title: Regulation of Coronaviral Poly(A) Tail Length during Infection

text: The positive-strand coronavirus genome of ~30 kilobase in length and subgenomic (sg) mRNAs of shorter lengths, are 5’ and 3’-co-terminal by virtue of a common 5’-capped leader and a common 3’-polyadenylated untranslated region. Here, by ligating head-to-tail viral RNAs from bovine coronavirus-infected cells and sequencing across the ligated junctions, it was learned that at the time of peak viral RNA synthesis [6 hours postinfection (hpi)] the 3’ poly(A) tail on genomic and sgmRNAs is ~65 nucleotides (nt) in length. Surprisingly, this length was found to vary throughout infection from ~45 nt immediately after virus entry (at 0 to 4 hpi) to ~65 nt later on (at 6 h to 9 hpi) and from ~65 nt (at 6 h to 9 hpi) to ~30 nt (at 120-144 hpi). With the same method, poly(U) sequences of the same lengths were simultaneously found on the ligated viral negative-strand RNAs. Functional analyses of poly(A) tail length on specific viral RNA species, furthermore, revealed that translation, in vivo, of RNAs with the longer poly(A) tail was enhanced over those with the shorter poly(A). Although the mechanisms by which the tail lengths vary is unknown, experimental results together suggest that the length of the poly(A) and poly(U) tails is regulated. One potential function of regulated poly(A) tail length might be that for the coronavirus genome a longer poly(A) favors translation. The regulation of coronavirus translation by poly(A) tail length resembles that during embryonal development suggesting there may be mechanistic parallels.


title: Ebola Risk and Preparedness: A National Survey of Internists

text: BACKGROUND: The 2014–2015 Ebola virus disease (Ebola) epidemic centered in West Africa highlighted recurring challenges in the United States regarding risk communication and preparedness during global epidemics. OBJECTIVE: To investigate perceptions, preparedness, and knowledge among U.S. internists with regard to Ebola risk. DESIGN: Cross-sectional Web-based national survey distributed by e-mail between December 2014 and January 2015. PARTICIPANTS: Practicing U.S. internists participating in a research panel representative of American College of Physicians (ACP) membership. MAIN MEASURES: Respondents’ perceptions of Ebola, reported sources of information, and reported management of possible Ebola cases. The primary predictor was the possibility of encountering Ebola (based on respondents’ geographic proximity to designated airports or confirmed Ebola cases, or on their patients’ travel histories). Pre-specified outcomes included reported management intensity in clinical vignettes involving patients at low risk of symptomatic Ebola as well as reported Ebola preparedness. KEY RESULTS: The survey response rate was 46.1 %. Among the 202 respondents, 9.9 % (95 % CI 6.2–14.9 %) reported that they had recently evaluated a patient who had traveled to West Africa. Seventy percent (95 % CI 63.0–76.0 %) reported a practice-level protocol. The Centers for Disease Control and Prevention (CDC) was the most popular source for Ebola information (75.2 %, 95 % CI 68.7–81.0 %). Most respondents felt very (45.0 %) or somewhat prepared (52.0 %) to communicate information about or diagnose Ebola, especially those with the possibility of encountering Ebola and those who reported medical journals, professional groups, or government as information sources. One-fifth of respondents (19.8 %, 95 % CI 14.5–26.0 %) reported overly intensive management for low-risk patients. Those with the possibility of encountering Ebola were less likely to report overly intensive management (3.1 vs. 22.9 %, p = 0.011). CONCLUSIONS: Internists had wide-ranging views and understanding of Ebola risk; those least likely to encounter Ebola were most likely to be overly aggressive in managing patients at low risk. Our findings underscore the need for better risk communication through various information channels to empower frontline providers in infectious disease outbreaks. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11606-015-3493-1) contains supplementary material, which is available to authorized users.


title: US school/academic institution disaster and pandemic preparedness and seasonal influenza vaccination among school nurses

text: BACKGROUND: School pandemic preparedness is essential, but has not been evaluated. METHODS: An online survey was sent to school nurses (from state school nurse associations and/or state departments of education) between May and July 2011. Overall school pandemic preparedness scores were calculated by assigning 1 point for each item in the school's pandemic plan; the maximum score was 11. Linear regression was used to describe factors associated with higher school pandemic preparedness scores. Nurse influenza vaccine uptake was assessed as well. RESULTS: A total of 1,997 nurses from 26 states completed the survey. Almost three-quarters (73.7%; n = 1,472) reported receiving the seasonal influenza vaccine during the 2010-11 season. Very few (2.2%; n = 43) reported that their school/district had a mandatory influenza vaccination policy. Pandemic preparedness scores ranged from 0 to 10 points, with an average score of 4.3. Determinants of school pandemic preparedness were as follows: planning to be a point of dispensing during a future pandemic (P < .001), having experienced multiple student or employee hospitalizations and/or deaths related to H1N1 during the pandemic (P = .01 or <.05, respectively), having a lead nurse complete the survey (P < .001), and having the school nurse study participant be a member of the school disaster planning committee (P < .001). CONCLUSIONS: US schools must continue to address gaps in pandemic planning.


Influence of hypokalemia upon the ultrastructure of rat gastrocnemius muscle. A semi-automatic morphometric study


title: HCAtk and pyHCA: A Toolkit and Python API for the Hydrophobic Cluster Analysis of Protein Sequences

text: Motivation: Detecting protein domains sharing no similarity to known domains, as stored in domain databases, is a challenging problem, particularly for unannotated proteomes, domains emerged recently, fast diverging proteins or domains with intrinsically disordered regions. Results: We developed pyHCA and HCAtk, a python API and standalone tool gathering together improved versions of previously developed methodologies, with new functionalities. The developed tools can be either used from command line or from a python API. Availability: HCAtk and pyHCA are available at https://github.com/T-B-F/pyHCA under the CeCILL-C license.


title: Innate immunity in the retina: Toll-like receptor (TLR) signaling in human retinal pigment epithelial cells

text: Abstract Toll-like receptors (TLRs) are crucial components of innate immunity that participate in host defense against microbial pathogens. We evaluated the expression and function of TLRs in human retinal pigment epithelial (RPE) cells. Real time PCR analysis revealed gene expression for TLRs 1–7, 9, and 10 in RPE cells. TLRs 1 and 3 were the most highly expressed TLRs. Protein expression for TLRs 2, 3, and 4 was observed on RPE cells and this expression was augmented by treatment with poly I:C or interferon-γ (IFN-γ). TLR 3 is the receptor for dsRNA, an intermediate of virus replication. Because RPE cells express TLR 3 and are frequently the site of virus replication within the retina, we evaluated TLR 3 signaling. RPE cells treated with poly I:C produced IFN-β but not IFN-α, and this was inhibited by the treatment of RPE cells with anti-TLR 3 antibody. Human recombinant IFN-β was shown to be biologically active on RPE cells by inhibiting viral replication. Poly I:C treatment of RPE resulted in an increase in the production of IL-6, IL-8, MCP-1, and sICAM-1. The presence of TLRs on RPE cells and the resultant TLR signaling in RPE cells suggest that these molecules may play an important role in innate and adaptive immune responses within the retina.


title: Functional motifs responsible for human metapneumovirus M2-2-mediated innate immune evasion

text: Abstract Human metapneumovirus (hMPV) is a major cause of lower respiratory infection in young children. Repeated infections occur throughout life, but its immune evasion mechanisms are largely unknown. We recently found that hMPV M2-2 protein elicits immune evasion by targeting mitochondrial antiviral-signaling protein (MAVS), an antiviral signaling molecule. However, the molecular mechanisms underlying such inhibition are not known. Our mutagenesis studies revealed that PDZ-binding motifs, 29-DEMI-32 and 39-KEALSDGI-46, located in an immune inhibitory region of M2-2, are responsible for M2-2-mediated immune evasion. We also found both motifs prevent TRAF5 and TRAF6, the MAVS downstream adaptors, to be recruited to MAVS, while the motif 39-KEALSDGI-46 also blocks TRAF3 migrating to MAVS. In parallel, these TRAFs are important in activating transcription factors NF-kB and/or IRF-3 by hMPV. Our findings collectively demonstrate that M2-2 uses its PDZ motifs to launch the hMPV immune evasion through blocking the interaction of MAVS and its downstream TRAFs.


title: A novel multiplex real-time RT-PCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses

text: Abstract Quantitative multiplex real-time RT-PCR assays utilizing fluorescence resonance energy transfer (FRET) hybridization probes were developed for the detection of 13 respiratory viruses, including well recognized viral causes (respiratory syncytial virus, influenza viruses A and B, parainfluenza viruses types 1, 2, and 3, adenovirus) as well as viruses described recently as causes of acute respiratory tract infections (human coronaviruses NL63, HKU1, 229E, and OC43, human bocavirus, and human metapneumovirus). FRET probes have an improved toleration for single base mismatches than other probe chemistries, reducing the chances of missing highly variable RNA viruses. The assay could detect 2.5–25 DNA/RNA copies/μl (2.5×103–2.5×104 copies/ml). Validation on 91 known positive respiratory specimens indicated similar specificity as commercial direct immunofluorescence assays (IFA) or single-round PCRs used in initial identification. Screening of 270 IFA negative respiratory specimens identified new viruses in 40/270 (14.8%) cases and additional 79/270 (29.3%) well recognized viruses missed by routine diagnostic assays including 6.7% co-infections. All viruses could be detected in the clinical screening panel. The assays demonstrates an improved sensitivity and scope of detecting respiratory viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the role of co-infections and viral load in respiratory virus pathogenesis.


