Titers Of Infectious Virus Research Articles

Absence of Vaccine-enhanced Disease With Unexpected Positive Protection Against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by Inactivated Vaccine Given Within 3 Days of Virus Challenge in Syrian Hamster Model.

BackgroundMass vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is ongoing amidst widespread transmission during the coronavirus disease-2019 (COVID-19) pandemic. Disease phenotypes of SARS-CoV-2 exposure occurring around the time of vaccine administration have not been described.MethodsTwo-dose (14 days apart) vaccination regimen with formalin-inactivated whole virion SARS-CoV-2 in golden Syrian hamster model was established. To investigate the disease phenotypes of a 1-dose regimen given 3 days prior (D-3), 1 (D1) or 2 (D2) days after, or on the day (D0) of virus challenge, we monitored the serial clinical severity, tissue histopathology, virus burden, and antibody response of the vaccinated hamsters.ResultsThe 1-dose vaccinated hamsters had significantly lower clinical disease severity score, body weight loss, lung histology score, nucleocapsid protein expression in lung, infectious virus titers in the lung and nasal turbinate, inflammatory changes in intestines, and a higher serum neutralizing antibody or IgG titer against the spike receptor-binding domain or nucleocapsid protein when compared to unvaccinated controls. These improvements were particularly noticeable in D-3, but also in D0, D1, and even D2 vaccinated hamsters to varying degrees. No increased eosinophilic infiltration was found in the nasal turbinate, lung, and intestine after virus challenge. Significantly higher serum titer of fluorescent foci microneutralization inhibition antibody was detected in D1 and D2 vaccinated hamsters at day 4 post-challenge compared to controls despite undetectable neutralizing antibody titer.ConclusionsVaccination just before or soon after exposure to SARS-CoV-2 does not worsen disease phenotypes and may even ameliorate infection.

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells.

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture–based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 μL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 μL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture–based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness.Key points • First MDCK suspension cell–based perfusion process for IAV produciton was established. • “Cell density effect” was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 μL).Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-020-11050-8.

K18-hACE2 mice develop respiratory disease resembling severe COVID-19.

SARS-CoV-2 emerged in late 2019 and resulted in the ongoing COVID-19 pandemic. Several animal models have been rapidly developed that recapitulate the asymptomatic to moderate disease spectrum. Now, there is a direct need for additional small animal models to study the pathogenesis of severe COVID-19 and for fast-tracked medical countermeasure development. Here, we show that transgenic mice expressing the human SARS-CoV-2 receptor (angiotensin-converting enzyme 2 [hACE2]) under a cytokeratin 18 promoter (K18) are susceptible to SARS-CoV-2 and that infection resulted in a dose-dependent lethal disease course. After inoculation with either 104 TCID50 or 105 TCID50, the SARS-CoV-2 infection resulted in rapid weight loss in both groups and uniform lethality in the 105 TCID50 group. High levels of viral RNA shedding were observed from the upper and lower respiratory tract and intermittent shedding was observed from the intestinal tract. Inoculation with SARS-CoV-2 resulted in upper and lower respiratory tract infection with high infectious virus titers in nasal turbinates, trachea and lungs. The observed interstitial pneumonia and pulmonary pathology, with SARS-CoV-2 replication evident in pneumocytes, were similar to that reported in severe cases of COVID-19. SARS-CoV-2 infection resulted in macrophage and lymphocyte infiltration in the lungs and upregulation of Th1 and proinflammatory cytokines/chemokines. Extrapulmonary replication of SARS-CoV-2 was observed in the cerebral cortex and hippocampus of several animals at 7 DPI but not at 3 DPI. The rapid inflammatory response and observed pathology bears resemblance to COVID-19. Additionally, we demonstrate that a mild disease course can be simulated by low dose infection with 102 TCID50 SARS-CoV-2, resulting in minimal clinical manifestation and near uniform survival. Taken together, these data support future application of this model to studies of pathogenesis and medical countermeasure development.

Development of Three-Dimensional Human Intestinal Organoids as a Physiologically Relevant Model for Characterizing the Viral Replication Kinetics and Antiviral Susceptibility of Enteroviruses.

