification of the index case, the box plots of MERS-CoV incidences in Korea were computed, and the results were analyzed via ANOVA.The box plots showed that there was a significant difference between the non-intervention and intervention groups (the 20th day, 40th day, and 60th day groups) and seemed to show no significant difference based on the time of intervention. However, the ANOVA revealed that early intervention was a good strategy to control the disease. Conclusion: Appropriate risk communication can secure the confidence of the general public in the public health authorities. Abstract Background: Early recognition of the signs and symptoms of clinical deterioration could diminish the incidence of cardiopulmonary arrest. The present study investigates outcomes with respect to cardiopulmonary arrest rates in institutions with and without rapid response systems (RRSs) and the current level of cardiopulmonary arrest rate in tertiary hospitals. Methods: This was a retrospective study based on data from 14 tertiary hospitals. Cardiopulmonary resuscitation (CPR) rate reports were obtained from each hospital to include the number of cardiopulmonary arrest events in adult patients in the general ward, the annual adult admission statistics, and the structure of the RRS if present. Results: Hospitals with RRSs showed a statistically significant reduction of the CPR rate between 2013 and 2015 (odds ratio [OR], 0.731; 95% confidence interval [CI], 0.577 to 0.927; P = 0.009). Nevertheless, CPR rates of 2013 and 2015 did not change in hospitals without RRS (OR, 0.988; 95% CI, 0.868 to 1.124; P = 0.854). National university-affiliated hospitals showed less cardiopulmonary arrest rate than private university-affiliated in 2015 (1.92 vs. 2.40; OR, 0.800; 95% CI, 0.702 to 0.912; P = 0.001). High-volume hospitals showed lower cardiopulmonary arrest rates compared with medium-volume hospitals in 2013 (1.76 vs. 2.63; OR, 0.667; 95% CI, 0.577 to 0.772; P < 0.001) and in 2015 (1.55 vs. 3.20; OR, 0.485; 95% CI, 0.428 to 0.550; P < 0.001). Conclusions: RRSs may be a feasible option to reduce the CPR rate. The discrepancy in cardiopulmonary arrest rates suggests further research should include a nationwide survey to tease out factors involved in in-hospital cardiopulmonary arrest and differences in outcomes based on hospital characteristics. Abstract Dengue virus (DENV) is a single-stranded, positive-sense RNA virus that belongs to the Flaviviridae family. There are four different serotypes of DENV [1] [2] [3] [4] . DENV is transmitted by Aedes aegypti and Aedes albopictus mosquitoes, and DENV infection causes dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DSS is fatal in 1%-2.5% of cases with intensive treatment, and the mortality rate increases to >20% in the absence of proper treatment [1]. Thus far, DENV infection has not been reported in South Korea, except in patients who have traveled to endemic areas, such as Asia-Pacific, Central and South America, and Africa, which are the major sited of dengue fever outbreaks. According to the World Health Organization, 40% of the world population lives in high-DENV areas. An estimated 390 million people are infected with DENV, and about 20,000 of them die each year [2]. In 2014, 113 cases of dengue fever were reported in Yoyogi park in central Tokyo [3] . Similarly, in Europe, from 2012 to 2013, more than 3,000 people were infected with DENV in Portuguese Madeira on the Atlantic Ocean. In addition, 2 and 17 cases of dengue fever in persons who had no previous travel abroad were reported in France and Croatia, respectively, which were the first known cases of self-occurrence in Europe since 1928 and suggest the possibility of a future DENV epidemic [4] . A. aegypti and A. albopictus, which spread DENV, mostly inhabit the tropics and subtropics. These mosquitoes also carry several other infectious viruses, such as Chikungunya, West Nile fever, and yellow fever viruses, which have already been imported into Europe and the Americas by cargo ships and airlines. In 2013, subtropical Dengue fever is a tropical endemic disease; however, because of climate change, it may become a problem in South Korea in the near future. Research on vaccines for dengue fever and outbreak preparedness are currently insufficient. In addition, because there are no appropriate animal models, controversial results from vaccine efficacy assessments and clinical trials have been reported. Therefore, to study the mechanism of dengue fever and test the immunogenicity of vaccines, an appropriate animal model is urgently needed. In addition to mouse models, more suitable models using animals that can be humanized will need to be constructed. In this report, we look at the current status of model