title: Anti-Recombinant Gametocyte 56 Protein IgY Protected Chickens from Homologous Coccidian Infection

text: Abstract Coccidiosis is caused by intra-cellular infection of Eimeria spp., which goes through a complex life cycle in the intestinal mucosa of infected hosts. Specific immunoglobulins (IgY) could be produced in egg yolk by immunizing hens with specific antigens. In the present study, we cloned the E. maxima gam56 gene, expressed the GST-GAM56 fusion protein and raised IgY to GST-GAM56 in hens. The anti-GST-GAM56 IgY antibody was isolated and used to treat chickens infected with E. maxima oocysts. Intramuscular injection of the antibodies provided minimal protection against parasite infection. However, oral dosing of the IgY 3 or 5 d after oocyst inoculation significantly improved body weight gain, reduced oocyst output and intestinal lesion score were reduced at 3 or 5 d after oocyst challenging, compared to the untreated control group. Our findings suggest that the IgY to gam56 could be an effective prophylactic or therapeutic agent against E. maxima infection in chickens and should have a practical application value.


title: Global use of asbestos - legitimate and illegitimate issues

text: BACKGROUND: Exposure to asbestos causes non-malignant and malignant diseases including asbestosis, lung cancer, and mesothelioma. The modern history of such diseases goes back more than a century. MAIN TEXT: While much is known about the ability of asbestos to cause disease, the carcinogenic mechanism is not yet understood. Continuing legitimate scientific questions include such issues as potential differential toxicity and carcinogenicity of different fiber types. Illegitimate issues include the supposed “safe” use of asbestos, and the chrysotile hypothesis. CONCLUSION: Asbestos disease issues are highly politicized and vested economic interests perpetuate false issues regarding the hazards of asbestos.


title: Trainee and attending perspectives on remote radiology readouts in the era of the COVID-19 pandemic

text: Abstract Rationale and Objectives Social distancing mandates due to COVID-19 have necessitated adaptations to radiology trainee workflow and educational practices, including the radiology ‘readout’. We describe how a large academic radiology department achieved socially distant ‘remote readouts’, provide trainee and attending perspectives on this early experience, and propose ways by which ‘remote readouts’ can be used effectively by training programs beyond COVID-19. Materials and Methods Beginning March 2020, radiologists were relocated to workspaces outside of conventional reading rooms. Information technologies were employed to allow for ‘remote readouts’ between trainees and attendings. An optional anonymous open-ended survey regarding remote readouts was administered to radiology trainees and attendings as a quality improvement initiative. From the responses, response themes were abstracted using thematic analysis. Descriptive statistics of the qualitative data were calculated. Results Radiologist workstations from 14 traditional reading rooms were relocated to 36 workspaces across the hospital system. Two models of remote readouts, synchronous and asynchronous, were developed, facilitated by commercially-available information technologies. 39/105 (37%) trainees and 42/90 (47%) attendings responded to the survey. Main response themes included: social distancing, technology, autonomy/competency, efficiency, education/feedback and atmosphere/professional relationship. 148 positive versus 97 negative comments were reported. Social distancing, technology and autonomy/competency were most positively rated. Trainees and attending perspectives differed regarding the efficiency of remote readouts. Conclusions ‘Remote readouts’, compliant with social distancing measures, are feasible in academic radiology practice settings. Perspectives from our initial experience provide insight into how this can be accomplished, opportunities for improvement and future application, beyond the COVID-19 pandemic.


title: Heparan sulfate is a binding molecule but not a receptor for CEACAM1-independent infection of murine coronavirus

text: Abstract A highly neurovirulent mouse hepatitis virus (MHV) JHMV strain (wt) with receptor (MHVR)-independent infection activity and its low-virulent mutant srr7 without such activity were found to attach to MHVR-negative, non-permissive BHK cells. To identify the molecule that interacts with JHMV, we focused on heparan sulfate (HS) since it works as a receptor of a mutant MHV-rec1 that infects in an MHVR-independent fashion. The present study indicates that HS interacts with both wt JHMV and srr7 but it does not function as an entry receptor as it apparently does for MHV-rec1. Furthermore, HS failed to serve as an entry receptor in the MHVR-independent infection of wt JHMV, indicating that HS is not a host factor that wt JHMV utilizes in an MHVR-independent infection.


United States Cutaneous Lymphoma Consortium Recommendations for Treatment of Cutaneous Lymphomas During the COVID-19 Pandemic


title: Cytokeratin 8/18 overexpression and complex formation as an indicator of GST-P positive foci transformation into hepatocellular carcinomas

text: Abstract Screening of the proteome of microdissected glutathione S-transferase placental form (GST-P) positive foci and normal-appearing liver on anionic (Q10), and cationic (CM10) surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip arrays demonstrated significant overexpression of cytokeratin 8 (CK8; m/z 54,020), cytokeratin 18 (CK18; m/z 47,760), microsomal cytochrome 5A (m/z 15,224) and histone type 2 H2aa3 (m/z 15,964) in the livers of rats initiated with diethylnitrosamine (DEN) followed by 10 weeks on phenobarbital (PB) at a dose of 500 ppm. Furthermore, formation of CK8 and CK18 complexes due to CK8 phosphorylation at Ser73 and Ser431 was found to be strongly associated with promotion of hepatocarcinogenesis by PB and the development of hepatocellular carcinomas. The data were confirmed by immunohistochemistry and real-time Q-PCR and profound overexpression of CK8 and CK18 (CK8/18) proteins and mRNAs were detected in several large size GST-P positive foci and liver tumors. A strong correlation between CK8/18 positive foci development and multiplicity of hepatocellular carcinomas was further observed. Moreover, elevation of CK8/18 was strongly associated with induction of cell proliferation in GST-P positive foci and tumors. In conclusion, our data imply that CK8/18 overexpression, those two cytokeratins complex formation associated with histone type 2 H2aa3 up-regulation and intermediate filament reorganization may drive neoplastic transformation of GST-P positive foci during rat hepatocarcinogenesis leading to the formation of hepatocellular carcinomas.


title: Phospho-Ser(784)-VCP Is Required for DNA Damage Response and Is Associated with Poor Prognosis of Chemotherapy-Treated Breast Cancer

text: Spatiotemporal protein reorganization at DNA damage sites induced by genotoxic chemotherapies is crucial for DNA damage response (DDR), which influences treatment response by directing cancer cell fate. This process is orchestrated by valosin-containing protein (VCP), an AAA+ ATPase that extracts polyubiquinated chromatin proteins and facilitates their turnover. However, because of the essential and pleiotropic effects of VCP in global proteostasis, it remains challenging practically to understand and target its DDR-specific functions. We describe a DNA-damage-induced phosphorylation event (Ser(784)), which selectively enhances chromatin-associated protein degradation mediated by VCP and is required for DNA repair, signaling, and cell survival. These functional effects of Ser(784) phosphorylation on DDR correlate with a decrease in VCP association with chromatin, cofactors NPL4/UFD1, and polyubiquitinated substrates. Clinically, high phospho-Ser(784)-VCP levels are significantly associated with poor outcome among chemotherapy-treated breast cancer patients. Thus, Ser(784) phosphorylation is a DDR-specific enhancer of VCP function and a potential predictive biomarker for chemotherapy treatments.


Mass gathering-related mask use during 2009 pandemic influenza A (H1N1) and Middle East respiratory syndrome coronavirus


title: Serologic and Molecular Biologic Methods for SARS-associated Coronavirus Infection, Taiwan

text: Severe acute respiratory syndrome (SARS) has raised a global alert since March 2003. After its causative agent, SARS-associated coronavirus (SARS-CoV), was confirmed, laboratory methods, including virus isolation, reverse transcriptase–polymerase chain reaction (RT-PCR), and serologic methods, have been quickly developed. In this study, we evaluated four serologic tests ( neutralization test, enzyme-linked immunosorbent assay [ELISA], immunofluorescent assay [IFA], and immunochromatographic test [ICT]) for detecting antibodies to SARS-CoV in sera of 537 probable SARS case-patients with correlation to the RT-PCR . With the neutralization test as a reference method, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.2%, 98.7%, 98.7%, and 98.4% for ELISA; 99.1%, 87.8%, 88.1% and 99.1% for IFA; 33.6%, 98.2%, 95.7%, and 56.1% for ICT, respectively. We also compared the recombinant-based western blot with the whole virus–based IFA and ELISA; the data showed a high correlation between these methods, with an overall agreement of >90%. Our results provide a systematic analysis of serologic and molecular methods for evaluating SARS-CoV infection.


title: Biowaiver Monographs for Immediate Release Solid Oral Dosage Forms: Ribavirin

text: Abstract Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate release solid oral dosage forms containing ribavirin are reviewed. Ribavirin is highly soluble, but its permeability characteristics are not well defined. Therefore according to the Biopharmaceutical Classification System, and taking a “worst case” approach, ribavirin should be assigned to class III. As ribavirin is transported across the brush border membrane of the human jejunum by hCNT2, it shows saturable uptake in the intestine. However, no common excipients have been shown to compete for ribavirin absorption, nor have problems with BE of immediate release ribavirin formulations containing different excipients and produced by different manufacturing methods been reported in the open literature. So the risk of bioinequivalence caused by these factors appears to be low. Ribavirin is considered a narrow therapeutic index drug, as judged by comparing the minimum effective concentration and minimum toxic concentrations in blood. Although ribavirin would not be eligible for approval via a Biopharmaceutical Classification System–based biowaiver procedure according to today's guidances due to its narrow therapeutic index, the risks of biowaiving should be weighed against the considerable risks associated with studying BE of ribavirin products in healthy subjects.


