niryuu/sni-each-converted huggingface dataset:
The importance of monospecific antisera for the experimental analysis of viral proteins is undisputed. They make it possible to identify and analyze the target protein against a background of a large number of other proteins, either in whole fixed cells or in cell lysates. This chapter describes our experience with the production of such rabbit antisera directed against proteins of coronaviruses and other nidoviruses. The use as antigens of either synthetic peptides (coupled to a carrier protein) or proteins expressed in Escherichia coli is described, and detailed protocols for immunization and preparation of test bleeds are provided. For screening of the immune response following immunization, detailed protocols for three commonly used techniques are described, all of which are based on the use of infected cells or cells expressing the protein of interest, side by side with appropriate controls. The in situ immunodetection of the target in fixed cells by immunofluorescence microscopy is described, as are protocols for techniques that can be applied to cell lysates containing the target protein (Western blotting and immunoprecipitation). The latter techniques are performed in combination with polyacrylamide gel electrophoresis, thus allowing confirmation of the molecular weight of the target that is recognized by the antiserum.
Output: Production of Monospecific Rabbit Antisera Recognizing Nidovirus Proteins
One of the most perplexing questions regarding the current COVID-19 coronavirus epidemic is the discrepancy between the severity of cases observed in the Hubei province of China and those occurring elsewhere in the world. One possible answer is antibody dependent enhancement (ADE) of SARS-CoV-2 due to prior exposure to other coronaviruses. ADE modulates the immune response and can elicit sustained inflammation, lymphopenia, and/or cytokine storm, one or all of which have been documented in severe cases and deaths. ADE also requires prior exposure to similar antigenic epitopes, presumably circulating in local viruses, making it a possible explanation for the observed geographic limitation of severe cases and deaths.
Output: Is COVID-19 receiving ADE from other coronaviruses?
Provision of an adequate mass of IgG from maternal colostrum is essential to health and survival of neonatal calves. Colostrum supplements (CS) have been developed to provide supplemental immunoglobulin when maternal colostrum is of poor quality. However, colostrum replacers (CR) that provide ≥100 g of IgG have not been formulated. Our objective was to determine the absorption of IgG in newborn calves fed CS derived from bovine serum or CR derived from bovine immunoglobulin concentrate. The CS were prepared by collecting, processing, and spray drying bovine serum and blending with other ingredients to provide 45 to 50 g of IgG per dose. The CR were prepared by further processing bovine serum to increase IgG concentration to >50% IgG and blending with other ingredients to provide 100 to 122 g of IgG per dose. Holstein calves (n = 160) were fed 90 to 244 g of IgG from CS or CR in 1 or 2 feedings in two experiments. Blood was collected from each calf by jugular venipuncture at 0 and 24 h of age and plasma IgG was determined by turbidimetric immunoassay. Apparent efficiency of IgG absorption was calculated. Plasma IgG concentrations at 24 h of age were indicative of IgG intake and averaged 5.5 to 14.1 g/L in calves fed CS and CR. Mean apparent efficiency of IgG absorption in calves fed CS was 25 and 28% in experiments 1 and 2, respectively. Mean apparent efficiency of IgG absorption in calves fed CR ranged from 19 to 32% and were affected by method of processing and number of times fed. Treatment of plasma with polyethylene glycol reduced the efficiency of IgG absorption in experiment 1. The addition of animal fat to CR had no effect on IgG absorption. A second feeding of CR increased plasma IgG, but efficiency of absorption was reduced. Mean body weights at 60 d of 2059 age were not affected by treatment and ranged from 64.3 to 78.2 kg. Plasma IgG concentration in calves fed ≥122 g of IgG from Ig concentrate approached (9.9 g/L) or exceeded 10 g/L, indicating successful transfer of passive immunity. Provision of IgG to prevent failure of passive transfer is possible with CR containing >20% IgG when fed at 454 g per dose. ( Abbreviation key: AEA = apparent efficiency of IgG absorption; CR = colostrum replacer; CS = colostrum supplement; MC = maternal colostrum.
Output: Formulation of Colostrum Supplements, Colostrum Replacers and Acquisition of Passive Immunity in Neonatal Calves 1
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.
Output: Protein folding liquid chromatography and its recent developments ଝ
Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development.
Output: Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays
A growing body of literature offers a framework for understanding geographic and ecological distributions of species; a few applications of this framework have treated disease transmission systems and their geography. The general framework focuses on interactions among abiotic requirements, biotic constraints, and dispersal abilities of species as determinants of distributional areas. Disease transmission systems have key differences from other sorts of biological phenomena: Interactions among species are particularly important, interactions may be stable or unstable, abiotic conditions may be relatively less important in shaping disease distributions, and dispersal abilities may be quite variable. The ways in which these differences may influence disease transmission geography are complex; I illustrate their effects by means of worked examples regarding West Nile Virus, plague, filoviruses, and yellow fever.