animal construction and discuss which models require further development. Abstract The location of autocrine interactions between the v-sis protein and PDGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constructed fusion proteins that anchor v-sis to specific intracellular membranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 glycoprotein to v-sis protein completely abolished its transforming ability when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimerize properly. Derivatives of some of these constructs were also constructed bearing the cytoplasmic tail from the glycoprotein of vesicular stomatitis virus (VSV-G). These constructs allowed examination of subcellular localization by double-label immunofluorescence, using antibodies that distinguish between the extraceUular PDGF-related domain and the VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi markers confirmed its targeting to the early Golgi complex. The sis-E1 constructs, targeted to the early Golgi complex, exhibited no proteo-lyric processing whereas the mutant forms of sis-E1 exhibited normal proteolytic processing. Treatment with suramin, a polyanionic compound that disrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 constructs described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Golgi complex do not result in functional signal transduction.Another v-sis fusion protein was constructed by attaching the transmembrane domain and COOHterminus of TGN38, a protein that localizes to the trans-Golgi network (TGN). This construct was primarily retained intracellularly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated the TGN-localization, as evidenced by very prominent cell surface localization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the properties of TGN38 itself, which is known to recycle from the cell surface to the TGN. Abstract A 68-year old man diagnosed with Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) presented with multiple pneumonic infiltrations on his chest X-ray, and the patient was placed on a mechanical ventilator because of progressive respiratory failure. Urinary protein excretion steadily increased for a microalbumin to creatinine ratio of 538.4 mg/g Cr and a protein to creatinine ratio of 3,025.8 mg/g Cr. The isotope dilution mass spectrometry traceable serum creatinine level increased to 3.0 mg/dL. We performed a kidney biopsy 8 weeks after the onset of symptoms. Acute tubular necrosis was the main finding, and proteinaceous cast formation and acute tubulointerstitial nephritis were found. There were no electron dense deposits observed with electron microscopy. We could not verify the virus itself by in situ hybridization and confocal microscopy (MERS-CoV costained with dipeptidyl peptidase 4). The viremic status, urinary virus excretion, and timely kidney biopsy results should be investigated with thorough precautions to reveal the direct effects of MERS-CoV with respect to renal complications. Abstract Cassiae semen (Leguminosae), a well-known traditional Chinese medicine, has been used for a number of centuries in areas of Southeast Asia, including Korea, Japan and China. The present review aims to provide updated and comprehensive information, on the botany, phytochemistry and pharmacology of Cassiae semen. The available information on Cassiae semen was collected using several different resources, including classic books on Chinese herbal medicine and a number of scientific databases, including the China Academic Journals full-text database, PubMed, SciFinder, the Web of Science and Science Direct. To date >70 chemical compounds have been isolated from Cassiae semen, and the major components have been determined to be anthraquinones, naphthopyrones and volatile oil. The crude extracts and pure compounds of Cassiae semen have been used as effective agents in preclinical and clinical practice due to their beneficial activities, including antihyperlipidemic, antidiabetic, neuroprotective, hepatoprotective, antibacterial, antioxidant and hypotensive activities. With the body of reported data, it has been suggested that Cassiae semen has convincing medicinal potential. However, the pharmacological mechanisms of the main bioactive compounds and the association between structure and activity require further investigation.C. obtusifolia is similar to C. tora in terms of botanical morphology. The two are an annual, erect, stout herb, ~1-2 m in length, and their leaves are paripinnate, typically pubescent and are 4-8 cm in length with a conical gland between each of the two lowest pairs of leaflets. Leaflets are formed of 3 leaf Abstract The world is becoming more urban every day, and the process has been ongoing since the industrial revolution in the 18th century. The