title: The presence of co-infections in pigs with clinical signs of PMWS in The Netherlands: a case-control study

text: Abstract In this study, 60 pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) from 20 different pig herds and 180 control pigs (without clinical signs of PMWS) were examined to get more insights into the frequencies of porcine circovirus 2 infections and the presence of co-infections in pigs with and without clinical signs of PMWS in the Netherlands. Porcine circovirus type 2 was detected in 100% of the pigs with clinical signs of PMWS by virus isolation and/or PCR and in 50% of the pigs from PMWS-free herds. There was an association between the levels of infectious PCV2 and/or PCV2 DNA load and the severity of clinical signs as described for PMWS. A high variation in PCV2 antibody titres was found in the clinically affected pigs, and 27% of these pigs did not mount PCV2 antibody titres higher than 1:200. A concurrent infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) was found in at least 83% of the pigs with clinical signs of PMWS and in 35% of the pigs from PMWS-free herds. Co-infections of European- and American-type PRRSV were detected only in PMWS herds and in one control herd with a history of PMWS clinical signs.


title: Comparison of suspension MDCK cells, adherent MDCK cells, and LLC‐MK2 cells for selective isolation of influenza viruses to be used as vaccine seeds

text: BACKGROUND: Cell‐based influenza vaccines can solve the problem of the frequent occurrence of egg adaptation–associated antigenic changes observed in egg‐based vaccines. Seed viruses for cell‐based vaccines can be prepared from clinical specimens by cell culture; however, clinical samples risk harboring respiratory viruses other than influenza virus. Therefore, it is necessary to investigate the patterns of co‐infection in clinical samples and explore whether cell culture technology can selectively propagate influenza viruses from samples containing other respiratory viruses. METHODS: A total of 341 clinical specimens were collected from patients with influenza or influenza‐like illness and analyzed by ResPlex II assay to detect 18 respiratory viruses. The patterns of co‐infection were statistically analyzed with Fisher's exact test. The samples with double or triple infections were passaged in suspension MDCK cells (MDCK‐S), adherent MDCK cells (MDCK‐A), and LLC‐MK2D cells. Cell‐passaged samples were analyzed by ResPlex II assay again to investigate whether each cell line could amplify influenza viruses and eliminate other respiratory viruses. RESULTS: Double infections were detected in 8.5% and triple infections in 0.9% of the collected clinical specimens. We identified four pairs of viruses with significant correlation. For all samples with double and triple infection, MDCK‐S and MDCK‐A could selectively propagate influenza viruses, while eliminating all contaminating viruses. In contrast, LLC‐MK2D showed lower isolation efficiency for influenza virus and higher isolation efficiency for coxsackievirus/echovirus than MDCK‐S and MDCK‐A. CONCLUSIONS: Both MDCK‐S and MDCK‐A are considered suitable for the preparation of influenza vaccine seed viruses without adventitious agents or egg‐adaptation mutations.


Coronaphobia and chronophobia – A psychiatric perspective


title: Comparison of asymptomatic and symptomatic rhinovirus infections in university students: incidence, species diversity, and viral load

text: Abstract Human rhinovirus (HRV) infections are common but poorly characterized in university students. Thus, we characterized asymptomatic and symptomatic HRV infections by incidence, species diversity, and viral load of 502 university students during September and October of 2010 and 2011 from nasal swabs and electronically submitted symptom questionnaires. We tested all symptomatic students and randomly sampled participants who remained asymptomatic (n=25/week, over 8weeks each study year) on a weekly basis by real-time PCR and sequenced HRV positives. HRV was identified in 33/400 (8.3%) and 85/92 (92.4%) of the asymptomatic and symptomatic students, respectively. We identified a higher than previously reported rate of HRV-B in both groups, although the distribution of HRV species was similar (P =0.37). Asymptomatic viral load averaged 1.2 log10 copies/mL lower than symptomatic HRV (P <0.001). In conclusion, asymptomatic HRV activity preceded peak symptomatic activity in September and October and was associated with lower viral load.


title: Susceptibility of Rats of Different Ages to Inoculation with Swine Haemagglutinating Encephalomyelitis Virus (a Coronavirus) by Various Routes

text: Abstract Haemagglutinating encephalomyelitis virus, strain 67N, was used to inoculate 1-, 2-, 4- and 8-week-old rats by the intracerebral (i.c.), intranasal (i.n.), intraperitoneal (i.p.), subcutaneous (s.c.), intravenous (i.v.) and oral routes with graded doses. The routes of infection, in descending order of efficacy, were: i.ci.ns.ci.pi.v. and oral. Rats aged 1 and 2 weeks were generally similar in terms of mortality and mean time to death, regardless of inoculation route, except for the oral route, which had little effect. In comparison with the 1- and 2-week-old rats, the 4-week-old rats were less susceptible to the virus by all routes. Eight-week-old rats inoculated by the i.ci.n. or s.c. routes died, but all those inoculated by other routes survived. To follow the spread of virus in the central nervous system, 4-week-old rats inoculated by the i.c. route were examined. The virus was first detected in the brain on day 1 and in the spinal cord on day 2. The viral titres in both tissues reached a plateau of 107plaque-forming units (PFU)/0·2 g by day 4, at which time clinical signs had developed. By immunohistochemical analysis, virus-specific antigen was found first in the pyramidal cells of the hippocampus and cerebral cortex, and later in the large-sized neurons of the pons and spinal cord. Still later (day 4) immunolabelling was found in Purkinje cells of the cerebellum, but not in the ependymal cells, choroid plexus or other glial cells.


title: Expression of β-defensins in the canine respiratory tract and antimicrobial activity against Bordetella bronchiseptica

text: Abstract β-Defensins are cationic peptides which form part of the innate immune response of the respiratory epithelium. Due to their antimicrobial properties and immunostimulatory activity, β-defensins are potential tools for the treatment and prevention of respiratory disease. In dogs, infectious respiratory disease is a common problem, particularly in housed animals. This study aimed to assess the presence of four β-defensins in the canine respiratory tract and to use quantitative real-time PCR to determine mRNA levels following microbial challenge. Three β-defensins, CBD1, CBD103 and CBD108, were detected in respiratory cells. All three defensins were also readily expressed in skin samples, while their expression in lymphoid tissues and the kidney was low and inconsistent. Treatment of primary tracheal epithelial cells with lipopolysaccharide (LPS) or infection with canine respiratory coronavirus led to decreased expression of CBD103 and CBD108, while cells infected with canine parainfluenza virus had lower levels of CBD1 and CBD108. Furthermore CBD103 was demonstrated to have antimicrobial activity against the respiratory pathogen Bordetella bronchiseptica.


title: Coronavirus JHM-Induced Demyelinating Encephalomyelitis in Rats: Influence of Immunity on the Course of Disease

text: Publisher Summary Disease processes of the central nervous system (CNS) accompanied by demyelination may be the result of a viral infection or the consequence of an immunopathological reaction directed against myelin. In acute viral infections, the infection of oligodendroglial cells, leading to cell destruction, may be the main mechanism for inducing this neuropathological lesion. In the case of a persistent virus infection in oligodendroglia cells, however, it is conceivable that functional impairment of oligodendroglia cells, or the induction of an immune reaction to the agent that may cross-react with brain antigens, could eventually cause demyelination. Therefore, pathogenic studies on subacute or chronic demyelinating encephalomyelitides in association with viral infections may provide information on the mechanisms involved in demyelination. In rats, depending on the biological property of the virus material used, the genetic background and immune response of the host, a subacute or late demyelinating encephalomyelitis can be induced, accompanied by primary demyelination. This provides a model for analysis of the virus and host factors interacting in the pathogenesis of these diseases.


HIV and SARS-Coronavirus-2 Epidemics: Possible Interactions and Need for Studies, Especially in Africa


title: Viruses Associated With Pneumonia in Adults

text: Viral pneumonia, which is typically associated with disease in childhood, is increasingly recognized as causing problems in adults. Certain viruses, such as influenza virus, can attack fully immunocompetent adults, but many viruses take advantage of more-vulnerable patients. The latter include patients receiving immunosuppressive therapy and elderly subjects, particularly those residing in long-term care facilities. The range of viruses producing pneumonia in adults includes common agents, such as varicella-zoster virus and influenza virus, as well as respiratory syncytial virus, human metapneumovirus, adenoviruses, picornaviruses, and coronaviruses. The roles played by other agents, such as rhinoviruses and human bocaviruses, in pneumonia are still under study. While therapy for most of theses agents, at least in adults, has not yet been fully clarified, it is reasonable to assume antivirals may work in certain situations if they are introduced early enough in the course of infection.


title: Delegation im Krisenfall

text: Die dramatische Verbreitung des SARS-CoV-2/Covid-19-Virus (Corona-Virus) ist vor allem für Entscheidende eine große Herausforderung. Denn ihnen obliegt die Verantwortung für effiziente und zielgerichtete Prozesse ebenso wie für ein Personalmanagement, welches den Kriterien der Ausführbarkeit, Erträglichkeit, Zumutbarkeit und der Zufriedenheit entspricht. Vor dem Hintergrund eines komplexen Versorgungsauftrages, bei welchem die Notfall- sowie die Intensivmedizin eine Schlüsselrolle einnehmen, kann das beschriebene Delegationsmodell eine zielgerichtete Aufgabenverteilung unterstützen und eine Grundlage für Entscheidungen sein. Zusatzmaterial online: Zu diesem Beitrag sind unter 10.1007/s41906-020-0721-z für autorisierte Leser zusätzliche Dateien abrufbar.