Output: Biogeography of diseases: a framework for analysis
These authors contributed equally to this work White powder, yield 49%, m. p. >300 o C; 1 H-NMR (DMSO-D 6 ) δ H 6.95 (1H, br, s, H10), 7.49-7.95 (5H, m. H15-H16), 8.58 (2H, s, H2 and H11), 12.41 (1H, s, H1); 13 C-NMR (DMSO-d 6 ) 121.9 (C15 ). QTOF-MS m/z calcd for C 12 H 9 KN 5 OS M+K + 310.39 found 310.33. 7 General procedure for the N-alkylation of acetamide-thioguanine The synthesis of 17 and 20 followed the general procedure for N-alkylation Salvatore et al., 2002 4 . Briefly, 138.23 mg of 4 Å Molecular Sieves (MS) was suspended into 3 mL of DMF. Cessium hydroxide monohydrate (CsOH.H 2 O) (140 µL) was added into the mixture and then stirred up for 10 minutes at the room temperature. The stirring was continued while adding N-(6-mercapto-9H-purin-2-yl)acetamide (MH022; 0.5 mmol) into the mixture for 30 minutes at the same temperature. Lastly, cyclopentyl bromide (0.6 mmol) was added into the mixture and the stirring was continued while monitoring the reaction's progress using TLC with CHCl 3 : acetone (3:1) as a solvent. After the reaction completed, the mixture was diluted with 70 mL of water and then extracted using 3 x 70 mL of EtO-Ac. The organic phase was collected, washed with 3 x 70 mL of water and then dried over anhydrous magnesium sulfate. This organic phase was then evaporated in vacuo and purified using PLC to afford the product as a white off powder. (15) White powder; yield 86%; m.p 154-156 o C; ʋ max / cm -1 (KBr) 3211 (NH), 1585 (C=O); 1 H-NMR (DMSO-D 6 ) δ H 1.73 (4H, m, H12 and H13), 2.19 (3H, s,H19), 2.26 (4H, m, H11 and H14), 2.65 (1H. m, H10), 8.03 (1H, s, H2), 12.85 (1H, br, s, H16); δ C 24.0 (MS m/z calcd for C 12 H 16 N 5 OS M+H + 278.35 found 278.13. White powder, yield 64%, m. p. >300 o C; 1 H-NMR (DMSO-D 6 ) δ H 1.09 (3H, t, H17), 1.19 (3H, t, H19), 2.55 (2H, q, H18), 3.29 (2H, q, H16), 8.61 (1H, s, H2), 11.97 (1H, br, s, H1); 13 C-NMR (DMSO-d 6 ) 8.
Output: Supporting Information Thioguanine-based DENV-2 NS2B/NS3 protease inhibitors: Virtual screening, synthesis, biological evaluation and molecular modelling
Calculation of infectious viral titers represents a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for strains 229E and OC43 of human coronavirus (HCoV). An alternative indirect immunoperoxidase assay (IPA) is herein described for the detection and titration of these viruses. Susceptible cells are inoculated with serial logarithmic dilutions of samples in a 96-well plate. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as "Tissue Culture Infectious Dose" (TCID 50 ). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain replicating virus. This technique is a reliable method for the titration of HCoV in biological samples (cells, tissues, or fluids).
Output: Titration of Human Coronaviruses, HCoV-229E and HCoV-OC43, by an Indirect Immunoperoxidase Assay
An epidemic of Ebola virus disease (EVD) from 2013-16 posed a serious risk of global spread during its early growth phase. A post-epidemic evaluation of the effectiveness of travel restrictions has yet to be conducted. The present study aimed to estimate the effectiveness of travel restrictions in reducing the risk of importation from mid-August to September, 2014, using a simple hazard-based statistical model. The hazard rate was modeled as an inverse function of the effective distance, an excellent predictor of disease spread, which was calculated from the airline transportation network. By analyzing datasets of the date of EVD case importation from the 15 th of July to the 15 th of September 2014, and assuming that the network structure changed from the 8 th of August 2014 because of travel restrictions, parameters that characterized the hazard rate were estimated. The absolute risk reduction and relative risk reductions due to travel restrictions were estimated to be less than 1% and about 20%, respectively, for all models tested. Effectiveness estimates among African countries were greater than those for other countries outside Africa. The travel restrictions were not effective enough to expect the prevention of global spread of Ebola virus disease. It is more efficient to control the spread of disease locally during an early phase of an epidemic than to attempt to control the epidemic at international borders. Capacity building for local containment and coordinated and expedited international cooperation are essential to reduce the risk of global transmission.