Enteroviruses are important causes of hand, foot, and mouth disease, respiratory infections, and neurological infections in human. A major hurdle for the development of anti-enterovirus agents is the lack of physiologically relevant evaluation platforms that closely correlate with the in vivo state. We established the human small intestinal organoids as a novel platform for characterizing the viral replication kinetics and evaluating candidate antivirals for enteroviruses. The organoids supported productive replication of enterovirus (EV)-A71, coxsackievirus B2, and poliovirus type 3, as evidenced by increasing viral loads, infectious virus titers, and the presence of cytopathic effects. In contrast, EV-D68, which mainly causes respiratory tract infection in humans, did not replicate significantly in the organoids. The differential expression profiles of the receptors for these enteroviruses correlated with their replication kinetics. Using itraconazole as control, we showed that the results of various antiviral assays, including viral load reduction, plaque reduction, and cytopathic effect inhibition assays, were highly reproducible in the organoids. Moreover, itraconazole attenuated virus-induced inflammatory response in the organoids, which helped to explain its antiviral effects and mechanism. Collectively, these data showed that the human small intestinal organoids may serve as a robust platform for investigating the pathogenesis and evaluating antivirals for enteroviruses.

Decontamination of common healthcare facility surfaces contaminated with SARS-CoV-2 using peracetic acid dry fogging

BackgroundThe SARS-CoV-2 pandemic has highlighted the urgent need for safe and effective surface decontamination methods, particularly in healthcare settings.AimTo evaluate the effectiveness of peracetic acid (PAA) dry fogging in decontaminating healthcare facility surfaces experimentally contaminated with SARS-CoV-2.MethodsNine materials (stainless steel, latex painted wood, unsealed hardwood, melamine countertop, vinyl flooring, clear plastic, faux leather, computer keyboard button, and smartphone touch screen) were surface contaminated with >106 median tissue culture infectious dose (TCID50) of SARS-CoV-2, and allowed to dry before exposing to PAA dry fogging.FindingsWhen fumigated with PAA dry fog for 1 h, no infectious SARS-CoV-2 virus was recovered from any of the experimentally inoculated surface types. By contrast, high titres of infectious virus were recovered from corresponding untreated drying controls of the same materials.ConclusionStandard surface decontamination processes, including sprays and wipes, are laborious and frequently cannot completely decontaminate sensitive electronic equipment. The ease of use, low cost, and overall effectiveness of a PAA dry fogging suggest that it should be considered for decontaminating healthcare settings, particularly intensive care units where severely ill SARS-CoV-2 patients are cared for.

A Single Intranasal or Intramuscular Immunization with Chimpanzee Adenovirus Vectored SARS-CoV-2 Vaccine Protects Against Pneumonia in Hamsters

The development of an effective vaccine against SARS-CoV-2, the etiologic agent of COVID-19, is a global priority. Here, we compared the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in Golden Syrian hamsters. While immunization with ChAd-SARS-CoV-2-S induced robust spike protein specific antibodies capable or neutralizing the virus, antibody levels in serum were higher in hamsters immunized by an intranasal compared to intramuscular route. Accordingly, ChAd-SARS-CoV-2-S immunized hamsters were protected against a challenge with a high dose of SARS-CoV-2. After challenge, ChAd-SARS-CoV-2-S-immunized hamsters had less weight loss and showed reductions in viral RNA and infectious virus titer in both nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-Control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provided superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission.Funding: This study was funded by NIH contracts and grants (R01 AI157155, 75N93019C00062, U01 AI151810, HHSN272201400018C, and HHSN272201700060C). J.B.C. is supported by a Helen Hay Whitney Foundation postdoctoral fellowship.Conflict of Interest: M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, andCarnival Corporation and on the Scientific Advisory Board of Moderna and Immunome. TheDiamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. The Boon laboratory has received unrelated funding support in sponsored research agreements from AI Therapeutics, 548 GreenLight Biosciences Inc., AbbVie Inc., and Nano targeting & Therapy Biopharma Inc. M.S.D. and A.O.H.have filed a disclosure with Washington University for possible commercial development of ChAd SARS-CoV-2.