United Nations now estimates that 3.9 billion people live in urban centres. The rapid influx of residents is however not universal and the developed countries are already urban, but the big rise in urban population in the next 30 years is expected to be in Asia and Africa. Urbanization leads to many challenges for global health and the epidemiology of infectious diseases. New megacities can be incubators for new epidemics, and zoonotic diseases can spread in a more rapid manner and become worldwide threats. Adequate city planning and surveillance can be powerful tools to improve the global health and decrease the burden of communicable diseases. Abstract Astroviruses are the principal causative agents of gastroenteritis in humans and have been associated with diarrhea in other mammals as well as birds. However, astroviral infection of animals had been poorly studied. In the present study, 211 rectal swabs collected from cattle and water buffalo calves with mild to severe diarrhea were tested for bovine astrovirus (BAstV) by RT-PCR. Results: 92/211 (43.6%) samples were positive for BAstV, at a rate of 46.10% (71/154) in cattle and 36.84% (21/57) in water buffalo. Phylogenetic analysis based on the partial and full-length of 25 ORF2 amino acid sequences obtained in this study classified the Guangxi BAstVs isolates into five subgroups under the genus of Mamastrovirus, genotype MAstV33, which suggested that the water buffalo was a new host of this genogroup that previously included only cattle and roe deer. Despite the origin of the host, the Guangxi BAstV isolates were closely related to the BAstV Hong Kong isolates (B18/HK and B76-2/HK), but highly divergent from the BAstV NeuroS1 isolate previously associated with neurologic disease in cattle in the U.S.A. Nucleotide sequence-based characterization of the ORF1b/ORF2 junction and corresponding overlapping regions showed distinctive properties, which may be common to BAstVs. Our results suggested that cattle and water buffalo are prone to infection of closely related astroviruses, which probably evolved from the same ancestor. The current study described astroviruses in water buffalo for the first time and is thus far among the largest epidemiological investigations of BAstV infection in cattle conducted in China. Abstract It has been proposed that cholesterol in host cell membranes plays a pivotal role for cell entry of HIV. However, it remains largely unknown why virions prefer cholesterol-rich heterogeneous membranes to uniformly fluid membranes for membrane fusion. Using giant plasma membrane vesicles containing cholesterol-rich ordered and cholesterol-poor fluid lipid domains, we demonstrate that the HIV receptor CD4 is substantially sequestered into ordered domains, whereas the co-receptor CCR5 localizes preferentially at ordered/disordered domain boundaries. We also show that HIV does not fuse from within ordered regions of the plasma membrane but rather at their boundaries. Ordered/disordered lipid domain coexistence is not required for HIV attachment but is a prerequisite for successful fusion. We propose that HIV virions sense and exploit membrane discontinuities to gain entry into cells. This study provides surprising answers to the long-standing question about the roles of cholesterol and ordered lipid domains in cell entry of HIV and perhaps other enveloped viruses. Abstract A nine-month-old girl from northern Manitoba presented in September 2014, with a three-day history of vesicular rash on the soles of her feet. She was diagnosed with hand, foot and mouth disease, and discharged from the local nursing station. Over the next 24 h, she developed cough and fever that led to progressive vomiting, diarrhea and increased work of breathing, causing significant oxygen requirements, which led her to be transferred to a tertiary care centre. On admission, she required intubation and fluid resuscitation for respiratory failure and hypotension. On physical examination, she was afebrile. On respiratory examination, there were diffuse crackles and coarse breath sounds bilaterally. Cardiac and abdominal examinations were normal. The only remaining rash consisted of vesicular lesions on an erythematous base on the sole of the right foot.Laboratory tests revealed a white blood cell count of 4.3×10 9 /L, including an absolute neutrophil count of 1.7×10 9 /L. The rest of the complete blood cell count, liver enzyme levels, renal function and electrolytes were normal. Blood and endotracheal tube aspirate cultures, obtained before vancomycin and ceftriaxone were administered, were negative. Urine analysis revealed no leukocytes or nitrites; however, urine culture was positive for pansusceptible Escherichia coli (2×10 6 CFU/L). Cerebrospinal fluid obtained