Virus Stocks Research Articles

Using Diploid Human Fibroblasts as a Model System to Culture, Grow, and Study Human Cytomegalovirus Infection.

Primary human diploid fibroblasts are used routinely to study host/pathogen interactions of human cytomegalovirus (HCMV). Fibroblasts' ease of culture and tremendous permissiveness for infection allow the study of all facets of infection, an abbreviated list of which includes ligand-receptor interactions, activation of cell signaling responses, and dysregulation of the cell cycle and DNA repair processes. Another advantage to fibroblasts' permissiveness for HCMV is the capability to grow high titer stocks of virus in them. This chapter will discuss the production of viral stocks of HCMV in primary human fibroblasts, commencing with culturing and infection of cells and continuing through harvest, titration (determining the infectious capacity of a particular virus preparation), and storage of viral stocks for use in downstream experiments.

Efficacy of a coxsackievirus A6 vaccine candidate in an actively immunized mouse model

ABSTRACT Coxsackievirus A6 (CV-A6) has been emerging as a major pathogen of hand, foot and mouth disease (HFMD). Study on the pathogenesis of CV-A6 infection and development of vaccines is hindered by a lack of appropriate animal models. Here, we report an actively immunized-challenged mouse model to evaluate the efficacy of a Vero-cell-based, inactivated CV-A6 vaccine candidate. The neonatal Kunming mice were inoculated with a purified, formaldehyde-inactivated CV-A6 vaccine on days 3 and 9, followed by challenging on day 14 with a naturally selected virulent strain at a lethal dose. Within 14 days postchallenge, all mice in the immunized groups survived, while 100% of the Alum-only inoculated mice died. Neutralizing antibodies (NtAbs) were detected in the serum of immunized suckling mice, and the NtAb levels correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak in the immunized mice compared with those in Alum-only inoculated control mice. Elevated levels of interleukin-4, 6, interferon γ and tumour necrosis factor α were also observed in Alum-only control mice compared with immunized mice. Importantly, the virulent CV-A6 challenge strain was selected quickly and conveniently from a RD cell virus stock characterized with the natural multi-genotypes. The virulent determinants were mapped to V124M and I242 V at VP1. Together, our results indicated that this actively immunized mouse model is invaluable for future studies to develop multivalent vaccines containing the major component of CV-A6 against HFMD.

Optimized production and immunogenicity of an insect virus-based chikungunya virus candidate vaccine in cell culture and animal models

ABSTRACT A chimeric Eilat/ Chikungunya virus (EILV/CHIKV) was previously reported to replicate only in mosquito cells but capable of inducing robust adaptive immunity in animals. Here, we initially selected C7/10 cells to optimize the production of the chimeric virus. A two-step procedure produced highly purified virus stocks, which was shown to not cause hypersensitive reactions in a mouse sensitization study. We further optimized the dose and characterized the kinetics of EILV/CHIKV-induced immunity. A single dose of 108 PFU was sufficient for induction of high levels of CHIKV-specific IgM and IgG antibodies, memory B cell and CD8+ T cell responses. Compared to the live-attenuated CHIKV vaccine 181/25, EILV/CHIKV induced similar levels of CHIKV-specific memory B cells, but higher CD8+ T cell responses at day 28. It also induced stronger CD8+, but lower CD4+ T cell responses than another live-attenuated CHIKV strain (CHIKV/IRES) at day 55 post-vaccination. Lastly, the purified EILV/CHIKV triggered antiviral cytokine responses and activation of antigen presenting cell (APC)s in vivo, but did not induce APCs alone upon in vitro exposure. Overall, our results demonstrate that the EILV/CHIKV vaccine candidate is safe, inexpensive to produce and a potent inducer of both innate and adaptive immunity in mice.

RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens

ObjectiveTo develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. MethodsA panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID50 per μL and copies per μL, and the newly established methods were tested in clinical specimens collected in recent years. ResultsThe sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID50 per μL and 10 virus copies per μL, and for the complete VP1 gene was 1 CCID50 per μL and 100 virus copies per μL, using serially-diluted virus stocks of five serotypes. As a proof-of-concept, 25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons. ConclusionThis RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate.

BackgroundDue to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus.MethodsHere, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated.FindingsWe report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.InterpretationThe results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2.FundingThis work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.

Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1.