Output: Reduced Risk of Importing Ebola Virus Disease because of Travel Restrictions in 2014: A Retrospective Epidemiological Modeling Study
Background. The emerging zoonotic paramyxovirus Nipah virus (NiV) causes severe respiratory and neurological disease in humans, with high fatality rates. Nipah virus can be transmitted via person-to-person contact, posing a high risk for epidemic outbreaks. However, a broadly applicable approach for human NiV outbreaks in field settings is lacking. Methods. We engineered new antiviral lipopeptides and analyzed in vitro fusion inhibition to identify an optimal candidate for prophylaxis of NiV infection in the lower respiratory tract, and we assessed antiviral efficiency in 2 different animal models. Results. We show that lethal NiV infection can be prevented with lipopeptides delivered via the respiratory route in both hamsters and nonhuman primates. By targeting retention of peptides for NiV prophylaxis in the respiratory tract, we avoid its systemic delivery in individuals who need only prevention, and thus we increase the safety of treatment and enhance utility of the intervention. Conclusions. The experiments provide a proof of concept for the use of antifusion lipopeptides for prophylaxis of lethal NiV. These results advance the goal of rational development of potent lipopeptide inhibitors with desirable pharmacokinetic and biodistribution properties and a safe effective delivery method to target NiV and other pathogenic viruses.
Output: Fusion Inhibitory Lipopeptides Engineered for Prophylaxis of Nipah Virus in Primates
Background: Accurate data on childhood pneumonia aetiology are essential especially from regions where mortality is high, in order to inform case-management guidelines and the potential of prevention strategies such as bacterial conjugate vaccines. Yield from blood culture is low, but lung aspirate culture provides a higher diagnostic yield. We aimed to determine if diagnostic yield could be increased further by polymerase chain reaction (PCR) detection of bacteria (Streptococcus pneumoniae and Haemophilus influenzae b) and viruses in lung aspirate fluid. Methods: A total of 95 children with radiological focal, lobar or segmental consolidation had lung aspirate performed and sent for bacterial culture and for PCR for detection of bacteria, viruses and Pneumocystis jirovecii. In children with a pneumococcal aetiology, pneumococcal bacterial loads were calculated in blood and lung aspirate fluid. Results: Blood culture identified a bacterial pathogen in only 8 patients (8%). With the addition of PCR on lung aspirate samples, causative pathogens (bacterial, viral, pneumocystis) were identified singly or as co-infections in 59 children (62%). The commonest bacterial organism was S.pneumoniae (41%), followed by H. influenzae b (6%), and the commonest virus identified was adenovirus (16%), followed by human bocavirus (HBoV) (4%), either as single or co-infection. In a select group of African children, lung aspirate PCR significantly improves diagnostic yield. Our study confirms a major role of S.pneumoniae and viruses in the aetiology of childhood pneumonia in Africa.
Output: PCR Improves Diagnostic Yield from Lung Aspiration in Malawian Children with Radiologically Confirmed Pneumonia
Coronaviruses and arteriviruses, members of the order Nidovirales, are positive strand RNA viruses that encode large replicase polyproteins that are processed by viral proteases to generate the nonstructural proteins which mediate viral RNA synthesis. The viral papain-like proteases (PLPs) are critical for processing the amino-terminal end of the replicase and are attractive targets for antiviral therapies. With the analysis of the papain-like protease of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), came the realization of the multifunctional nature of these enzymes. Structural and enzymatic studies revealed that SARS-CoV PLpro can act as both a protease to cleave peptide bonds and also as a deubiquitinating (DUB) enzyme to cleave the isopeptide bonds found in polyubiquitin chains. Furthermore, viral DUBs can also remove the protective effect of conjugated ubiquitin-like molecules such as interferon stimulated gene 15 (ISG15). Extension of these studies to other coronaviruses and arteriviruses led to the realization that viral protease/DUB activity is conserved in many family members. Overexpression studies revealed that viral protease/DUB activity can modulate or block activation of the innate immune response pathway. Importantly, mutations that alter DUB activity but not viral protease activity have been identified and arteriviruses expressing DUB mutants stimulated higher levels of acute inflammatory cytokines after infection. Further understanding of the multifunctional nature of the Nidovirus PLP/DUBs may facilitate vaccine development. Here, we review studies describing the PLPs' enzymatic activity and their role in virus pathogenesis.