Inhibition of African swine fever virus in liquid and feed by medium-chain fatty acids and glycerol monolaurate

BackgroundThe ongoing African swine fever virus (ASFv) epidemic has had a major impact on pig production globally and biosecurity efforts to curb ASFv infectivity and transmission are a high priority. It has been recently identified that feed and feed ingredients, along with drinking water, can serve as transmission vehicles and might facilitate transboundary spread of ASFv. Thus, it is important to test the antiviral activity of regulatory compatible, antiviral feed additives that might inhibit ASFv infectivity in feed. One promising group of feed additive candidates includes medium-chain fatty acids (MCFA) and monoglyceride derivatives, which are known to disrupt the lipid membrane surrounding certain enveloped viruses and bacteria.ResultsThe antiviral activities of selected MCFA, namely caprylic, capric, and lauric acids, and a related monoglyceride, glycerol monolaurate (GML), to inhibit ASFv in liquid and feed conditions were investigated and suitable compounds and inclusion rates were identified that might be useful for mitigating ASFv in feed environments. Antiviral assays showed that all tested MCFA and GML inhibit ASFv. GML was more potent than MCFA because it worked at a lower concentration and inhibited ASFv due to direct virucidal activity along with one or more other antiviral mechanisms. Dose-dependent feed experiments further showed that sufficiently high GML doses can significantly reduce ASFv infectivity in feed in a linear manner in periods as short as 30 min, as determined by infectious viral titer measurements. Enzyme-linked immunosorbent assay (ELISA) experiments revealed that GML treatment also hinders antibody recognition of the membrane-associated ASFv p72 structural protein, which likely relates to protein conformational changes arising from viral membrane disruption.ConclusionTogether, the findings in this study indicate that MCFA and GML inhibit ASFv in liquid conditions and that GML is also able to reduce ASFv infectivity in feed, which may help to curb disease transmission.

Coinfection by Severe Acute Respiratory Syndrome Coronavirus 2 and Influenza A(H1N1)pdm09 Virus Enhances the Severity of Pneumonia in Golden Syrian Hamsters.

BackgroundClinical outcomes of the interaction between the co-circulating pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and seasonal influenza viruses are unknown.MethodsWe established a golden Syrian hamster model coinfected by SARS-CoV-2 and mouse-adapted A(H1N1)pdm09 simultaneously or sequentially. The weight loss, clinical scores, histopathological changes, viral load and titer, and serum neutralizing antibody titer were compared with hamsters challenged by either virus.ResultsCoinfected hamsters had more weight loss, more severe lung inflammatory damage, and tissue cytokine/chemokine expression. Lung viral load, infectious virus titers, and virus antigen expression suggested that hamsters were generally more susceptible to SARS-CoV-2 than to A(H1N1)pdm09. Sequential coinfection with A(H1N1)pdm09 one day prior to SARS-CoV-2 exposure resulted in a lower lung SARS-CoV-2 titer and viral load than with SARS-CoV-2 monoinfection, but a higher lung A(H1N1)pdm09 viral load. Coinfection also increased intestinal inflammation with more SARS-CoV-2 nucleoprotein expression in enterocytes. Simultaneous coinfection was associated with delay in resolution of lung damage, lower serum SARS-CoV-2 neutralizing antibody, and longer SARS-CoV-2 shedding in oral swabs compared to that of SARS-CoV-2 monoinfection.ConclusionsSimultaneous or sequential coinfection by SARS-CoV-2 and A(H1N1)pdm09 caused more severe disease than monoinfection by either virus in hamsters. Prior A(H1N1)pdm09 infection lowered SARS-CoV-2 pulmonary viral loads but enhanced lung damage. Whole-population influenza vaccination for prevention of coinfection, and multiplex molecular diagnostics for both viruses to achieve early initiation of antiviral treatment for improvement of clinical outcome should be considered.

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2−infected hamsters, whereas hydroxychloroquine lacks activity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production.