Disnea y dolor torácico pleurítico durante la pandemia de la COVID-19


title: Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens

text: Abstract It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman®-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5′-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.


Answering to the Call of Critically Ill Patients: Limiting Sonographer Exposure to COVID-19 With Focused Protocols


title: Whole genome characterisation of quail deltacoronavirus detected in Poland

text: Quail deltacoronavirus (QdCoV) described for the first time in the United Arab Emirates in 2018 belongs to the same deltacoronavirus species as viruses discovered in swine and tree sparrows. The full-length genome of QdCoV detected in quails with enteritis in Poland has similar organization as Middle Eastern viruses although there is no NSP7c gene. The overall degree of nucleotide sequence identity was 92.4–92.6% between Polish PL/G032/2015 and Middle Eastern UAE-HKU30 QdCoV isolates. The sequences of the individual genes show similar nucleotide identities in the range of 91.4–94.7% with the exception of the S gene with lower identity of 85.6–85.7%. The most variable part of the S gene is its fragment encoding the N-terminal domain of the S protein which is responsible for receptor binding. The amino acid homology in this region between PL/G032/2015 and UAE-HKU30 QdCoVs was 74.5–74.7%. In contrast, the C-terminal domain of the S protein which is responsible for membrane fusion had an amino acid homology of 96.9%. In the phylogenetic tree, PL/G032/2015 branched separately but clustered with the UAE-HKU30 QdCoV isolates. These data suggest that PL/G032/2015 could be a new genetic/serologic variant of QdCoV. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11262-019-01639-1) contains supplementary material, which is available to authorized users.


title: Challenges in the conduct of large simple trials of important generic questions in resource-poor settings: The CREATE and ECLA trial program evaluating GIK (glucose, insulin and potassium) and low-molecular-weight heparin in acute myocardial infarction

text: Abstract Background Approximately 15.5 million deaths from cardiovascular diseases occur every year. About half are due to acute myocardial infarction (AMI), and 80% occur in low- and middle-income countries. Therefore, low-cost therapies would be invaluable. Although glucose-insulin-potassium (GIK) infusion and low-molecular-weight heparin (LMWH) appear to be promising in AMI, the available trials are inconclusive and these treatments require rigorous evaluation. Methods The Clinical Trial of Reviparin and Metabolic Modulation in Acute Myocardial Infarction Treatment and Evaluation-Estudios Clínicos Latino America (CREATE-ECLA) study is a randomized controlled trial in ST-elevation AMI patients evaluating a 24-hour infusion of Glucose-Insulin-Potassium (GIK) intravenous vs usual care (control) on 30-day mortality in 20 000 patients from 21 countries. Patients from India and China (n = 15 000) are also randomized using a factorial design to receive low-molecular-weight heparin (Reviparin) or placebo injection twice daily for 7 days to assess the impact on the composite outcomes of death, reinfarction or stroke (first co-primary outcome) or the composite + refractory ischemia (second co-primary outcome). Results Twenty thousand two hundred and one (20,201) GIK/control patients and 15,570 Reviparin/placebo patients have been included, with results expected in November 2004. Conclusions The CREATE-ECLA trial will provide definitive answers to the role of 2 practical, promising and low-cost therapies, LMWH and GIK, in AMI patients. If effective, these therapies could be used in small medical centers in low- and middle- income countries. The experiences in this trial indicate that large trials of important questions can be successfully conducted in resource-poor settings, by academic groups without industry involvement.


title: A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

text: Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF)-alpha through p38 mitogen activated protein kinase (MAPK). However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1) and protein phosphatase type 2A (PP2A) in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac) infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.


title: Effective inhibition of porcine transmissible gastroenteritis virus replication in ST cells by shRNAs targeting RNA-dependent RNA polymerase gene

text: Abstract Transmissible gastroenteritis virus (TGEV) is identified as one of the most important pathogenic agents during swine enteric infection, leading to high mortality in neonatal pigs and severe annual economic loss in swine-producing areas. Up to date, various vaccines developed against TGEV still need to be improved. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, two shRNA-expressing plasmids (pEGFP-U6/P1 and pEGFP-U6/P2) targeting the RNA-dependent RNA polymerase (RdRp) gene of TGEV were constructed and transfected into swine testicular (ST) cells. The cytopathic effect (CPE) and MTS assays demonstrated that both shRNAs were capable of protecting cells against TGEV invasion with very high specificity and efficiency. A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the two plasmids were reduced by 95.2% and up to 100%, respectively. Our results suggest that RNAi might be a promising new strategy against TGEV infection.


title: Angiotensin Converting Enzyme 2 May Mediate Disease Severity In COVID-19

text: Identification of vulnerability to severe coronavirus disease 2019 (COVID-19) is extremely important and might allow optimised shielding and easing of lockdown. The disease is attributed to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which enters host cells through binding to angiotensin converting enzyme 2 (ACE2) on the cell surface. Clinical syndromes such as hypertension that display reduced ACE2 expression tend to correlate with a more severe disease course, whereas treatments which upregulate ACE2 such as the use of angiotensin converting enzyme inhibitors (ACE-i) appear to have a protective effect against COVID-19. Pre-clinical studies have shown that plasma soluble ACE2 could render SARS-CoV-2 inactive in a dose-dependent manner. The association of clinical syndromes or treatments that impact ACE2 expression and clinical severity of COVID-19 infection combined with the reduction in viral load with human recombinant serum ACE2 shown in pre-clinical studies indicate a key role for ACE2 in determining COVID-19 severity. In conclusion, we propose that measurement of ACE2 level may help identify individuals at risk of severe infection where targeted shielding can be used and could provide a novel therapeutic target.


title: Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs

text: Abstract Two genotype-specific fluorogenic RT-PCR assays were developed for the detection and quantitation of canine coronavirus (CCoV) type I and type II RNA in the faeces of dogs with diarrhoea. Both the fluorogenic assays showed high specificity, sensitivity and reproducibility, allowing a precise quantitation of CCoV type I and type II RNA over a linear range of about eight orders of magnitude (from 101 to 108 copies of standard RNA). Comparison with genotype-specific gel-based RT-PCR assays revealed that the fluorogenic assays were more sensitive and more rapid than conventional amplifications, with a large increase in throughput. The genotype-specific fluorogenic assays were then used to detect and measure viral loads in the faecal samples collected from dogs naturally or experimentally infected with type I, type II, or both genotypes. Of 174 samples collected from naturally infected dogs, 77 were positive for CCoV type I and 46 for CCoV type II. Thirty-eight dogs were found to be infected naturally by both genotypes, with viral RNA titres generally higher for type I in comparison to type II. At the same time, dogs infected experimentally shed type I RNA with higher titres with respect to type II.


title: A method for evaluating health care workers’ personal protective equipment technique

text: BACKGROUND: Given the potential for the transfer of infectious diseases among patients in isolation, health care workers (HCWs), and other patients in the hospital environment, the proper use of personal protective equipment (PPE) is paramount. The literature is limited regarding studies of HCWs’ use of PPE in patient care tasks. METHODS: A pilot study was conducted to examine the feasibility of using a simulated health care environment to assess HCWs’ technique when implementing standard airborne and contact isolation precautions. The participants (n = 10) were assigned patient care tasks based on their specific professional roles. The encounters were digitally recorded during donning and doffing of PPE, as well as during interactions with the simulated patient. Powdered fluorescent marker was used as a measure of contamination. RESULTS: The pilot data show various inconsistencies in the HCWs’ PPE technique. Each of the 10 participants committed at least one breach of standard airborne and contact isolation precautions. CONCLUSION: An expanded research study of HCW behaviors is needed to properly examine these contamination and exposure pathways. Training programs should be developed that emphasize the common errors in HCWs’ PPE technique.


title: Responses of three urban U.S. children’s hospitals to COVID-19: Seattle, new York and new orleans

text: Since January 2020, there has been a worldwide pandemic of COVID-19, caused by a novel coronavirus--severe acute respiratory syndrome coronavirus 2. The United States has been particularly affected, with the largest number of confirmed cases in a single country in the world. Healthcare systems for adults as well as children have dealt with challenges. This article will reflect on the experiences of selected children’s hospitals in Seattle, New York City, and New Orleans, three of the “hotspots” in the US and share common aspects and lessons learned from these experiences. This article discusses testing and cohorting of patients, personal protective equipment utilization, limiting workplace exposure, and information sharing.


title: A novel delivery system for supraglottic atomization allows increased lung deposition rates of pulmonary surfactant in newborn piglets

text: BACKGROUND: Earlier attempts to deliver effective lung doses of surfactant by aerosolization were unsuccessful, mostly because of technical shortcomings. We aimed at quantifying the lung deposition of poractant alfa with a new supraglottic delivery system for surfactant atomization in an experimental neonatal model. METHODS: The method involved six sedated 1-day-old piglets lying in the lateral decubitus, spontaneously breathing on nasal-mask continuous positive airway pressure (nCPAP). A pharyngeal cannula housing a multi-channel air-blasting atomization catheter was placed through the mouth with its tip above the glottis entrance. In all, 200 mg kg(−1) of a (99m)Tc-surfactant mixture was atomized through the catheter synchronously with inspiration. Six intubated control piglets received an equal amount of intratracheally instilled (99m)Tc-surfactant mixture. The percentage of the (99m)Tc-surfactant mixture deposited in the lungs was estimated by scintigraphy. RESULTS: Median (range) deposition in the lungs was 40% (24–68%) after atomization and 87% (55–95%) after instillation (p < 0.001). Overall, almost 80% of the deposited surfactant was in the dependent lung. Effective atomization time (atomizer on) was 28 (17–52) min, yielding an output rate of 0.1–0.2 mL min(−1). CONCLUSIONS: Without endotracheal intubation, in spontaneously breathing newborn piglets, this new supraglottic atomizer delivery system attained a median lung deposition of 40% of the nominal dose of surfactant.