Output: Nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deISGylating activities
A 19-day-old female infant was admitted to a neonatal ICU in Ankara, Turkey. A product of an unremarkable gestation and vaginal delivery at 39 weeks, she began to have symptoms of an upper respiratory infection at the age of 15 days. The illness abruptly worsened 3 days later with cough, cyanosis, periods of apnea, and progressive respiratory failure precipitating her admission. The mother, who had received DPT (diphtheria, pertussis, and tetanus) vaccination as a child, reported a mild cough beginning in the 2 weeks prior to delivery. Upon arrival, the infant was having apneic episodes with cyanosis and her O 2 saturation dropped to 36% during a coughing spasm. An initial white blood cell count was 27,200/µL with 60% lymphocytes and a chest X-ray did not reveal any infiltrates. Despite ampicillin and gentamicin, she deteriorated further and endotracheal intubation was necessary but was discontinued on day 2 of her admission. Nasopharyngeal washings were tested by PCR and were negative for respiratory syncytial virus, influenza A and B, adenovirus, parainfluenza virus, and coronavirus. Based on the infant's symptom complex and lymphocytosis, she had erythromycin therapy added on day 4. Nasopharyngeal aspirates obtained before erythromycin treatment was begun were positive for pertussis by PCR and by culture on BordetÀGengou agar. The infant's serum antibody against pertussis toxin was undetectable. The infant slowly improved with fewer bouts of paroxysmal coughing and she was discharged on hospital day 10. The mother was the only contact who reported cough and was the only contact that was seropositive for pertussis although her culture was negative. Emerging Infectious Diseases.
Output: Pertussis CASE PRESENTATION 1
Background-Graphics are increasingly used to represent the spread of infectious diseases (e.g., influenza, Zika, Ebola); however, the impact of using graphics to adequately inform the general population is unknown. Objective-To examine whether three ways of visually presenting data (heat map, dot map, or picto-trendline)-all depicting the same information regarding the spread of a hypothetical outbreak of influenza-influence intent to vaccinate, risk perception, and knowledge. Design-Survey with participants randomized to receive a simulated news article accompanied by one of the three graphics that communicated prevalence of influenza and number of influenzarelated deaths. Participants-16,510 adults living in 11 countries selected using stratified random sampling based on age and gender Measurements-After reading the article and viewing the presented graphic, participants completed a survey that measured interest in vaccination, perceived risk of contracting disease, knowledge gained, interest in additional information about the disease, and perception of the graphic. Results-Heat maps and picto-trendlines were evaluated more positively than dot maps. Heat maps were more effective than picto-trendlines and no different from dot maps at increasing interest in vaccination, perceived risk of contracting disease, and interest in additional information about the disease. Heat maps and picto-trendlines were more successful at conveying knowledge than dot maps. Overall, heat maps were the only graphic to be superior in every outcome. Conclusion-Heat maps are a viable option to promote interest in and concern about infectious diseases.
Output: Communicating infectious disease prevalence through graphics: results from an international survey
KEYWORDS Cox proportional hazard model; dentist; mortality; standardized mortality ratios (SMRs) Background/Purpose: Controversy exists in the literature regarding whether dentists with multiple occupational exposures suffer from premature mortality. A cohort mortality study was conducted to evaluate the survival outcome and determine if potential exposure to harmful agents leads to premature mortality among dentists. Methods: Using the Life Table Analysis System, we calculated standardized mortality ratios (SMRs) for a cohort of 11,700 dentists affiliated with the Taiwan Dental Association. These dentists were followed from 1985e2009. Reference rates were derived from cause-, gender-, and age-specific mortality rates of the general population of Taiwan and 18,664 Taiwanese internists, who were considered to be more socioeconomically proximal to dentists. A Cox proportional hazard model was also constructed to determine multiple risk factors associated with mortality. Results: Compared with the general population, dentists in Taiwan consistently demonstrated reduced from all-cause mortality. However, compared with internists, significant and excess mortality were observed in dentists for overall mortality (SMR Z 1.13; 95% confidence interval [CI] Z 1.00 e1.26), drowning (SMR Z 6.62; 95% CI Z 2.15 e15.45), and heart diseases (SMR Z 1.66; 95% CI Z 1.22e2.21). After adjusting for other risk factors, the Cox model showed an increased hazard ratio of 1.17 (95% CI Z 1.01e1.37) for dentists. Conclusion: Taiwanese dentists demonstrated significant elevated SMRs for overall causes, drowning, and heart diseases. Careful precaution should be taken to reduce these trends. Future studies are also needed for in-depth exploration of the mechanisms regarding how professional stress and exposure contribute to the increased risk of mortality in Taiwanese dentists.