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA‐CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred‐tank bioreactors. Furthermore, the authors study hollow‐fiber bioreactors and an orbital‐shaken bioreactor in perfusion mode, both available for single‐use. Productivity for the virus strain MVA‐CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred‐tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1–16.5 × 109 virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high‐yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single‐use perfusion platforms.

Efficient acute and chronic infection of stem cell-derived hepatocytes by hepatitis C virus

Objective and designHuman stem cell-derived hepatocyte-like cells (HLCs) have shown high potential as authentic model for dissection of the HCV life cycle and virus-induced pathogenesis. However, modest HCV replication, possibly...

The role of ozonized oil and a combination of tobramycin/dexamethasone eye drops in the treatment of viral conjunctivitis: a randomized clinical trial

PurposeTo determine whether topical tobramycin 0.3%/dexamethasone 0.1% plus ozonized oil eye drops reduces clinical signs and infectious viral titers of presumed viral conjunctivitis more than tobramycin/dexamethasone eye drops alone.MethodsProspective, single-blind, randomized, parallel-groups trial. Eighty patients with a clinical diagnosis of presumed viral conjunctivitis were randomizedly divided into two treatment groups: a study group and a control group, 40 for each group. Patients in the study group received topical tobramycin 0.3%/dexamethasone 0.1% eye drops, plus ozonized oil eye drops, both four times daily; patients in the control group received only topical tobramycin 0.3%/dexamethasone eye drops four times daily. The treatment was for seven days in both groups. Swabs were taken from the conjunctival fornix for adenovirus PCR analysis on the day of recruitment and at seven days follow-up. Clinical signs were also recorded on the day of recruitment and at follow-up examination: the main outcomes were conjunctival injection and conjunctival chemosis, graded on a 4-point clinical scale, presence or absence of superficial punctate keratitis and subepithelial corneal infiltrates.ResultsNo statistically significant difference was reached in adenoviral infection negativization between the two groups, although the study group showed a higher number of PCR negative results at seven days follow-up. PCR real time detected adenoviral infection in 17 of 24 patients on the day of recruitment and it was positive in 4 patients on the seventh day (viral positivity reduction of 76%). In the control group PCR was positive for adenovirus in 18 of 24 patients on the day of recruitment and in 7 patients at seven days follow-up (reduction of 61%). There was statistically significant difference on conjunctival clinical signs between the study and control groups. Significant difference was also found on superficial punctate keratitis resolution between the study and the control group. In the former superficial punctate keratitis was detected in 14 eyes on the first day and in 5 eyes after seven days while in the latter superficial punctate keratitis was found in 124 eyes on the first day and in 6 eyes on the seventh day. No difference was found in subepithelial corneal infiltrates appearance between the two groups.ConclusionsThe use of ozonized-oil containing eye drops in combination with topical tobramycin 0.3%/dexamethasone 0.1% eye drops four times daily seems to reduce the signs of conjunctivitis, and the duration of viral infection, although it does not affect the subepithelial corneal infiltrates appearance.

Minimum Infective Dose of a Lumpy Skin Disease Virus Field Strain from North Macedonia

Infection with Lumpy Skin Disease virus (LSDV), as well as infections with other Capripox virus species, are described as the most severe pox diseases of production animals and are therefore listed as notifiable diseases under the guidelines of the World Organization for Animal Health (OIE). To our knowledge there is only a single study examining dose dependency, clinical course, viremia, virus shedding, as well as serological response following experimental LSDV “Neethling” inoculation. Here, we inoculated cattle with four different doses of LSDV strain “Macedonia2016”, a recently characterized virulent LSDV field strain, and examined clinical symptoms, viremia, viral shedding, and seroconversion. Interestingly, around 400 cell culture infectious dose50 (CCID50) of LSDV-“Macedonia2016” were sufficient to induce generalized Lumpy Skin Disease (LSD) in two out of six cattle but with a different incubation time, whereas the other animals of this group showed only a mild course of LSD. However, differences in incubation time, viral loads, serology, and in the clinical scoring could not be observed in the other three groups. In summary, we concluded that experimental LSDV infection of cattle with an infectious virus titer of 105 to 106 CCID50/mL of “Macedonia2016” provides a robust and sufficient challenge model for future studies.