The COVID-19 pandemic and its impact on addiction treatment


title: Who shares? Profiling consumers in the sharing economy

text: Abstract Sharing platforms are becoming increasingly common, transforming how organisations and customers interact across diverse categories. While there is clear demand for the sharing economy, less is known about heterogeneity of consumer preferences and the varying demand that exists for sharing experiences across different categories of consumption. In order to help brands better understand who shares, this research takes a step forward in the profiling of users of the sharing economy. Drawing on social psychology, this research investigates how social norms can be employed as a form of social influence and nudge consumers to engage in higher levels of shared consumption. We find three clear segments of sharing consumers, representing 86% of all consumers: the mobility-focused sharer, the diverse-platform sharer, and the power-platform sharer. The last segment (accounting for 14%) comprises consumers who do not engage with sharing platforms. Moreover, social norms influenced the future behaviours of only one segment of consumers: the diverse-platform sharer. We discuss how sharing platform providers can better understand, target, and convert consumers to engage in sharing.


title: Putting Synthesis into Biology: A Viral View of Genetic Engineering through De Novo Gene and Genome Synthesis

text: Summary The rapid improvements in DNA synthesis technology hold the potential to revolutionize biosciences in the near future. Traditional genetic engineering methods are template dependent and make extensive but laborious use of site-directed mutagenesis to explore the impact of small variations on an existing sequence “theme.” De novo gene and genome synthesis frees the investigator from the restrictions of the pre-existing template and allows for the rational design of any conceivable new sequence theme. Viruses, being among the simplest replicating entities, have been at the forefront of the advancing biosciences since the dawn of molecular biology. Viral genomes, especially those of RNA viruses, are relatively short, often less than 10,000 bases long, making them amenable to whole genome synthesis with the currently available technology. For this reason viruses are once again poised to lead the way in the budding field of synthetic biology—for better or worse.


title: A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

text: Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-κB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems. DOI: http://dx.doi.org/10.7554/eLife.02910.001


title: Antibody-mediated protection against MERS-CoV in the murine model

text: Abstract Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. The murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the MERS coronavirus (MERS-CoV). To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. Subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the MERS-CoV resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. The murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of RBD with a human IgG Fc tag (RBD-Fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. The data gained indicate that this dual-route vaccination with novel formulations of the RBD-Fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV.


title: Newly emerging C group enteroviruses may elude diagnosis due to a divergent 5′-UTR

text: Summary Human enterovirus (HEV) 105 was first reported in 2012 in children from Peru and Congo. We report on the identification of a novel HEV-C105 strain in a pediatric patient in Cyprus with an upper respiratory tract infection. Sequence alignment and phylogenetic analysis of 5′-UTRs of all known HEVs revealed that our isolate belongs to a group of recently identified HEV-C viruses exhibiting a 5′-UTR distinct from all other previously known enteroviruses. This has important implications for diagnosis, as this region is the primary target for diagnostic assays. Increased awareness in laboratories may thus increase the rate of detection of enteroviruses belonging to this subspecies, or lead to the discovery of further genotypes.


title: Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene

text: Abstract The highly contagious and deadly porcine epidemic diarrhea virus (PEDV) first appeared in the US in April 2013. Since then the virus has spread rapidly nationwide and to Canada and Mexico causing high mortality among nursing piglets and significant economic losses. Currently there are no efficacious preventive measures or therapeutic tools to control PEDV in the US. The isolation of PEDV in cell culture is the first step toward the development of an attenuated vaccine, to study the biology of PEDV and to develop in vitro PEDV immunoassays, inactivation assays and screen for PEDV antivirals. In this study, nine of 88 US PEDV strains were isolated successfully on Vero cells with supplemental trypsin and subjected to genomic sequence analysis. They differed genetically mainly in the N-terminal S protein region as follows: (1) strains (n =7) similar to the highly virulent US PEDV strains; (2) one similar to the reportedly US S INDEL PEDV strain; and (3) one novel strain most closely related to highly virulent US PEDV strains, but with a large (197aa) deletion in the S protein. Representative strains of these three genetic groups were passaged serially and grew to titers of ∼5–6log10 plaque forming units/mL. To our knowledge, this is the first report of the isolation in cell culture of an S INDEL PEDV strain and a PEDV strain with a large (197aa) deletion in the S protein. We also designed primer sets to detect these genetically diverse US PEDV strains.


title: Prototype of a Smart Microfluidic Platform for the Evaluation of SARS-Cov-2 Pathogenesis, Along with Estimation of the Effectiveness of Potential Drug Candidates and Antigen–Antibody Interactions in Convalescent Plasma Therapy

text: Originating in China during December 2019, the novel corona-virus, SARS-CoV-2, has created mayhem worldwide in a very short time. The outbreak has been so rapid and widespread that the only option to treat the patients was administering drugs already available in the market like chloroquine/hydroxychloroquine (an antimalarial drug) and remedesivir. A large number of patients have been cured but the attribution to survival by these drugs has been controversial. Till date, we do not have any specific drug or vaccine available for COVID-19 and the pandemic seems to be far from over. To handle the current challenges posed by the outbreak effectively, we need to employ innovative interdisciplinary approaches. Organ-on-chip (OOC), particularly lung-on-chip, is one such approach which combines the potential of microfluidics, cell culture and molecular biology into a single miniaturised platform. The device is realized to be capable of simulating in-vivo physiological responses of an organ. In the current study, an OOC, which is a multichannel 3D cell culture microfluidic device, is made via soft lithography technique, using polydimethylsiloxane-polymer and diverse polymeric porous/semipermeable membranes. Several polymer membranes i.e. PDMS, polyvinylidene fluoride (PVDF), nitrocellulose, polyester etc., integrated into the microdevices, were efficiently explored to realize their better cell-adhesion and viability property. We also propose for the application of a simple, smart and cost-effective lung-on-chip platform to study the SARS-CoV-2 pathogenesis in humans, drug toxicity testing and provide insights into antigen–antibody interactions. This platform will enable us to study multiple phenomena at a micro-level generating more reliable data and a better understanding of the underlying mechanisms of SARS-CoV-2 infection and pathogenesis.


title: A Brief Review: The Z-curve Theory and its Application in Genome Analysis

text: 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.


title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods

text: Abstract Virome (viral megagenomics) detection using next generation sequencing has been widely applied in virology, but its methods remain complicated and need optimization. In this study, we detected the viromes of RNA viruses of one mock sample, one pooled duck feces sample and one pooled mink feces sample on the Personal Genome Machine platform using the sequencing libraries prepared by three methods. The sequencing primers were added through random hybridization and ligation to fragmented viral RNA using a RNA-Seq kit in method 1, through random reverse transcription (RT) and polymerase chain reaction (PCR) in method 2 which was developed in our laboratory, and through hybridization and ligation to fragmented amplicons of random RT-PCR using a single primer in method 3. Although the results of these three samples (nine libraries) all showed that more classified viral families and genera were identified using methods 2 and 3 than using method 1, and more classified viral families and genera were identified using method 2 than using method 3, most of the differences were of no statistical significance. Moreover, 11 mammalian viral genera in minks were possibly identified for the first time through this study.


title: The major subunit ClpG of Escherichia coli CS31A fibrillae as an expression vector for different combinations of two TGEV coronavirus epitopes

text: Abstract Previously, two B-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (TGEV), namely the C epitope (TGEV-C) amino acids (aa) 363–371 and the A epitope (TGEV-A) aa 522–531 of the spike S protein (TGEV-S), have been separately expressed on the CS31A fibrillae at the surface of Escherichia coli following insertion into a same region of ClpG. However, the resulting chimeras induced a marginal TGEV-neutralizing antibody (Ab) response in mice. Here, with the view to improving this response, we introduced TGEV-C alone or in different tandem association with TGEV-A (A::C or C::A) in twelve putatively exposed regions of ClpG. Among the 28 resulting engineered proteins only 15, carrying up to 51 extra aa, had not essentially disturbed the correct CS31A fibrillae formation process. Six partially permissive sites accepting only TGEV-C and three highly permissive sites tolerating A::C or C::A tandem peptide, were identified throughout ClpG. Intact bacteria or extracted CS31A hybrid fibrillae expressing TGEV epitopes at any of the permissive sites, were recognized by Ab directed against the foreign parent protein, providing a direct argument for exposure of the corresponding ClpG region at the cell surface and for antigenicity of the epitopes in the polymeric CS31A fibrillae context. The potential of CS31A fibrillae as carriers of the TGEV peptides indicates that there may be three positions (N terminus, aa 202–204 and 202–218) in ClpG which may turn out to be important fusion sites and therefore be relevant for the eventual design of TGEV vaccines. Unexpectedly, TGEV-A, whatever its position in ClpG, mediated the partial proteolytic degradation of the hybrid proteins, suggesting that it functions as a substrate for a cellular protease, and thereby that its suitability as a vaccine antigen candidate is doubtful.