Output: Mortality among dentists in Taiwan, 1985e2009
The microarray is a platform with wide-ranging potential in biodefence. Owing to the high level of throughput attainable through miniaturization, microarrays have accelerated the ability to respond in an epidemic or crisis. Extending beyond diagnostics, recent studies have applied microarrays as a research tool towards understanding the etiology and pathogenicity of dangerous pathogens, as well as in vaccine development. The original emphasis was on DNA microarrays, but the range now includes protein, antibody and carbohydrate microarrays, and research groups have exploited this diversity to further extend microarray applications in the area of biodefence. Here, we discuss the impact and contributions of the growing range of microarrays and emphasize the concepts that might shape the future of biodefence research. 0167-7799/$ -see front matter ß
Output: Applications of microarrays in pathogen detection and biodefence
Emerging infectious diseases arising from livestock and wildlife pose serious threats to global human health, as shown by a series of continuous outbreaks involving highly pathogenic influenza, SARS, Ebola and MERS. The risk of pandemics and bioterrorism threats is ever present and growing, but our ability to combat them is limited by the lack of available vaccines, therapeutics and rapid diagnostics. The use of high bio-containment facilities, such as the CSIRO Australian Animal Health Laboratory, plays a key role studying these dangerous pathogens and facilitates the development of countermeasures. To combat diseases like MERS, we must take a holistic approach that involves the development of early biomarkers of infection, a suite of treatment options (vaccines, anti-viral drugs and antibody therapeutics) and appropriate animal models to test the safety and efficacy of candidate treatments.
Output: Overview of the CSIRO Australian Animal Health Laboratory
Coronaviruses (CoVs) are a group of enveloped, single-stranded positive genomic RNA viruses and some of them are known to cause severe respiratory diseases in human, including Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and the ongoing coronavirus disease-19 (COVID-19). One key element in viral infection is the process of viral entry into the host cells. In the last two decades, there is increasing understanding on the importance of the endocytic pathway and the autophagy process in viral entry and replication. As a result, the endocytic pathway including endosome and lysosome has become important targets for development of therapeutic strategies in combating diseases caused by CoVs. In this mini-review, we will focus on the importance of the endocytic pathway as well as the autophagy process in viral infection of several pathogenic CoVs inclusive of SARS-CoV, MERS-CoV and the new CoV named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and discuss the development of therapeutic agents by targeting these processes. Such knowledge will provide important clues for control of the ongoing epidemic of SARS-CoV-2 infection and treatment of COVID-19.
Output: Targeting the Endocytic Pathway and Autophagy Process as a Novel Therapeutic Strategy in COVID-19
Since late 2010, the outbreak of porcine epidemic diarrhea (PED) in China has resulted in the deaths of millions of suckling piglets. The main cause of the disease outbreak was unknown. In this study, partial spike (S), ORF3, and membrane (M) genes amplified from these variants were sequenced and analyzed. The results showed that the variants could be clustered into one to three subgroups and suggested that S genes were variable, while M genes were relatively conserved. Moreover, in comparison with the vaccine strain CV777, sequence alignment analyses revealed that the S genes of the newly isolated strains contained several mutations at the aa level. It is possible that these mutations have changed the hydrophobicity of the S protein and influenced the viral antigenicity and virulence. Interestingly, homology analyses based on ORF3 demonstrated that the isolates had an intact opening reading frame (ORF), which were different from the attenuated DR13 strain. In conclusion, the widespread PED virus (PEDV) isolates had virulent characteristics. Additionally, the high degree of variation in the genes, particularly S genes, might provide an explanation for the poor immunity and rapid spread of the disease.
Output: Genetic Variability and Phylogeny of Current Chinese Porcine Epidemic Diarrhea Virus Strains Based on Spike, ORF3, and Membrane Genes
Studies on several viral pathogens have been hampered by the lack of appropriate in vitro systems for their propagation and amplification. Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus containing a single-stranded positive-sense RNA genome (∼15 kb), was served as a model virus and its genomic cDNA was recombinated into baculovirus. We investigated whether infectious virus particles could be generated by expression of the full-length cloned genome from the modified baculovirus vector. The recombinant baculovirus, AcAPRRS, was used to infect sf9 cells. Immunofluorescence assay demonstrated the presence of PRRSV nonstructural protein (nsp) 2 and nucleocapsid (N) protein and electron microscopy revealed PRRSV particles in the culture supernatant. Infectious PRRSV particles were also produced in susceptible MARC-145 cells inoculated with AcAPRRS, and the growth characteristics of the PRRSV generated were similar to those of the parental PRRSV strain. Infectious PRRSV particles were also generated following AcAPRRS transduction of BHK-21 cells and Vero cells that are not sensitive to PRRSV. Titers of PRRSV obtained from BHK-21 and Vero cells were up to 10 4.05 TCID 50 /ml. These findings open a new route to the propagation of the virus in vitro and will be of utility in vaccine development.