Neuraminidase antigenic drift of H3N2 clade 3c.2a viruses alters virus replication, enzymatic activity and inhibitory antibody binding.

In the 2014–2015 influenza season a novel neuraminidase (NA) genotype was detected in global human influenza A surveillance. This novel genotype encoded an N-linked glycosylation site at position 245–247 in the NA protein from clade 3c.2a H3N2 viruses. In the years following the 2014–2015 season, this novel NA glycosylation genotype quickly dominated the human H3N2 population of viruses. To assess the effect this novel N-linked glycan has on virus fitness and antibody binding, recombinant viruses with (NA Gly+) or without (NA Gly-) the 245 NA glycan were created. Viruses with the 245 NA Gly+ genotype grew to a significantly lower infectious virus titer on primary, differentiated human nasal epithelial cells (hNEC) compared to viruses with the 245 NA Gly- genotype, but growth was similar on immortalized cells. The 245 NA Gly+ blocked human and rabbit monoclonal antibodies that target the enzymatic site from binding to their epitope. Additionally, viruses with the 245 NA Gly+ genotype had significantly lower enzymatic activity compared to viruses with the 245 NA Gly- genotype. Human monoclonal antibodies that target residues near the 245 NA glycan were less effective at inhibiting NA enzymatic activity and virus replication of viruses encoding an NA Gly+ protein compared to ones encoding NA Gly- protein. Additionally, a recombinant H6N2 virus with the 245 NA Gly+ protein was more resistant to enzymatic inhibition from convalescent serum from H3N2-infected humans compared to viruses with the 245 NA Gly- genotype. Finally, the 245 NA Gly+ protected from NA antibody mediated virus neutralization. These results suggest that while the 245 NA Gly+ decreases virus replication in hNECs and decreases enzymatic activity, the 245 NA glycan blocks the binding of monoclonal and human serum NA specific antibodies that would otherwise inhibit enzymatic activity and virus replication.

Two Detailed Plaque Assay Protocols for the Quantification of Infectious SARS-CoV-2.

Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) has been identified as the causal agent of COronaVIrus Disease‐19 (COVID‐19), an atypical pneumonia‐like syndrome that emerged in December 2019. While SARS‐CoV‐2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS‐CoV‐2 can be achieved by tissue culture infectious dose−50 (TCID50), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS‐CoV‐2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication‐competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS‐CoV‐2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS‐CoV‐2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS‐CoV‐2 plaque assay using a solid double overlay method Alternate Protocol: SARS‐CoV‐2 plaque assay using a liquid overlay and fixation‐staining method

Hepatitis E virus is effectively inactivated in platelet concentrates by ultraviolet C light

As previous investigations have shown, THERAFLEX UV-Platelets, a UVC-based pathogen inactivation (PI) system, is effective against non-enveloped transfusion-relevant viruses such as hepatitis A virus (HAV), which are insensitive to most PI treatments for blood products. This study investigated the PI efficacy of THERAFLEX UV-Platelets against HEV in platelet concentrates (PCs). Buffy coat-derived PCs in additive solution were spiked with cell culture-derived HEV and treated with the THERAFLEX UV-Platelets system using various doses of UVC (0·05, 0·10, 0·15 and 0·20 (standard) J/cm2 ). Titres of infectious virus in pre- and post-treatment samples were determined using a large-volume plating assay to improve the detection limit of the virus assay. THERAFLEX UV-Platelets dose-dependently inactivated HEV in PCs. The standard UVC dose inactivated the virus to below the limit of detection, corresponding to a mean log reduction of greater than 3·5. Our study demonstrates that the THERAFLEX UV-Platelets system effectively inactivates HEV in PCs.

Resolution of herpes simplex virus reactivation in vivo results in neuronal destruction.