one day after admission, when her hemodynamic status improved, revealed a total white blood cell count of 28×10 6 cells/L, including 66% lymphocytes, 8% neutrophils, 26% monocytes, glucose 5 mmol/L, protein 0.21 g/L and a few red blood cells observed on microscopy. Bacterial cultures and polymerase chain reaction (PCR) for enterovirus and herpes simplex virus were negative. Stool sent for electron microscopy was negative, but stool for viral culture was pending. Chest x-ray revealed a right middlelobe infiltration. A nasopharyngeal aspirate and bronchoalveolar lavage sent for respiratory virus panel PCR, which included adenovirus, human coronavirus OC43, 229E and NL63, repiratory syncytial virus A and B, influenza A and B, parainfluenza 1, 2, 3 and 4, enterovirus, rhinovirus A, B and C, bocavirus 1 and 2, and human metapneumovirus, were both negative. Acid-fast bacilli smear and culture, bacterial culture and fungal culture from the bronchoalveolar lavage were negative. The patient became unable to move her upper extremities on day 2 of admission. Magnetic resonance imaging of the head and spine was performed on the same day, and revealed restricted diffusion at the dorsal brain stem and patchy areas of prolonged signal intensity involving the cranial-cervical junction, cervical and, to a lesser extent, the thoracic and lumbar spinal cord, suggestive of transverse myelitis with rhomboencephalitis.An autoimmune-mediated process was suspected due to the absence of detection of infectious agents. A three-day course of pulse methylprednisolone led to no improvement. The patient developed increased ventilator requirements, including high frequency jet ventilation on days 6 to 11 of admission. The patient slowly recovered with supportive care over the next five days. She was extubated on day 14 of admission and discharged on day 37, with residual impaired mobility of her right arm. She was seen two months after discharge with improved, but ongoing, difficulty lifting her right arm to her face, and was unable to lift her right arm above her shoulder. She is followed by physiotherapy, occupational therapy and pediatric neurology. Abstract Background: Streptococcus equi subspecies equi infection elicits M protein antibody titers in equids. Interpretation of titers is not generally accepted. Hypothesis: The magnitude of S. equi M protein (SeM) antibody titer after infection (titer ≥1:12 800) will be useful to monitor for the presence of complications or the risk of development of complications. Animals: Forty-eight horses on 1 farm involved in strangles outbreak. Methods: Clinical and observational study. S. equi M protein antibody titers were measured on all horses 8 weeks after infection and select horses 12 and 28 weeks after infection. Horses were categorized: no disease, uncomplicated case, persistent guttural pouch (GP) infection, or complicated cases (metastatic abscesses, purpura hemorrhagica, secondary infections, and dysphagia). Category was compared to titer. Results: Twenty-eight of 48 (58%) developed clinical signs of S. equi infection. Of those, 11 (39%) had uncomplicated strangles, 9 (21%) had persistent GP infection, 5 (18%) were complicated cases, and 3 (11%) had both persistent GP infection and complications. Thirty-three percent of horses (16 of 48) had SeM antibody titers ≥1:12 800 eight weeks after infection. Of horses with titers ≥1:12 800, 6 of 16 had evidence of complications. Of complicated cases, 6 of 8 had titers ≥1:12 800. In this outbreak, the sensitivity (75%; 95% CI [confidence interval] 45-105) for a SeM antibody titer ≥1:12 800 detecting complications was higher than the specificity (43%; 95% CI 23-64).Conclusions and Clinical Importance: This outbreak demonstrates that SeM antibody titers can be increased after infection (≥1:12 800) in the absence of complications of strangles. Abstract Chlamydia pecorum (designated 22-58) was isolated in 2010 in HmLu-1 cells from the jejunum of a calf which died of necrotizing enterocolitis in Yamaguchi Prefecture, Japan. Immunohistochemical staining identified C. pecorum positive reactions in the jejunal villi. C. pecorum, designated 24-100, was isolated from the feces of a calf with diarrhea in another farm in Yamaguchi Prefecture in 2012. A significant increase in neutralizing antibody titers against C. pecorum was confirmed in paired sera. Nucleotide sequence identities of omp1 genes of the 2 isolates were 100%. The isolates were genetically and antigenically more closely related to C. pecorum Bo/Yokohama strain isolated from cattle with enteritis in Japan than to the other prototype strains, Bo/Maeda isolated from cattle with pneumonia and Ov/ IPA isolated from sheep with polyarthritis. These results indicate that C. pecorum strains