title: China’s ongoing battle against the coronavirus: a scholar-practitioner's experiences and reflections

text: In this communication essay, I respond to the three questions the journal editor Wei-Ning Xiang asked about my experiences in, and reflections on, the ongoing battle against the coronavirus pandemic in China. At this critical juncture when the people in China are progressing on the battleground, I dedicate the article to all the people fighting against the coronavirus around the world.


title: An explorative study on coping flexibility with behavioral approach system-activating stimuli: A comparison of people with and without bipolar disorder

text: Abstract Life events play a significant role in affecting mood symptoms of people with bipolar disorder (BD). However, we lack empirical data about the associations among disorder, mood state, behavioral activation system (BAS) sensitivity, and psychosocial functioning level. Thus, the present study aimed to identify the role of coping flexibility with BAS activating stimuli in relation to mood states among a sample of individuals with BD (n = 90) and a healthy control group (n = 90). Through multiple regressions, the moderating role of coping flexibility was determined. Findings showed that coping flexibility had an additional value in predicting mood states beyond BAS sensitivity and psychosocial functioning level. Specifically, perceived controllability was considerably important for the BD group, whereas fit index was crucial in the controls. In addition, a moderation analysis showed that perceived controllability alleviated the effects of BD diagnosis, BAS sensitivity, and psychosocial functioning level on mood states. Theoretically, this study helps integrate the concept of coping flexibility into the BAS dysregulation theory as it applies to BD. The practical implication for enhancing mindfulness practice is also discussed.


title: Autophagy in the regulation of pathogen replication and adaptive immunity

text: Autophagy is an evolutionary conserved homeostatic process by which cells deliver cytoplasmic material for degradation into lysosomes. Autophagy may have evolved as a nutrient-providing homeostatic pathway induced upon starvation, but with the acquisition of cargo-receptors autophagy has become an important cellular defence mechanism as well as a generator of antigenic peptides for MHC presentation. We propose that autophagy efficiently protects against microbes encountering the cytosolic environment accidentally, for example upon phagosomal damage, while pathogens routinely accessing the host cytosol have evolved to avoid or even benefit from autophagy.


title: Tropism and pathological findings associated with canine respiratory coronavirus (CRCoV)

text: Abstract Canine infectious respiratory disease (CIRD) occurs frequently in densely housed dog populations. One of the common pathogens involved is canine respiratory coronavirus (CRCoV), however little is known regarding its pathogenesis and the role it plays in the development of CIRD. The pathogenesis of five geographically unrelated canine respiratory coronavirus (CRCoV) isolates was investigated. Following experimental infection in dogs, all five CRCoV isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. The presence of CRCoV was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. Viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. The successful re-isolation of CRCoV from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of CRCoV for respiratory tissues and fulfils the final requirement for Koch's postulates. By study day 14 dogs had seroconverted to CRCoV and the antibodies raised were neutralising against both homologous and heterologous strains of CRCoV in vitro, thus demonstrating antigenic homogeneity among CRCoV strains from the two continents. Defining the role that CRCoV and other agents play in CIRD is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. Here we have successfully developed a model for studying the pathogenicity and the role of CRCoV in CIRD.


title: Features and drivers for energy-related carbon emissions in mega city: The case of Guangzhou, China based on an extended LMDI model

text: Based on the apparent energy consumption data, a systematic and comprehensive city-level total carbon accounting approach was established and applied in Guangzhou, China. A newly extended LMDI method based on the Kaya identity was adopted to examine the main drivers for the carbon emissions increments both at the industrial sector and the residential sector. Research results are listed as follow: (1) Carbon emissions embodied in the imported electricity played a significant important role in emissions mitigation in Guangzhou. (2) The influences and impacts of various driving factors on industrial and residential carbon emissions are different in the three different development periods, namely, the 10(th) five-year plan period (2003–2005), the 11(th) five-year plan period (2005–2010), and the 12(th) five-year plan period (2010–2013). The main reasons underlying these influencing mechanisms were different policy measures announced by the central and local government during the different five-year plan periods. (3) The affluence effect (g-effect) was the dominant positive effect in driving emissions increase, while the energy intensity effect of production (e-effect-Production), the economic structure effect (s-effect) and the carbon intensity effect of production (f-effect-Production) were the main contributing factors suppressing emissions growth at the industrial sector. (4) The affluence effect of urban (g-effect-AUI) was the most dominant positive driving factor on emissions increment, while the energy intensity effect of urban (e-effect-Urban) played the most important role in curbing emissions growth at the residential sector.


title: Fever with Rash in COVID‐19: Viral Exanthema or Secondary Lesions?

text: COVID‐19 originated in Wuhan, China and has developed into a pandemic since late 2019. The virus possesses powerful pathogenicity as well as transmissibility. Many open questions remain, including the description of potential involvement of other organs than the respiratory tract. Most patients have mild influenza‐like symptoms. A minority, especially patients with chronic lung disease, develops lethal disease including severe pneumonia, pulmonary edema, rapidly developing acute respiratory distress syndrome (ARDS), multiple organ failure and septic shock.


title: Diagnostic Testing for SARS-Coronavirus-2 in the Nursing Facility: Recommendations of a Delphi Panel of Long-Term Care Clinicians

text: With the COVID-19 pandemic progressing, guidance on strategies to mitigate its devastating effects in nursing facilities (NFs) is critical to preventing additional tragic outcomes. Asymptomatic spread of COVID-19 from nursing facility staff and residents is a major accelerator of infection. Facility-wide point-prevalence testing is an emerging strategy in disease mitigation. Because time is not available to await the results of randomized controlled trials before implementing strategies in this high-risk setting, an expert Delphi panel composed of experienced long-term care medicine professionals has now met to provide testing guidance for SARS-Coronavirus-2 to NFs. After many email and telephone discussions, the panel responded to a questionnaire that included six different scenarios, based on varying availability of Polymerase Chain Reaction (RT-PCR) testing and personal protective equipment (PPE). The panel endorsed facility-wide testing of staff and residents without dissent when diagnostic RT-PCR was available. While the panel recognized the limitations of RT-PCR testing, it strongly recommended this testing for both staff and residents in NFs that were either COVID-19 naive or had limited outbreaks. There was also consensus on testing residents with atypical symptoms in a scenario of limited testing capability. The panel favored testing every 1 to 2 weeks if testing was readily available, reducing the frequency to every month as community prevalence declined or as the collection of additional data further informed clinical critical thinking and decision-making. The panel recognized that frequent testing would have consequences in terms of potential staff shortages due to quarantine after positive tests and increased PPE use. However, the panel felt that not testing would allow new clusters of infection to form. The resulting high mortality rate would outweigh the potential negative consequences of testing. The panel also recognized the pandemic as a rapidly evolving crisis, and that new science and increasing experience might require an updating of its recommendations. The panel hopes that its recommendations will be of value to the long-term care industry and to policy makers as we work together to manage through this challenging and stressful time.


title: Telestroke in the Time of COVID-19: The Mayo Clinic Experience

text: Abstract On March 11, 2020, the World Health Organization declared the coronavirus disease 2019 (COVID-19) a pandemic, and in the weeks following, public health organizations, medical associations, and governing bodies throughout the world recommended limiting contact with others to “flatten the curve” of COVID-19. Although both ischemic and hemorrhagic strokes have been reported with COVID-19, there has been anecdotal suggestion of an overall decrease in stroke admissions. To date, the effects of any pandemic on telestroke service lines have not been described. The purpose of this cross-sectional analysis of telestroke activations in the 30 days before and after the declaration of the COVID-19 pandemic is to describe the difference in case volumes of telestroke activations, the characteristics of patients, and treatment recommendations between the two time frames. We found a 50% reduction in total telestroke activations between the pre- and post- groups, 142 and 71, respectively. There were no statistically significant differences in age, sex, diagnosis, or regional variations in activation volumes. The percentage of patients in whom we recommended acute stroke treatment with intravenous alteplase, mechanical thrombectomy, or both, decreased from 44% to 33%. The reasons for the significant decrease in telestroke activations and acute stroke treatment recommendations are likely multifactorial, regardless, underscoring the importance of continued public health measures to encourage patients and families to seek emergent medical care at the time of symptom onset.


title: Molecular Alterations Prompted by SARS-CoV-2 Infection: Induction of Hyaluronan, Glycosaminoglycan and Mucopolysaccharide Metabolism

text: Abstract Background The SARS-CoV-2 is the etiological agent causing COVID-19 which has infected more than 2 million people with more than 200000 deaths since its emergence in December 2019. In the majority of cases patients are either asymptomatic or show mild to moderate symptoms and signs of a common cold. A subset of patients, however, develop a severe atypical pneumonia, with the characteristic ground-glass appearance on chest x-ray and computerized tomography, which evolves into an acute respiratory distress syndrome, that requires mechanical ventilation and eventually results in multiple organ failure and death. The Molecular pathogenesis of COVID-19 is still unknown. Aim of the study In the present work we performed a stringent metanalysis from the publicly available RNAseq data from bronchoalveolar cells and peripheral blood mononuclear cells to elucidate molecular alterations and cellular deconvolution to identify immune cell profiles. Results Alterations in genes involved in hyaluronan, glycosaminoglycan and mucopolysaccharides metabolism were over-represented in bronchoalveolar cells infected by SARS-CoV-2, as well as potential lung infiltration with neutrophils, NK cells, T CD4+ cell and macrophages. The blood mononuclear cells presented a proliferative state. Dramatic reduction of neutrophils, NK and T lymphocytes, whereas an exacerbated increase in monocytes. Conclusions In summary our results revealed molecular pathogenesis of the SARS-CoV-2 infection to bronchoalveolar cells inducing the hyaluronan and glycosaminoglycan metabolism that could shape partially the components of the ground-glass opacities observed in CT. And the potential immune response profile in COVID-19.