A fundamental question in herpes simplex virus (HSV) pathogenesis is the consequence of viral reactivation to the neuron. Evidence supporting both post-reactivation survival and demise is published. The exceedingly rare nature of this event at the neuronal level in the sensory ganglion has limited direct examination of this important question. In this study, an in-depth in vivo analysis of the resolution of reactivation was undertaken. Latently infected C57BL/6 mice were induced to reactivate in vivo by hyperthermic stress. Infectious virus was detected in a high percentage (60-80%) of the trigeminal ganglia from these mice at 20 hours post-reactivation stimulus, but declined by 48 hours post-stimulus (0-13%). With increasing time post-reactivation stimulus, the percentage of reactivating neurons surrounded by a cellular cuff increased, which correlated with a decrease in detectable infectious virus and number of viral protein positive neurons. Importantly, in addition to intact viral protein positive neurons, fragmented viral protein positive neurons morphologically consistent with apoptotic bodies and containing cleaved caspase-3 were detected. The frequency of this phenotype increased through time post-reactivation. These fragmented neurons were surrounded by Iba1+ cells, consistent with phagocytic removal of dead neurons. Evidence of neuronal destruction post-reactivation prompted re-examination of the previously reported non-cytolytic role of T cells in controlling reactivation. Latently infected mice were treated with anti-CD4/CD8 antibodies prior to induced reactivation. Neither infectious virus titers nor neuronal fragmentation were altered. In contrast, when viral DNA replication was blocked during reactivation, fragmentation was not observed even though viral proteins were expressed. Our data demonstrate that at least a portion of reactivating neurons are destroyed. Although no evidence for direct T cell mediated antigen recognition in this process was apparent, inhibition of viral DNA replication blocked neuronal fragmentation. These unexpected findings raise new questions about the resolution of HSV reactivation in the host nervous system.

Inactivation of severe fever with thrombocytopenia syndrome virus for improved laboratory safety

Summary Severe fever with thrombocytopenia syndrome (SFTS), a tick-borne infectious disease with high mortality, is diagnosed by the serological testing of serum samples from patients in the acute and convalescent phases and/or by direct detection of the SFTS virus (SFTSV). Conventionally, heat and UV treatments have been used to inactivate the viruses present in serum samples before performing the serological assays. Here, we examined the inactivation conditions optimal for SFTSV-containing serum samples to ensure the safety of laboratory workers while maintaining the accuracy of serological assay results simultaneously. Heating human serum samples spiked with SFTSV to 60 °C for 30 min or exposing them to UV irradiation for 30 min, reduced the infectious virus titer below the limit of detection. SFTSV in sera from patients in the acute phase of SFTS was completely inactivated by heating to 60 °C for 30 min, followed by UV irradiation for 10 min. This inactivation procedure had minimal impact on the performance of SFTSV antibody detection tests. The data provided herein can serve as a guide for laboratory workers and researchers working with SFTS serological tests.

Priming Melon Defenses with Acibenzolar-S-methyl Attenuates Infections by Phylogenetically Distinct Viruses and Diminishes Vector Preferences for Infected Hosts.

Plant virus management is mostly achieved through control of insect vectors using insecticides. However, insecticides are only marginally effective for preventing virus transmission. Furthermore, it is well established that symptoms of virus infections often encourage vector visitation to infected hosts, which exacerbates secondary spread. Plant defense elicitors, phytohormone analogs that prime the plant immune system against attack, may be a viable approach for virus control that complements insecticide use by disrupting pathologies that attract vectors. To explore this, we tested the effect of a commercial plant elicitor, acibenzolar-S-methyl (ASM), on infection rates, virus titers, and symptom development in melon plants inoculated with one of two virus species, Cucumber mosaic virus (CMV) and Cucurbit yellow stunting disorder virus (CYSDV). We also conducted behavioral assays to assess the effect of ASM treatment and virus inoculation on vector behavior. For both pathogens, ASM treatment reduced symptom severity and delayed disease progression. For CYSDV, this resulted in the attenuation of symptoms that encourage vector visitation and virion uptake. We did observe slight trade-offs in growth vs. defense following ASM treatment, but these effects did not translate into reduced yields or plant performance in the field. Our results suggest that immunity priming may be a valuable tool for improving management of insect-transmitted plant viruses.