similar to 22-58 and 24-100 might be endemic in Yamaguchi Prefecture and cause enteric disease in cattle. Abstract # Contributed equally J o u r n a l P r e -p r o o f Highlights We reviewed discovery and development process of broad-spectrum antiviral agents. We summarized the information on 119 safe-in-man agents in freely accessible database. Further studies will increase the number of broad-spectrum antivirals, expand spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections.: Viral diseases are one of the leading causes of morbidity and mortality in the world. Virus-specific vaccines and antiviral drugs are the most powerful tools to combat viral diseases. However, broad-spectrum antiviral agents (BSAAs, i.e. compounds targeting viruses belonging to two or more viral families) could provide additional protection of general population from emerging and reemerging viral diseases reinforcing the arsenal of available antiviral options. Here, we reviewed discovery and development of BSAAs and summarized the information on 119 safe-in-man agents in freely accessible database (https://drugvirus.info/). Future and ongoing pre-clinical and clinical studies will increase the number of BSAAs, expand spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections as well as co-infections. Abstract Graphical Abstract Highlights d We describe cooperative neutralization and in vivo protection d Cooperativity turns non-neutralizing ebolavirus antibodies into potent neutralizers d A hotspot for antibody cooperativity identified on Ebola virus glycoprotein Abstract Ramsay Hunt syndrome (RHS) is characterized by varicella zoster virus (VZV) infection in the geniculate ganglion of the facial nerve. It typically presents with ipsilateral facial palsy and vesicles in the external auditory canal [1] . Recognizing atypical clinical manifestations of RHS, though not frequently reported, is of clinical importance for clinicians. For instance, RHS without vesicles, condition known as zoster sine herpete [2] , is difficult to distinguish itself from Bell's palsy. Although the vestibulocochlear nerve is frequently co-involved during the course of RHS, multiple lower cranial nerve involvement has rarely been described in the literature [5] [6] [7] [8] [9] [10] [11] [12] . Additionally, laryngitis due to VZV associated with RHS is not a well-recognized clinical entity. We herein report a case of Ramsay Hunt syndrome with zoster pharyngitis involving multiple lower cranial nerves.A 66-year-old woman with a history of hypertension was hospitalized because of progressive dizziness, right-side facial weakness and dysphagia. The patient was in her usual state of health until 6 days before admission, when sore throat and odynophagia developed, associated with subjective fevers and anorexia. Two days before admission, the patient reported dizziness, right-side facial weakness associated with pain radiating to the right ear, and dysphagia. The patient denied headache, photophobia, neck stiffness, nausea or vomiting. On the morning of admission, the patient developed right-side tinnitus, unsteady gait and inability to close the right eye.On physical examination, the patient appeared to be in mild distress. The temperature was 101.8 8F, blood pressure 176/93 mm Hg, pulse 102 beats per minute, respirations 20 breaths per minute and oxygen saturation 97% on room air. Her heart sounds were regular without murmurs and her lungs were clear to auscultation. On otologic examination, a few vesicles were observed in the external auditory canal of the right ear with a normal tympanic membrane. Neurological examination revealed peripheral facial nerve palsy (VII), with involvement of the left vestibulocochlear nerve (VIII), glossopharyngeal nerve (IX) and vagus nerve (X) ( Table 1) . Spinal accessory nerve (XI) and hypoglossal nerve (XII) were intact. Flexible laryngoscopic examination revealed erythema and erosive lesions on the right side of the epiglottis and arytenoid. There was an asymmetry of the soft palate. A biopsy of the erosive lesions was not performed. A non-contrast computed tomography of the head and temporal bone revealed no abnormalities.Ramsay Hunt syndrome with laryngitis due to varicella zoster virus was suspected and the patient was started on acyclovir and steroid therapy. After the completion of a 7-day course of the combination therapy, the patient's neurological symptoms improved except for the facial nerve palsy. Healed erosions on the IDCases 2 (2015) 47-48 A B S T R A C T Ramsay Hunt syndrome is characterized by varicella zoster virus infection affecting the geniculate ganglion of the facial nerve. It typically presents with vesicles in the external auditory canal associated with auricular pain and peripheral