title: Breaking the Enigma Code of Angiotensin II Type 2 Receptor Signaling

text: In this issue of Structure, Asada et al. (2019) present angiotensin receptor AT2R structure in complex with its main endogenous agonist, AngII peptide. Complementing the previous structural studies, the new complex structure sheds light on the AT2R activation mechanism and opens new avenues for drug discovery targeting this enigmatic receptor.


title: Murine coronaviruses: the histopathology of disease induced by intranasal inoculation

text: Mice inoculated intranasally with murine coronaviruses (mouse hepatitus viruses) were killed daily for seven days. Lung and liver sections stained by the immunopcroxidasc technique indicated that with three of the four strains examined viral localisation and replication in the lung preceded that found in (he liver. Thus, infection by the respiratory route may be of importance in the Iransmission of these viruses.


title: Heparanase, cell signaling, and viral infections

text: Heparanase (HPSE) is a multifunctional protein endowed with many non-enzymatic functions and a unique enzymatic activity as an endo-β-d-glucuronidase. The latter allows it to serve as a key modulator of extracellular matrix (ECM) via a well-regulated cleavage of heparan sulfate side chains of proteoglycans at cell surfaces. The cleavage and associated changes at the ECM cause release of multiple signaling molecules with important cellular and pathological functions. New and emerging data suggest that both enzymatic as well as non-enzymatic functions of HPSE are important for health and illnesses including viral infections and virally induced cancers. This review summarizes recent findings on the roles of HPSE in activation, inhibition, or bioavailability of key signaling molecules such as AKT, VEGF, MAPK–ERK, and EGFR, which are known regulators of common viral infections in immune and non-immune cell types. Altogether, our review provides a unique overview of HPSE in cell-survival signaling pathways and how they relate to viral infections.


Crops from U.S. food supply chains will never look nor taste the same again


title: Frontline nurses’ burnout, anxiety, depression, and fear statuses and their associated factors during the COVID-19 outbreak in Wuhan, China: A large-scale cross-sectional study

text: BACKGROUND: During the Coronavirus Disease 2019 (COVID-19) pandemic, frontline nurses face enormous mental health challenges. Epidemiological data on the mental health statuses of frontline nurses are still limited. The aim of this study was to examine mental health (burnout, anxiety, depression, and fear) and their associated factors among frontline nurses who were caring for COVID-19 patients in Wuhan, China. METHODS: A large-scale cross-sectional, descriptive, correlational study design was used. A total of 2,014 eligible frontline nurses from two hospitals in Wuhan, China, participated in the study. Besides sociodemographic and background data, a set of valid and reliable instruments were used to measure outcomes of burnout, anxiety, depression, fear, skin lesion, self-efficacy, resilience, and social support via the online survey in February 2020. FINDINGS: On average, the participants had a moderate level of burnout and a high level of fear. About half of the nurses reported moderate and high work burnout, as shown in emotional exhaustion (n = 1,218, 60.5%), depersonalization (n = 853, 42.3%), and personal accomplishment (n = 1,219, 60.6%). The findings showed that 288 (14.3%), 217 (10.7%), and 1,837 (91.2%) nurses reported moderate and high levels of anxiety, depression, and fear, respectively. The majority of the nurses (n = 1,910, 94.8%) had one or more skin lesions, and 1,950 (96.8%) nurses expressed their frontline work willingness. Mental health outcomes were statistically positively correlated with skin lesion and negatively correlated with self-efficacy, resilience, social support, and frontline work willingness. INTERPRETATION: The frontline nurses experienced a variety of mental health challenges, especially burnout and fear, which warrant attention and support from policymakers. Future interventions at the national and organisational levels are needed to improve mental health during this pandemic by preventing and managing skin lesions, building self-efficacy and resilience, providing sufficient social support, and ensuring frontline work willingness.


title: VII Digestive System 1

text: Publisher Summary This chapter deals with the digestive system. The major and minor salivary glands and their secretions also represent and integral part of the protective mechanism of the oral cavity, and derangement of saliva production may lead to loss of integrity of the oral mucosa. Drug-induced abnormalities of taste sensation are also well-described phenomena occurring in man although human studies are necessary for the detection of these effects. Inflammation of the oral cavity may involve the buccal mucosa, the gingiva (gingivitis), the tongue (glossitis), and the peridontal tissues (peridontitis). Therapeutic agents can induce inflammatory lesions in the tongue. Moreover, a protective layer of mucus, a visco-elastic material containing high molecular weight glycoproteins produced by the major and minor salivary glands, covers the stratified squamous mucosa of the oral cavity. Salivary secretions also possess digestive enzyme activity although in herbivores and carnivores, it is usually low in contrast to high digestive enzyme activity in omnivorous species.


title: Genome Signatures, Self-Organizing Maps and Higher Order Phylogenies: A Parametric Analysis

text: Genome signatures are data vectors derived from the compositional statistics of DNA. The self-organizing map (SOM) is a neural network method for the conceptualisation of relationships within complex data, such as genome signatures. The various parameters of the SOM training phase are investigated for their effect on the accuracy of the resulting output map. It is concluded that larger SOMs, as well as taking longer to train, are less sensitive in phylogenetic classification of unknown DNA sequences. However, where a classification can be made, a larger SOM is more accurate. Increasing the number of iterations in the training phase of the SOM only slightly increases accuracy, without improving sensitivity. The optimal length of the DNA sequence k-mer from which the genome signature should be derived is 4 or 5, but shorter values are almost as effective. In general, these results indicate that small, rapidly trained SOMs are generally as good as larger, longer trained ones for the analysis of genome signatures. These results may also be more generally applicable to the use of SOMs for other complex data sets, such as microarray data.


title: Development of a rapid test kit for SARS-CoV-2: an example of product design

text: We present an example of applying ‘need-driven’ product design principle to the development of a rapid test kit to detect SARS-COV-2 (COVID-19). The tests are intended for use in the field and, longer term, for home use. They detect whether a subject is currently infected with the virus and is infectious. The urgent need for large numbers of tests in field setting imposes constraints such as short test time and lack of access to specialist equipment, laboratories and skilled technicians to perform the test and interpret results. To meet these needs, an antigen test based on RT-LAMP with colorimetric readout was chosen. Direct use of swab sample with no RNA extraction was explored. After extensive experimental study (reported elsewhere), a rapid test kit has been fabricated to satisfy all design criteria.


title: A possible role for B cells in COVID-19?: Lesson from patients with Agammaglobulinemia

text: Summary COVID-19 had a mild clinical course in patients with Agammaglobulinemia lacking B lymphocytes, whereas it developed aggressively in Common Variable Immune Deficiency. Our data offer mechanisms for possible therapeutic targets.


title: MHV-A59 Enters Polarized Murine Epithelial Cells through the Apical Surface but Is Released Basolaterally

text: Abstract Coronaviruses have a marked tropism for epithelial cells. Entry and release of the porcine transmissible gastroenteritis virus (TGEV) is restricted to apical surfaces of polarized epithelial cells, as we have recently shown (J. W. A. Rossen, C. P. J. Bekker, W. F. Voorhout, G. J. A. M. Strous, A. van der Ende, and P. J. M. Rottier, 1994, J. Virol. 68, 7966-7973). In this paper we analyze the interactions of mouse hepatitis coronavirus A59 (MHV-A59) with polarized murine kidney cells (mTAL) grown on permeable supports. After inoculation from the apical or basolateral side, virus entry was found to take place only through the apical membrane. The virus utilized a protein of the carcinoembryonic antigen family as its receptor. In contrast to TGEV, MHV-A59 was released preferentially from the basolateral plasma membrane domain, as evidenced by the accumulation of viral proteins and infectivity in the basolateral culture fluid as well as by electron microscopical observations in the mouse, MHV initially replicates in the nasal epithelium before being disseminated throughout the body; the basolateral release of MHV from epithelial cells into the animal's circulation may be the first step in the establishment of a systemic infection.