Since its emergence in the United States in 2014, enterovirus D68 (EV-D68) has been and is associated with severe respiratory diseases and acute flaccid myelitis. Even though EV-D68 has been shown to replicate in different neuronal cells in vitro, it is currently poorly understood which viral factors contribute to the ability to replicate efficiently in cells of the central nervous system and whether this feature is a clade-specific feature. Here, we determined the replication kinetics of clinical EV-D68 isolates from (sub)clades A, B1, B2, B3, and D1 in human neuroblastoma cells (SK-N-SH). Subsequently, we compared sequences to identify viral factors associated with increased viral replication. All clinical isolates replicated in SK-N-SH cells, although there was a large difference in efficiency. Efficient replication of clinical isolates was associated with an amino acid substitution at position 271 of VP1 (E271K), which was acquired during virus propagation in vitro Recognition of heparan sulfate in addition to sialic acids was associated with increased attachment, infection, and replication. Removal of heparan sulfate resulted in a decrease in attachment, internalization, and replication of viruses with E271K. Taken together, our study suggests that the replication kinetics of EV-D68 isolates in SK-N-SH cells is not a clade-specific feature. However, recognition of heparan sulfate as an additional receptor had a large effect on phenotypic characteristics in vitro. These observations emphasize the need to compare sequences from virus stocks with clinical isolates in order to retrieve phenotypic characteristics from original virus isolates.IMPORTANCE Enterovirus D68 (EV-D68) causes mild to severe respiratory disease and is associated with acute flaccid myelitis since 2014. Currently, the understanding of the ability of EV-D68 to replicate in the central nervous system (CNS), and whether it is associated with a specific clade of EV-D68 viruses or specific viral factors, is lacking. Comparing different EV-D68 clades did not reveal clade-specific phenotypic characteristics. However, we did show that viruses which acquired a cell culture-adapted amino acid substitution in VP1 (E271K) recognized heparan sulfate as an additional receptor. Recognition of heparan sulfate resulted in an increase in attachment, infection, and replication in neuroblastoma cells compared with viruses without this specific amino acid substitution. The ability of EV-D68 viruses to acquire cell culture-adaptive substitutions which have a large effect in experimental settings emphasizes the need to sequence virus stocks.

Propagation of oncolytic Newcastle Disease Virus in Embryonated Chicken Eggs and its Research Applications in Cell lines

Newcastle Disease Virus (NDV) can modulate cancer cell signaling pathways and induce apoptosis in cancer cells. The laboratory-based studies of the oncolytic NDV requires a reliable protocol for the propagation of the oncolytic NDV. A comprehensive protocol is provided for virus propagation in fertile chicken eggs, which consistently yields high titer viral stock. Aim: Propagation of oncolytic NDV AMHA1 attenuated strain in Embryonated Chicken Eggs (ECE) and tissue culture infective dose 50% (TCID50) determination protocol of the virus. Method: Specific pathogen-free fertilized chicken eggs were incubated at 37 °C and 55-60% humidity for 9’ 10 days. Over this period, embryo death was monitored using an egg candle regularly. Virus inoculation is carried out by injection of the diluted virus stock into the allantoic cavity using a needle. embryo death was recorded every two hours and the egg rushed to the refrigerator and fluids collected after four to six hours. Hemagglutination assay (HA) was used to determine the preliminary titer of the virus to collect the high titer egg fluids only which is about 128 to 256HAU. The Vero cell line was exposed to NDV at tenfold serial dilutions to determine TCID50 of the virus. The number of viruses in 1 ml of allantoic fluid was measured of embryonated chicken eggs. Results: NDV Iraqi virulent strain has the ability to kill all the chicken embryos through (24-72) h of inoculation. A high titer of NDV was achieved from the infected eggs.Conclusion: Oncolytic NDV propagated in embryonated chicken eggs in high titers as indicated by TCID50 value.