facial nerve paralysis. Although vestibulocochlear nerve is frequently co-involved during the course of Ramsay Hunt syndrome, multiple lower cranial nerve involvement has rarely been described in the literature. In addition, laryngitis due to varicella zoster virus is a diagnostic challenge due to its unfamiliarity among clinicians. We report a case of Ramsay Hunt syndrome with laryngitis involving multiple lower cranial nerves. Abstract Viruses from the Filoviridae family, as many other virus families, require an acidic pH for successful infection and are therefore susceptible to the actions of 4-aminoquinolines, such as chloroquine.Although the mechanisms of action of chloroquine clearly indicate that it might inhibit filoviral infections, several clinical trials that attempted to use chloroquine in the treatment of other acute viral infectionsincluding dengue and influenza A and Bcaused by low pH-dependent viruses, have reported that chloroquine had no clinical efficacy, and these results demoted chloroquine from the potential treatments for other virus families requiring low pH for infectivity.The present review is aimed at investigating whether chloroquine could combat the present Ebola virus epidemic, and also at exploring the main reasons for the reported lack of efficacy. Literature was sourced from PubMed, Scopus, Google Scholar, reference list of articles and textbooks -Fields Virology (Volumes 1and 2), the cytokine handbook, Pharmacology in Medicine: Principles and Practice, and hydroxychloroquine and chloroquine retinopathy.The present analysis concludes that (1) chloroquine might find a place in the treatment of Ebola, either as a monotherapy or in combination therapies; (2) the ineffectiveness of chloroquine, or its analogue, hydroxychloroquine, at treating infections from low pH-dependent viruses is a result of the failure to attain and sustain a steady state concentration sufficient to increase and keep the pH of the acidic organelles to approximately neutral levels; (3) to successfully treat filoviral infectionsor other viral infections that emerge or emerged from low pH-dependent virusesa steady state chloroquine plasma concentration of at least 1 μg/mL(~3.125 μM/L) or a whole blood concentration of 16 μM/L must be achieved and be sustained until the patients' viraemia becomes undetectable. These concentrations, however, do not rule out the efficacy of other, higher, steady state concentrationsalthough such concentrations might be accompanied by severe adverse effects or toxicities. The feasibility of the conclusion in the preceding texts has recently been supported by a subsequent study that shows that amodiaquine, a derivative of CQ, is able to protect humans infected with Ebola from death. Abstract Abbreviations used in this paper: CSF, cytostatic factor; esiRNA, endoribonucleaseprepared siRNA; rRNA, ribosomal RNA; snoRNA, small nucleolar RNA; wlm, washed light membrane. Abstract Retrovirus Moloney murine leukemia virus (M-MuLV) matures by budding at the cell surface.Central to the budding process is the myristoylated viral core protein precursor Gag which, even in the absence of all other viral components, is capable of associating with the cytoplasmic leaflet of the plasma membrane and assembling into extracellular virus-like particles. In this paper we have used heterologous, Semliki Forest virus-driven, expression of M-MuLV Gag to study the mechanism by which this protein is targeted to the cell surface. In pulse-chase experiments, BFA, monensin, and 20°C block did not affect incorporation of Gag into extracellular particles thereby indicating that the secretory pathway is not involved in targeting of Gag to the cell surface. Subcellular fractionation studies demonstrated that newly synthe- Abstract Web-based social media is increasingly being used across different settings in the health care industry. The increased frequency in the use of the Internet via computer or mobile devices provides an opportunity for social media to be the medium through which people can be provided with valuable health information quickly and directly. While traditional methods of detection relied predominately on hierarchical or bureaucratic lines of communication, these often failed to yield timely and accurate epidemiological intelligence. New web-based platforms promise increased opportunities for a more timely and accurate spreading of information and analysis. This article aims to provide an overview and discussion of the availability of timely and accurate information. It is especially useful for the rapid identification of an outbreak of an infectious disease that is necessary to promptly and effectively develop public health responses. These web-based platforms include search queries, data