Output: Baculovirus expression of cloned porcine arterivirus generates infectious particles in both insect and mammalian cells
Background: The diagnosis of ventilator-associated pneumonia (VAP) remains time-consuming and costly, the clinical tools lack specificity and a bedside test to exclude infection in suspected patients is unavailable. Breath contains hundreds to thousands of volatile organic compounds (VOCs) that result from host and microbial metabolism as well as the environment. The present study aims to use breath VOC analysis to develop a model that can discriminate between patients who have positive cultures and who have negative cultures with a high sensitivity. Methods/design: The Molecular Analysis of Exhaled Breath as Diagnostic Test for Ventilator-Associated Pneumonia (BreathDx) study is a multicentre observational study. Breath and bronchial lavage samples will be collected from 100 and 53 intubated and ventilated patients suspected of VAP. Breath will be analysed using Thermal Desorption -Gas Chromatography -Mass Spectrometry (TD-GC-MS). The primary endpoint is the accuracy of cross-validated prediction for positive respiratory cultures in patients that are suspected of VAP, with a sensitivity of at least 99% (high negative predictive value). Discussion: To our knowledge, BreathDx is the first study powered to investigate whether molecular analysis of breath can be used to classify suspected VAP patients with and without positive microbiological cultures with 99% sensitivity. Trial registration: UKCRN ID number 19086, registered May 2015; as well as registration at www.trialregister.nl under the acronym 'BreathDx' with trial ID number NTR 6114 (retrospectively registered on 28 October 2016).
Output: BreathDx -molecular analysis of exhaled breath as a diagnostic test for ventilator- associated pneumonia: protocol for a European multicentre observational study
Metagenomics was used to identify viral sequences in the plasma and CSF (cerobrospinal fluid) of 13 horses with unexplained neurological signs and in the plasma and respiratory swabs of 14 horses with unexplained respiratory signs. Equine hepacivirus and two copiparvoviruses (horse parvovirus-CSF and a novel parvovirus) were detected in plasma from neurological cases. Plasma from horses with respiratory signs contained the same two copiparvoviruses plus equine pegivirus D and respiratory swabs contained equine herpes virus 2 and 5. Based on genetic distances the novel copiparvovirus qualified as a member of a new parvovirus species we named Eqcopivirus. These samples plus another 41 plasma samples from healthy horses were tested by real-time PCRs for multiple equine parvoviruses and hepacivirus. Over half the samples tested were positive for one to three viruses with eqcopivirus DNA detected in 20.5%, equine hepacivirus RNA and equine parvovirus-H DNA in 16% each, and horse parvovirus-CSF DNA in 12% of horses. Comparing viral prevalence in plasma none of the now three genetically characterized equine parvoviruses (all in the copiparvovirus genus) was significantly associated with neurological and respiratory signs in this limited sampling.
Output: Viruses in Horses with Neurologic and Respiratory Diseases
Background: The interplay between IFN-γ, IL-17 and neutrophils during CNS inflammatory disease is complex due to cross-regulatory factors affecting both positive and negative feedback loops. These interactions have hindered the ability to distinguish the relative contributions of neutrophils, Th1 and Th17 cell-derived effector molecules from secondary mediators to tissue damage and morbidity. Methods: Encephalitis induced by a gliatropic murine coronavirus was used as a model to assess the direct contributions of neutrophils, IFN-γ and IL-17 to virus-induced mortality. CNS inflammatory conditions were selectively manipulated by adoptive transfer of virus-primed wild-type (WT) or IFN-γ deficient (GKO) memory CD4 + T cells into infected SCID mice, coupled with antibody-mediated neutrophil depletion and cytokine blockade. Results: Transfer of GKO memory CD4 + T cells into infected SCID mice induced rapid mortality compared to recipients of WT memory CD4 + T cells, despite similar virus control and demyelination. In contrast to recipients of WT CD4 + T cells, extensive neutrophil infiltration and IL-17 expression within the CNS in recipients of GKO CD4 + T cells provided a model to directly assess their contribution(s) to disease. Recipients of WT CD4 + T cells depleted of IFN-γ did not express IL-17 and were spared from mortality despite abundant CNS neutrophil infiltration, indicating that mortality was not mediated by excessive CNS neutrophil accumulation. By contrast, IL-17 depletion rescued recipients of GKO CD4 + T cells from rapid mortality without diminishing neutrophils or reducing GM-CSF, associated with pathogenic Th17 cells in CNS autoimmune models. Furthermore, co-transfer of WT and GKO CD4 + T cells prolonged survival in an IFN-γ dependent manner, although IL-17 transcription was not reduced. Conclusions: These data demonstrate that IL-17 mediates detrimental clinical consequences in an IFN-γ-deprived environment, independent of extensive neutrophil accumulation or GM-CSF upregulation. The results also suggest that IFN-γ overrides the detrimental IL-17 effector responses via a mechanism downstream of transcriptional regulation.