In-vitro Evaluation of Antiviral Activity of Moringa oleifera Extracts against Polio virus

Background: Moringa oleifera plant parts extract have been utilized tremendously in traditional medicine, having various pharmaceutical activities such as antifungal, antibacterial and antiviral properties.
 Aim: This study was carried out to evaluate antiviral activity of aqueous extract of Moringa leaves, seeds and flowers against Polio virus isolates (Vaccine Strains P1&P3), MDG-17-04852; MDG-17-04881.
 Study Design: This is a baseline study carried out to determine the efficacy of Moringa oleifera in the treatment of poliomyelitis.
 Place and Duration of Study: This study was carried out at the University of Maiduguri Teaching Hospital Maiduguri and Abubakar Tafawa Balewa University, Bauchi from September 2018 to October, 2019.
 Methods: Phytochemical substances was extracted and screened from the Moringa using standard laboratory techniques. Continuous cell line L20B cells was used to isolate polio virus. Polio virus stock was prepared and titration was carried out to determine TCID50 by Kerber’s formula, L-d(S - 0.5). Cytotoxicity of the extracts was evaluated using the end-point cytopathic effect assay. Antiviral assay and Polio virus Intratypic Differentiation (ITD) was performed using Real-Time PCR.
 Results: The results revealed present of Tannins in leaves and flowers, Alkaloids and Saponins in all the extracts, Glycosides and Steroids in leaves and Flavonoids in flowers only. Cytotoxicity of the extracts shows 50% effects in 100mg and 50mg concentrations, while 33.3% and 16.7% was observed in 25mg and 12.5mg (P>.05). The titration of the tissue culture infective dose 50% (TCID50) shows that Polio virus type 1and 3 had a titre value of 106.5 and 106.25 respectively. The ITD results observed presence of Sl1 and Sl3 from the extracts.
 Conclusion: This study found that the Moringa extracts did not neutralized the Polio virus strains studied, as the phytochemicals show no antiviral activity. Therefore further study is needed in this area to ascertain its antiviral potentials against poliomyelitis.

In Vitro Efficacy of a Povidone-Iodine Nasal Antiseptic for Rapid Inactivation of SARS-CoV-2

Research is needed to demonstrate the efficacy of nasal povidone-iodine (PVP-I) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To evaluate the in vitro efficacy of PVP-I nasal antiseptic for the inactivation of SARS-CoV-2 at clinically significant contact times of 15 and 30 seconds. The SARS-CoV-2, USA-WA1/2020 strain, virus stock was tested against nasal antiseptic solutions consisting of aqueous PVP-I as the sole active ingredient. Povidone-iodine was tested at diluted concentrations of 0.5%, 1.25%, and 2.5% and compared with controls. The test solutions and virus were incubated at mean (SD) room temperature of 22 (2) °C for time periods of 15 and 30 seconds. This controlled in vitro laboratory research study used 3 different concentrations of study solution and ethanol, 70%, as a positive control on test media infected with SARS-CoV-2. Test media without virus were added to 2 tubes of the compounds to serve as toxicity and neutralization controls. Ethanol, 70%, was tested in parallel as a positive control and water only as a negative control. The primary study outcome measurement was the log reduction value after 15 seconds and 30 seconds of given treatment. Surviving virus from each sample was quantified by standard end point dilution assay, and the log reduction value of each compound was compared with the negative (water) control. Povidone-iodine nasal antiseptics at concentrations (0.5%, 1.25%, and 2.5%) completely inactivated SARS-CoV-2 within 15 seconds of contact as measured by log reduction value of greater than 3 log10 of the 50% cell culture infectious dose of the virus. The ethanol, 70%, positive control did not completely inactivate SARS-CoV-2 after 15 seconds of contact. The nasal antiseptics tested performed better than the standard positive control routinely used for in vitro assessment of anti-SARS-CoV-2 agents at a contact time of 15 seconds. No cytotoxic effects on cells were observed after contact with each of the nasal antiseptics tested. Povidone-iodine nasal antiseptic solutions at concentrations as low as 0.5% rapidly inactivate SARS-CoV-2 at contact times as short as 15 seconds. Intranasal use of PVP-I has demonstrated safety at concentrations of 1.25% and below and may play an adjunctive role in mitigating viral transmission beyond personal protective equipment.

In Vitro Virucidal Effect of Intranasally Delivered Chlorpheniramine Maleate Compound Against Severe Acute Respiratory Syndrome Coronavirus 2.