mining of web and social media, process and analysis of blogs containing epidemic key words, text mining, and geographical information system data analyses. These new sources of analysis and information are intended to complement traditional sources of epidemic intelligence. Despite the attractiveness of these new approaches, further study is needed to determine the accuracy of blogger statements, as increases in public participation may not necessarily mean the information provided is more accurate.The shift in human interaction and media consumption to online communication has changed the way people seek information. The increased frequency in the use of the Internet via computer or mobile devices provides an opportunity for social media to be the means of providing people with valuable health information directly and quickly. The development of web-based social media analytics has given public health officers and physicians an earlier insight into the emergence and spread of infectious diseases. Availability of timely and accurate information is especially useful for the rapid identification of an outbreak of an infectious disease necessary to promptly and effectively develop public health responses. Abstract All positive-strand RNA viruses induce membrane structures in their host cells which are thought to serve as suitable microenvironments for viral RNA synthesis. The structures induced by enteroviruses, which are members of the family Picornaviridae, have so far been described as either single-or double-membrane vesicles (DMVs). Aside from the number of delimiting membranes, their exact architecture has also remained elusive due to the limitations of conventional electron microscopy. In this study, we used electron tomography (ET) to solve the three-dimensional (3-D) ultrastructure of these compartments. At different time points postinfection, coxsackievirus B3-infected cells were high-pressure frozen and freeze-substituted for ET analysis. The tomograms showed that during the exponential phase of viral RNA synthesis, closed smooth single-membrane tubules constituted the predominant virus-induced membrane structure, with a minor proportion of DMVs that were either closed or connected to the cytosol in a vase-like configuration. As infection progressed, the DMV number steadily increased, while the tubular single-membrane structures gradually disappeared. Late in infection, complex multilamellar structures, previously unreported, became apparent in the cytoplasm. Serial tomography disclosed that their basic unit is a DMV, which is enwrapped by one or multiple cisternae.IMPORTANCE Positive-strand RNA viruses hijack specific intracellular membranes and remodel them into special structures that support viral RNA synthesis. The ultrastructural characterization of these "replication structures" is key to understanding their precise role. Here, we resolved the three-dimensional architecture of enterovirus-induced membranous compartments and their transformation in time by applying electron tomography to cells infected with coxsackievirus B3 (CVB3). Our results show that closed single-membrane tubules are the predominant initial virus-induced structure, whereas double-membrane vesicles (DMVs) become increasingly abundant at the expense of these tubules as infection progresses. Additionally, more complex multilamellar structures appear late in infection. Based on compelling intermediate structures in our tomograms, we propose a model for transformation from the tubules to DMVs and multilamellar structures via enwrapping events. Our work provides an in-depth analysis of the development of an unsuspected variety of distinct replication structures during the course of CVB3 infection.Citation Limpens RWAL, et al. 2011. The transformation of enterovirus replication structures: a three-dimensional study of single-and double-membrane compartments. mBio 2(5):e00166-11. Abstract J Jo ou ur rn na al l o of f C Ca an nc ce er r 2015; 6(1): 54-65.The advancement of high throughput omic technologies during the past few years has made it possible to perform many complex assays in a much shorter time than the traditional approaches. The rapid accumulation and wide availability of omic data generated by these technologies offer great opportunities to unravel disease mechanisms, but also presents significant challenges to extract knowledge from such massive data and to evaluate the findings. To address these challenges, a number of pathway and network based approaches have been introduced. This review article evaluates these methods and discusses their application in cancer biomarker discovery using hepatocellular carcinoma (HCC) as an example. Abstract The One Health initiative is increasingly becoming a prominent