Output: IFN-γ protects from lethal IL-17 mediated viral encephalomyelitis independent of neutrophils
As a key factor in endemic and epidemic dynamics, the geographical distribution of viruses has been frequently interpreted in the light of their genetic histories. Unfortunately, inference of historical dispersal or migration patterns of viruses has mainly been restricted to model-free heuristic approaches that provide little insight into the temporal setting of the spatial dynamics. The introduction of probabilistic models of evolution, however, offers unique opportunities to engage in this statistical endeavor. Here we introduce a Bayesian framework for inference, visualization and hypothesis testing of phylogeographic history. By implementing character mapping in a Bayesian software that samples time-scaled phylogenies, we enable the reconstruction of timed viral dispersal patterns while accommodating phylogenetic uncertainty. Standard Markov model inference is extended with a stochastic search variable selection procedure that identifies the parsimonious descriptions of the diffusion process. In addition, we propose priors that can incorporate geographical sampling distributions or characterize alternative hypotheses about the spatial dynamics. To visualize the spatial and temporal information, we summarize inferences using virtual globe software. We describe how Bayesian phylogeography compares with previous parsimony analysis in the investigation of the influenza A H5N1 origin and H5N1 epidemiological linkage among sampling localities. Analysis of rabies in West African dog populations reveals how virus diffusion may enable endemic maintenance through continuous epidemic cycles. From these analyses, we conclude that our phylogeographic framework will make an important asset in molecular epidemiology that can be easily generalized to infer biogeogeography from genetic data for many organisms.
Output: Bayesian Phylogeography Finds Its Roots
To gain insight into the potential for aerosolization of viruses in wastewater systems, we investigated the partitioning of MS2 and Phi6 bacteriophages in synthetic sludge and anaerobically digested sludge from a wastewater treatment plant. We evaluated partitioning among the liquid, solids, and material surfaces of porcelain, concrete, polyvinyl chloride (PVC), and polypropylene. In all cases, at least 94% of the virions partitioned into the liquid fraction. In real sludge, no more than 0.8% of virions partitioned to the solids and no more than 6% to the material surface. Both MS2 and Phi6 partitioned more to the surface of concrete and polypropylene than to the surface of porcelain or PVC. Partitioning of viruses in wastewater among the liquid, biosolids, and material surface does not appear to mitigate the potential for aerosolization of virus, as most of the virus remains in the liquid phase. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Figure 4 . Concentration of Phi6 bacteriophage, determined by qPCR and reported in PFU equivalents per milliliter, in anaerobically digested sludge diluted 5-fold in water in each of three phases: 46 mL of liquid, 4 mL of wetted solids, and 1 mL of material surface extract for porcelain, concrete, PVC, and polypropylene. Each point represents an experimental replicate, and there are three nearly overlapping points for solids in concrete.
Output: Partitioning of Viruses in Wastewater Systems and Potential for Aerosolization
Theoretically, there are several ways of preventing the common cold: quarantine, immunisation (or vaccination); early treatment of effected individuals; or physical barriers to reduce transmission. All these methods can be dismissed after considering the epidemiology of the common cold, apart from the last. Evidence for effectiveness for physical barriers (which include masks to reduce aerosol transmission; handwashing; and gloves and gowns) come from a variety of empirical studies. The chance of bias for these studies is variable, but we can conclude that all of these barrier methods have important potential for preventing transmission of the common cold, although some methods will not be acceptable to the community currently.
Output: Common Cold 211 Interventions to prevent transmission of the common cold
Background: Chronic kidney disease (CKD) has typically a non-immune mediated origin in cats and immunecomplex glomerulonephritis (ICGN) is scarcely described. Aims of this study were to characterize ICGN by light and electron microscopy and identify associations with clinico-pathological findings. In addition, comparisons between cats with ICGN and non immune-complex glomerulonephritis (non-ICGN) were performed. Renal samples examined between 2010 and 2019 were considered if both light and electron microscopy were performed. Signalment, feline immunodeficiency virus (FIV) and leukemia virus (FeLV) status, serum creatinine concentration, urine protein-to-creatinine (UPC) ratio, systolic blood pressure (SBP) and International Renal Interest Society (IRIS) stage were retrieved and used for comparisons. Results: Sixty-eight client-owned cats were included. Thirty-seven cats (54.4%) had ICGN and 31 (45.6%) non-ICGN. Eighteen (48.6%) with ICGN had membranous glomerulonephropathy (MGN), 14 (37.8%) membranoproliferative glomerulonephritis (MPGN), and 5 (13.5%) mesangioproliferative glomerulonephritis (MeGN). Clinico-pathological data were not associated with any type of ICGN. Among cats with non-ICGN, 11 (35.5%) had end-stage CKD, 9 (29%) focal segmental glomerulosclerosis, 6 (19.4%) global and multifocal mesangiosclerosis, 2 (6.5%) glomerular atrophy, 2 (6.5%) renal dysplasia and 1 (3.1%) amyloidosis. Eight (25.8%) cats with non-ICGN had chronic interstitial nephritis (CIN) grade 1, 13 (41.9%) grade 2 and 10 (32.3%) grade 3; creatinine and UPC ratio increased with CIN grades (p = 0.001, p < 0.001). Cats with ICGN were more frequently FIV or FeLV-infected (OR:11.4; 95%CI:1.4-94.4; p = 0.024), had higher UPC ratio (OR:6.8; 95%CI:2.5-18.2; p < 0.001) and were younger (OR:0.9; 95%CI:0.7-1.0; p = 0.042) than cats with non-ICGN. Conclusions: MGN and MPGN were the most common morphological diagnoses of ICGN in cats. Unfortunately, none of the investigated findings differentiated ICGN morphological diagnoses. Serum creatinine concentration and UPC ratio were directly associated with grades of CIN (p = 0.001 and p < 0.001, respectively), confirming previous literature. More ICGN than non-ICGN was observed in cats with retroviral infections, younger cats and higher UPC ratio.