BackgroundThe initial global outbreak of the novel coronavirus disease 2019 (COVID-2019) pandemic, which is responsible for the severe acute respiratory syndrome 2 (SARS-CoV-2), shares similarities with the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) and behaves similarly to influenza with a high intranasal viral load. The genome sequence of COVID-19 opened the opportunity for multiple in vitro and clinical trials, but we still do not have a clear path to treatment. Chlorpheniramine maleate (CPM) is a safe and effective antihistamine with potent antiviral activity against various strains of influenza A/B, thus suggesting that CPM has broad antiviral activity. We tested the virucidal potential of CPM in a nasal spray composition currently in development as an anti-allergy medication.MethodsThe virucidal activity of CPM was tested using viral stock of SARS-CoV-2, USA-WA1/2020 strain in Vero 76 infected cells. The endpoint titer 50% cell culture infection dose (CCID50) values were calculated using the Reed-Muench (1948) equation. Three independent replicates of each sample were tested, and the average and standard deviation were calculated. Results were compared with untreated controls using one-way ANOVA (analysis of variance) with Dunnett’s multiple comparison test in GraphPad Prism (version 8) software.ResultsAfter 25 minutes of contact time, the nasal spray reduced the levels of the virus from 4.2 to 1.7 log10 CCID50 per 0.1 mL, a statistically significant 2.5 log reduction value or 99.7% reduction in the viral load.ConclusionsThis study demonstrates the strong virucidal effect against SARS-CoV-2 of a nasal spray containing CPM. Given that CPM has broad antiviral effects against influenza and virucidal effect against SARS-CoV-2, we propose two further studies: a randomized placebo-controlled study of intranasally delivered chlorpheniramine in patients with mild-to-moderate SARS-CoV-2 and a second study aiming to determine the potential antiviral and adjuvant effects of CPM plus hydroxychloroquine, versus hydroxychloroquine alone, in hospitalized patients with SARS-CoV-2.

Rapid, convenient and efficient kit-independent detection of SARS-CoV-2 RNA

Pandemic SARS-CoV-2 infection has rapidly developed into a socioeconomic and humanitarian catastrophe. Basic principles to prevent SARS-CoV-2 transmission are social distancing, face masks, contact tracing and early detection of SARS-CoV-2. To meet these requirements, virtually unlimited test capacities delivering results in a rapid and reliable manner are a prerequisite. Here, we provide and validate such a rapid, convenient and efficient kit-independent detection of SARS-CoV-2 RNA, termed COVID-quick-DET. This straightforward method operates with simple proteinase K treatment and repetitive heating steps with a sensitivity of 94.6% in head-to-head comparisons with kit-based isolation methods. This result is supported by data obtained from serially diluted SARS-CoV-2 virus stocks. Given its cost- and time-effective operation, COVID-quick-DET might be best suited for countries with general shortage or temporary acute scarcity of resources and equipment.

The effect of ultraviolet C radiation against different N95 respirators inoculated with SARS-CoV-2

ObjectivesThere are currently no studies that have examined whether one dosage can be uniformly applied to different respirator types to effectively decontaminate SARS-CoV-2 on N95 filtering facepiece respirators (FFRs). Health care workers have been using this disinfection method during the pandemic. Our objective was to determine the effect of UVC on SARS-CoV-2 inoculated N95 respirators and whether this was respirator material/model type dependent. MethodsFour different locations (facepiece and strap) on five different N95 FFR models (3M 1860, 8210, 8511, 9211; Moldex 1511) were inoculated with a 10 μL drop of SARS-CoV-2 viral stock (8 × 107 TCID50/mL). The outside-facing and wearer-facing surfaces of the respirators were each irradiated with a dose of 1.5 J/cm2 UVC (254 nm). Viable SARS-CoV-2 was quantified by a median tissue culture infectious dose assay (TCID50). ResultsUVC delivered using a dose of 1.5 J/cm2, to each side, was an effective method of decontamination for the facepieces of 3M 1860 and Moldex 1511, and for the straps of 3M 8210 and the Moldex 1511. ConclusionThis dose is an appropriate decontamination method to facilitate the reuse of respirators for healthcare personnel when applied to specific models/materials. Also, some straps may require additional disinfection to maximize the safety of frontline workers. Implementation of widespread UVC decontamination methods requires careful consideration of model, material type, design, and fit-testing following irradiation.