discussion topic in animal and human health, with its focus on prevention of spread of zoonotic diseases, both in animals, and from animals to humans. An important part of One Health is that diagnostics and vaccines for diseases may be the same thingand be used for both humans and animals. One potential problem standing in the way of wider adoption of One Health principles, though, is that use of conventional cell fermentation systems for production of the recombinant proteins that could be used as diagnostics or vaccines is often expensive and is not easily scalable. A solution to this may be the use of plants or plant cells as bioreactors: molecular farming, or the production of biologics in plants, is now a well-established science with many proofs of principle and important proofs of efficacy for especially animal vaccines. This review discusses how molecular farming could enable important advances in One Health, using as examples plant-made vacccines, reagents and therapeutics for influenza viruses, ebolaviruses, rabies virus, bunyaviruses and flaviviruses. Abstract The Feline coronavirus (FCoV) can lead to Feline infectious peritonitis (FIP), which the precise cause is still unknown. The theory of internal mutation suggests that a less virulent biotype of FCoV (FECV) would lead to another more pathogenic biotype (FIPV) capable of causing FIP. In this work, the 7b gene was amplified from 51 domestic cat plasma samples by semi-nested PCR and tested through phylogenetic and phylogeographical approaches. The 7b gene of Brazilian isolates displayed high conservation, a strong correlation between the geographic origin of the viral isolates and their genealogy, and its evolution was possibly shaped by a combination of high rates of nucleotide substitution and purifying selection.The Feline coronavirus (FCoV) is an important pathogen of domestic and wild felids, which can cause subclinical infection, mild enteritis or lead to feline infectious peritonitis (FIP), a fatal disease characterized by inflammatory lesions of serous membranes and systemic granulomatous lesions of parenchymatous organs [23] .Although the precise cause of FIP pathogenesis is still unknown, several hypotheses have been suggested [20] . The most accepted hypothesis, called internal mutation theory, suggests that during the replication of FCoV in the intestinal epithelium, a mutation occurs that makes the virus more pathogenic and able to infect monocytes and macrophages and cause FIP [23, 27] . This virulent mutant variant was designated Feline infectious peritonitis virus (FIPV), while a variant that leads to enteric infection has been termed Feline enteric coronavirus (FECV) [25] . The precise nature of the mutation responsible for the pathogenesis has not been identified in the FCoV genome [10] . Nevertheless, it has been deduced that the non-structural glycoprotein 7b, codified by ORF7b, plays a determinative role in FCoV virulence [31], besides having a strong phylogenetic sign for the differentiation between FECV and FIPV [3] .To better understand the molecular epidemiology of FCoV in Brazilian domestic cats, phylogenetic hypothesis and viral population dynamics were inferred from the 7b gene. A phylogenetic hypothesis and the reconstructed population history of FCoV isolates are presented in this work, providing insights into the origins of FCoV in Brazil. Furthermore, the molecular analysis of 7b gene dispenses considerations about the internal mutation theory, regarding to the virulence of the serotypes of FCoV.This study included samples from 210 domestic cats (Felis catus) of various breeds, random selected from different local animal hospitals (Minas Gerais, Brazil) during 2003-2010. One hundred twenty-nine animals were healthy and taken to veterinary clinics for vaccinations and/or elective surgery. Eighty-one of them showed clinical symptoms of FIP such as anorexia, weight loss, Abstract With over 4,500 deaths and counting, and new cases identified in two developed countries that are struggling and faltering in their handling of the epidemic, the 2014 Ebola Virus Disease (EVD) epidemic is unlike any of its kind ever encountered. The ability of some poor, resource-limited, developing countries in sub-Saharan Africa to efficiently handle the epidemic within their shores provides some lessons learned for the global health community. Among others, the 2014 EVD epidemic teaches us that it is time to put the "P" back in public and population health around the world. The global health community must support a sustainable strategy to mitigate Ebola virus and other epidemics both within and outside their shores, even after the cameras are gone. Ebola virus must not be called the disease of the poor and developing world.