Virtual clinics: Need of the hour, a way forward in the future. Adapting practice during a healthcare crisis.


title: SARS-CoV-2 screening test for Japanese returnees from Wuhan, China, January 2020

text: BACKGROUND: Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) was found to be the causative microorganism of coronavirus disease 2019 (COVID-19), which started to spread in Wuhan, China. This study was to evaluate the effectiveness of questionnaire, symptoms-based screening, and PCR screening of returnees from COVID-19 endemic areas on a chartered flight, to examine the proportion of infected persons and the proportion of asymptomatic persons among infected persons who returned from Wuhan. METHODS: A retrospective cohort study was done in seven tertiary medical institutions in Japan. A total of 566 Japanese who returned from Wuhan participated in the study. RESULTS: Overall, 11 of the 566 passengers had a positive SARS-CoV-2 PCR result for pharyngeal swabs and six were asymptomatic. Only fever differed between SARS-CoV-2-positive and -negative individuals (p < 0.043). Six of the eleven PCR-positive individuals were asymptomatic and four remained positive on day 10 and One asymptomatic person tested positive up to day 27. Two of the eleven were negative on the first PCR test and positive on the second. CONCLUSIONS: Our results will be important insights on screening returnees from locked down cities, as well as important data on the proportion of asymptomatic individuals infected with SARS-CoV-2. A 13-day observation period and a second round of PCR may be effective to screen patients, including asymptomatic infections.


title: Knowledge, attitudes and practices towards pandemic influenza among cases, close contacts, and healthcare workers in tropical Singapore: a cross-sectional survey

text: BACKGROUND: Effective influenza pandemic management requires understanding of the factors influencing behavioral changes. We aim to determine the differences in knowledge, attitudes and practices in various different cohorts and explore the pertinent factors that influenced behavior in tropical Singapore. METHODS: We performed a cross-sectional knowledge, attitudes and practices survey in the Singapore military from mid-August to early-October 2009, among 3054 personnel in four exposure groups - laboratory-confirmed H1N1-2009 cases, close contacts of cases, healthcare workers, and general personnel. RESULTS: 1063 (34.8%) participants responded. The mean age was 21.4 (SE 0.2) years old. Close contacts had the highest knowledge score (71.7%, p = 0.004) while cases had the highest practice scores (58.8%, p < 0.001). There was a strong correlation between knowledge and practice scores (r = 0.27, p < 0.01) and knowledge and attitudes scores (r = 0.21, p < 0.01). The significant predictors of higher practice scores were higher knowledge scores (p < 0.001), Malay ethnicity (p < 0.001), exposure group (p < 0.05) and lower education level (p < 0.05). The significant predictors for higher attitudes scores were Malay ethnicity (p = 0.014) and higher knowledge scores (p < 0.001). The significant predictor for higher knowledge score was being a contact (p = 0.007). CONCLUSION: Knowledge is a significant influence on attitudes and practices in a pandemic, and personal experience influences practice behaviors. Efforts should be targeted at educating the general population to improve practices in the current pandemic, as well as for future epidemics.


title: Prophylactic efficacy of orally administered Bacillus poly-γ-glutamic acid, a non-LPS TLR4 ligand, against norovirus infection in mice

text: Poly-gamma-glutamic acid (γ-PGA), an extracellular biopolymer produced by Bacillus sp., is a non-canonical toll-like receptor 4 (TLR4) agonist. Here we show its antiviral efficacy against noroviruses. γ-PGA with a molecular mass of 2,000-kDa limited murine norovirus (MNV) replication in the macrophage cell line RAW264.7 by inducing interferon (IFN)-β and conferred resistance to viral infection-induced cell death. Additionally, γ-PGA interfered with viral entry into cells. The potent antiviral state mounted by γ-PGA was not attributed to the upregulation of TLR4 or TLR3, a sensor known to recognize norovirus RNA. γ-PGA sensing by TLR4 required the two TLR4-associated accessory factors MD2 and CD14. In ex vivo cultures of mouse ileum, γ-PGA selectively increased the expression of IFN-β in villi. In contrast, IFN-β induction was negligible in the ileal Peyer’s patches (PPs) where its expression was primarily induced by the replication of MNV. Oral administration of γ-PGA, which increased serum IFN-β levels without inducing proinflammatory cytokines, reduced MNV loads in the ileum with PPs and mesenteric lymph nodes in mice. Our results disclose a γ-PGA-mediated non-conventional TLR4 signaling in the ileum, highlighting the potential use of γ-PGA as a prophylactic antiviral agent against noroviruses.


title: Severe cytomegalovirus (CMV) community-acquired pneumonia (CAP) in a nonimmunocompromised host

text: Background Community-acquired pneumonia (CAP) in an immunocompetent host may be severe because of a variety or combination of host and microbial factors. In patients with severe cardiopulmonary dysfunction, even relatively avirulent pathogens, that is, Mycoplasma pneumoniae, Moraxella catarrhalis, may compromise borderline cardiac/heart function and present clinically as severe CAP. Alternately, patients with Streptococcus pneumoniae and impaired humoral immunity/splenic dysfunction may present as severe CAP. With the exception of Legionnaire's disease, influenza, and adenovirus, pathogen virulence is not a key determinant of CAP severity. Methods Diagnostically, patients with severe CAP may be approached based on the pattern of infiltrates on chest x-ray together with the severity of hypoxemia (ie, increased A-a gradient: >35). Results We present the case of an immunocompetent adult who presented with severe CAP during peak influenza season. Direct fluorescent antibody testing of his respiratory secretions was negative for influenza, adenovirus, and other respiratory viruses. Diagnostic bronchoscopy was negative for bacterial and fungal pathogens. The only clues to the cause of his severe CAP was the presence of relative lymphopenia, atypical lymphocytosis and elevated serum transaminases. After influenza and adenovirus were ruled out, cytomegalovirus (CMV) CAP was considered. The diagnosis of CMV CAP was made serologically by demonstrating highly elevated IgM CMV titers. Because the diagnosis was made during the patient's recovery late in hospitalization, he did not receive CMV antiviral therapy. Conclusion This case should remind clinicians that influenza and adenovirus are diagnostic considerations in patients presenting with severe CAP with diffuse bilateral interstitial infiltrates accompanied by severe hypoxemia in normal hosts. If influenza and adenovirus are ruled out, then CMV CAP, although rare, should be considered, particularly when viral CAP is accompanied by relative lymphopenia, atypical lymphocytosis and increased serum transaminases.


title: Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay for Highly Sensitive Detection of SARS-CoV-2

text: [Image: see text] Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.


title: Review article: prevention, diagnosis and management of COVID‐19 in the IBD patient

text: BACKGROUND: The current COVID‐19 pandemic, caused by SARS‐CoV‐2, has emerged as a public health emergency. All nations are seriously challenged as the virus spreads rapidly across the globe with no regard for borders. The primary management of IBD involves treating uncontrolled inflammation with most patients requiring immune‐based therapies. However, these therapies may weaken the immune system and potentially place IBD patients at increased risk of infections and infectious complications including those from COVID‐19. AIM: To summarise the scale of the COVID‐19 pandemic, review unique concerns regarding IBD management and infection risk during the pandemic and assess COVID‐19 management options and drug interactions in the IBD population. METHODS: A literature review on IBD, SARS‐CoV‐2 and COVID‐19 was undertaken and relevant literature was summarised and critically examined. RESULTS: IBD patients do not appear to be more susceptible to SARS‐CoV‐2 infection and there is no evidence of an association between IBD therapies and increased risk of COVID‐19. IBD medication adherence should be encouraged to prevent disease flare but where possible high‐dose systemic corticosteroids should be avoided. Patients should exercise social distancing, optimise co‐morbidities and be up to date with influenza and pneumococcal vaccines. If a patient develops COVID‐19, immune suppressing medications should be withheld until infection resolution and if trial medications for COVID‐19 are being considered, potential drug interactions should be checked. CONCLUSION: IBD patient management presents a challenge in the current COVID‐19 pandemic. The primary focus should remain on keeping bowel inflammation controlled and encouraging medication adherence.


The Solitary Confinement of Incarcerated American Youth During COVID-19


title: 9-[2-(R)-(Phosphonomethoxy)propyl]-2,6-diaminopurine (R)-PMPDAP and its prodrugs: Optimized preparation, including identification of by-products formed, and antiviral evaluation in vitro

text: Abstract New large-scale synthetic approach to antiretroviral agent 9-[2-(R)-(phosphonomethoxy)propyl]-2,6-diaminopurine, (R)-PMPDAP, was developed. Reaction of (R)-propanediol carbonate with 2,6-diaminopurine afforded exclusively (R)-9-(2-hydroxypropyl)-2,6-diaminopurine which was subsequently used for introduction of a phosphonomethyl residue using TsOCH2P(O)(OiPr)2 or BrCH2P(O)(OiPr)2 followed by deprotection of ester groups. All minor ingredients and by-products formed during the process were identified and further studied. The final product was obtained in high yield and its high enantiomeric purity (>99%) was confirmed by chiral capillary electrophoretic analysis using β-cyclodextrin as a chiral selector. Antiretroviral activity data of (R)-PMPDAP and its diverse prodrugs against HIV and FIV were investigated. Akin to (R)-PMPDAP, both prodrugs inhibit FIV replication in a selective manner. Compared to the parent molecule, the amidate prodrug was 10-fold less active against FIV in cell culture, whereas the alkoxyalkyl ester prodrug was 200-fold more potent in inhibiting FIV replication in vitro.


title: Experiences after Twenty Months with Pandemic Influenza A (H1N1) 2009 Infection in the Naïve Norwegian Pig Population

text: Pandemic (H1N1) 2009 influenza A virus was detected in Norwegian pigs in October 2009. Until then, Norway was regarded free of swine influenza. Intensified screening revealed 91 positive herds within three months. The virus was rapidly transmitted to the susceptible population, including closed breeding herds with high biosecurity. Humans were important for the introduction as well as spread of the virus to pigs. Mild or no clinical signs were observed in infected pigs. Surveillance of SIV in 2010 revealed that 41% of all the Norwegian pig herds had antibodies to pandemic (H1N1) 2009 virus. Furthermore, this surveillance indicated that pigs born in positive herds after the active phase did not seroconvert, suggesting no ongoing infection in the herds. However, results from surveillance in 2011 show a continuing spread of the infection in many herds, either caused by new introduction or by virus circulation since 2009.