A Model for the Production of Regulatory Grade Viral Hemorrhagic Fever Exposure Stocks: From Field Surveillance to Advanced Characterization of SFTSV.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging human pathogen, endemic in areas of China, Japan, and the Korea (KOR). It is primarily transmitted through infected ticks and can cause a severe hemorrhagic fever disease with case fatality rates as high as 30%. Despite its high virulence and increasing prevalence, molecular and functional studies in situ are scarce due to the limited availability of high-titer SFTSV exposure stocks. During the course of field virologic surveillance in 2017, we detected SFTSV in ticks and in a symptomatic soldier in a KOR Army training area. SFTSV was isolated from the ticks producing a high-titer viral exposure stock. Through the use of advanced genomic tools, we present here a complete, in-depth characterization of this viral stock, including a comparison with both the virus in its arthropod source and in the human case, and an in vivo study of its pathogenicity. Thanks to this detailed characterization, this SFTSV viral exposure stock constitutes a quality biological tool for the study of this viral agent and for the development of medical countermeasures, fulfilling the requirements of the main regulatory agencies.

Low pathogenic avian influenza virus isolates with different levels of defective genome segments vary in pathogenicity and transmission efficiency

Defective interfering particles (DIPs) of influenza virus are generated through incorporation of highly truncated forms of genome segments, mostly those coding polymerase complex proteins (PB2, PB1, PA). Such particles are able to replicate only in the presence of a virus with the complete genome, thus DIPs may alter the infection outcome by suppressing production of standard virus particles, but also by stimulating the immune response. In the present study we compared the clinical outcome, mortality and transmission in chickens and turkeys infected with the same infectious doses of H7N7 low pathogenic avian influenza virus containing different levels of defective gene segments (95/95(DVG-high) and 95/95(DVG-low)). No clinical signs, mortality or transmission were noted in SPF chickens inoculated with neither virus stock. Turkeys infected with 95/95(DVG-high) showed only slight clinical signs with no mortality, and the virus was transmitted only to birds in direct contact. In contrast, more severe disease, mortality and transmission to direct and indirect contact birds was observed in turkeys infected with 95/95(DVG-low). Apathy, lower water and food intake, respiratory system disorders and a total mortality of 60% were noted. Shedding patterns in contact turkeys indicated more efficient within- and between-host spread of the virus than in 95/95(DVG-high) group. Sequencing of virus genomes showed no mutations that could account for the observed differences in pathogenicity. The results suggest that the abundance of DIPs in the inoculum was the factor responsible for the mild course of infection and disrupted virus transmission.

The Propagation, Quantification, and Storage of Vesicular Stomatitis Virus

Vesicular stomatitis virus (VSV) is the prototypical member of the Rhabdoviridae family of negative-sense single-stranded RNA viruses. This virus has been used as a powerful model system for decades and is currently being used as a vaccine platform and an oncolytic agent. Here, we present methods to propagate, quantitate, and store VSV. We also review the proper safety protocol for the handling of VSV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation, purification, and storage of vesicular stomatitis virus stocks Basic Protocol 2: Quantification of vesicular stomatitis virus by plaque assay Support Protocol: Propagation of Vero cells.

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Susceptibility of SARS-CoV-2 to UV irradiation

The coronavirus SARS-CoV-2 pandemic became a global health burden. We determined the susceptibility of SARS-CoV-2 to irradiation with ultraviolet light. The virus was highly susceptible to ultraviolet light. A viral stock with a high infectious titer of 5 × 106 TCID50/mL was completely inactivated by UVC irradiation after nine minutes of exposure. The UVC dose required for complete inactivation was 1,048 mJ/cm2. UVA exposure demonstrated only a weak effect on virus inactivation over 15 minutes. Hence, inactivation of SARS-CoV-2 by UVC irradiation constitutes a reliable method for disinfection purposes in health care facilities and for preparing SARS-CoV-2 material for research purpose.

Butyrate Reprograms Expression of Specific Interferon-Stimulated Genes.