Output: Immune-complex glomerulonephritis in cats: a retrospective study based on clinico- pathological data, histopathology and ultrastructural features
Aim. We presented the case of a child with central hypoventilation syndrome (CHS) to highlight issues that need to be considered in planning long-haul flight and problems that may arise during the flight. Case. The pediatric intensive care unit (PICU) received a child with central hypoventilation syndrome (Ondine's curse) on nocturnal ventilatory support who travelled to Hong Kong on a make-a-wish journey. He was diagnosed with central hypoventilation and had been well managed in Canada. During a long-haul aviation travel, he developed respiratory symptoms and desaturations. The child arrived in Hong Kong and his respiratory symptoms persisted. He was taken to a PICU for management. The child remained well and investigations revealed no pathogen to account for his respiratory infection. He went on with his make-a-wish journey. Conclusions. Various issues of travel medicine such as equipment, airline arrangement, in-flight ventilatory support, travel insurance, and respiratory infection are explored and discussed. This case illustrates that long-haul air travel is possible for children with respiratory compromise if anticipatory preparation is timely arranged.
Output: Case Report Central Hypoventilation: A Case Study of Issues Associated with Travel Medicine and Respiratory Infection
Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virusbased SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.
Output: Reverse Genetics of SARS-Related Coronavirus Using Vaccinia Virus-Based Recombination
FP7). Particular attention is given to the statutory role of the European institutions in the design and orientation of the successive biotechnology programmes, compared to the more informal -yet visionary -role of key individuals upstream to any legislative decision. Examples of success stories and of the role of the biotechnology programmes in addressing societal issues and industrial competitiveness are also presented. Finally, an outline of Horizon 2020, the successor of FP7, is described, together with the role of biotechnology in building the bioeconomy.
Output: Thirty years of European biotechnology programmes: from biomolecular engineering to the bioeconomy
Respiratory virus infections (RVIs) are increasingly recognized as a cause of significant morbidity and mortality in recipients of hematologic stem cell transplant (HCT) and patients with hematologic malignancy (HM). 1,2 With now widespread use of molecular diagnostics, the epidemiology and spectrum of clinical disease of these infections can be better characterized. Apart from influenza, the currently available antivirals are limited in efficacy and/or associated with potential for toxicity, thus emphasizing the importance of prevention strategies. This article provides a review of the epidemiology, clinical characteristics, management, and prevention of RVIs in HCT recipients and HM patients. No disclosures. KEYWORDS Respiratory virus infection Hematopoietic stem cell transplant RSV Influenza Parainfluenza Human metapneumovirus Rhinovirus Coronavirus The morbidity and associated complications of respiratory virus infections are greater in hematopoietic stem cell transplant recipients and patients with hematologic malignancy than in immunocompetent individuals, with severity of illness related to the degree of immunosuppression. Molecular microbiologic testing is the gold standard for diagnosis, allowing differentiation of what are largely overlapping clinical syndromes. Most of the respiratory viruses, apart from influenza and in some circumstances respiratory syncytial virus and adenovirus, are managed supportively. Prevention is key and should focus on vaccination for influenza, avoidance of ill contacts, and compliance with principles of infection control. Infect Dis Clin N Am 33 (2019) 523-544 a Studies are a combination of PCR and traditional laboratory methods (eg, culture, direct fluorescent antibody, and enzyme immunoassay). b PCR-based studies. c Includes all-cause and attributable mortality with variable timeframe to death. d One mortality in a coinfected patient attributed to enterovirus/rhinovirus infection.
Output: Respiratory Virus Infections of the Stem Cell Transplant Recipient and the Hematologic Malignancy Patient