Butyrate is an abundant metabolite produced by gut microbiota. While butyrate is a known histone deacetylase inhibitor that activates expression of many genes involved in immune system pathways, its effects on virus infections and on the antiviral type I interferon (IFN) response have not been adequately investigated. We found that butyrate increases cellular infection with viruses relevant to human and animal health, including influenza virus, reovirus, HIV-1, human metapneumovirus, and vesicular stomatitis virus. Mechanistically, butyrate suppresses levels of specific antiviral IFN-stimulated gene (ISG) products, such as RIG-I and IFITM3, in human and mouse cells without inhibiting IFN-induced phosphorylation or nuclear translocation of the STAT1 and STAT2 transcription factors. Accordingly, we discovered that although butyrate globally increases baseline expression of more than 800 cellular genes, it strongly represses IFN-induced expression of 60% of ISGs and upregulates 3% of ISGs. Our findings reveal that there are differences in the IFN responsiveness of major subsets of ISGs depending on the presence of butyrate in the cell environment, and overall, they identify a new mechanism by which butyrate influences virus infection of cells.IMPORTANCE Butyrate is a lipid produced by intestinal bacteria. Here, we newly show that butyrate reprograms the innate antiviral immune response mediated by type I interferons (IFNs). Many of the antiviral genes induced by type I IFNs are repressed in the presence of butyrate, resulting in increased virus infection and replication. Our research demonstrates that metabolites produced by the gut microbiome, such as butyrate, can have complex effects on cellular physiology, including dampening of an inflammatory innate immune pathway resulting in a proviral cellular environment. Our work further suggests that butyrate could be broadly used as a tool to increase growth of virus stocks for research and for the generation of vaccines.

An enzyme-based immunodetection assay to quantify SARS-CoV-2 infection

SARS-CoV-2 is a novel pandemic coronavirus that caused a global health and economic crisis. The development of efficient drugs and vaccines against COVID-19 requires detailed knowledge about SARS-CoV-2 biology. Several techniques to detect SARS-CoV-2 infection have been established, mainly based on counting infected cells by staining plaques or foci, or by quantifying the viral genome by PCR. These methods are laborious, time-consuming and expensive and therefore not suitable for a high sample throughput or rapid diagnostics. We here report a novel enzyme-based immunodetection assay that directly quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized cells. This in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter format, regardless of the virus isolate or target cell culture. It follows the established method of performing ELISA assays and does not require expensive instrumentation. Utilization of the in-cell ELISA allows to e.g. determine TCID50 of virus stocks, antiviral efficiencies (IC50 values) of drugs or neutralizing activity of sera. Thus, the in-cell spike ELISA represents a promising alternative to study SARS-CoV-2 infection and inhibition and may facilitate future research.

Assessing the Efficacy of Human Papillomavirus Disinfection and the Risk of Transmission from Clinical Lesions

<h3>Background</h3> HPV genomes can be detected on medical devices following hospital disinfectant procedures. Recent reports concluded that oncogenic HPVs derived from laboratory tissue-based models are not susceptible to commonly used high-level disinfectants, intensifying concerns that medical instruments may provide transmission of nosocomial HPVs infections. Therefore, we determined the infectious load of HPVs from clinical lesions and investigated the effectiveness of disinfectants on HPV virions derived from laboratory model systems. <h3>Methods</h3> Infectious HPV virions were isolated from cell culture, organotypic epithelial tissue cultures, and mouse xenografts. Clinical samples from recurrent respiratory papillomas (RRPs) and anogenital warts were obtained under IRB approval. The infectivity of HPV virion stocks was measured by the detection of spliced viral E1^E4 mRNAs in infected keratinocytes. Infections were validated by time-dependent detection of E1^E4 mRNAs, resistance to ribonuclease treatment and susceptibility to antibody-mediated neutralization. Our infectivity assay demonstrated a dynamic range of >3-5 log10. Suspension-based disinfection assays employed diluted ortho-phthalaldehyde (OPA) and 0.825% hypochlorite. <h3>Results</h3> In contrast to prior reports, we found that validated HPV virions obtained from a variety of sources were susceptible to a 2.5 to 4 log10 reduction in infectious titer when exposed as directed to OPA or hypochlorite. Some HPV virion stocks failed to meet the infectivity criteria of time-dependent detection of E1^E4 mRNAs, resistance to ribonuclease and susceptibility to antibody-mediated neutralization. Such unvalidated virus stocks are likely to produce spurious results and lead to confounding conclusions. Assessment of HPV infectious titers from clinical lesions suggests that compared to common warts, clinical RRP and anogenital warts have lower levels of virions present at apical surfaces. <h3>Conclusions</h3> We conclude that laboratory sourced HPVs are susceptible to disinfection by OPA and hypochlorite. As the levels of infectious virions recovered from HPV-induced lesions fall below those used in our assays, proper cleaning and disinfection processing is likely to be effective