What is in a Viral Stock: Perspectives and Current Limitations of Flow Virometry

Herpesviruses produce a plethora of pleomorphic and heterogeneous particle populations. The composition and biological role of these is not understood. Detailed analysis has been challenging due to the lack of multidimensional identification and purification methodologies. Fluorescence-activated cell sorting (FACS), originally developed to sort objects with at least ten thousand-fold larger volumes, has recently been applied to cellular exosomes as well as viral particles and has been dubbed nanoscale flow cytometry or “flow virometry”. In comparison to other nanoparticles, herpesvirus concentrations can be measured with high precision using simple culturing methods. Here, we used this unique capability to evaluate a standard FACS sorter. We demonstrate that detection and separation capabilities were insufficient to distinguish infectious fluorescent viral populations from populations lacking fluorescence and infectivity. Moreover, fluorescent populations did not contain single virus particles but mostly aggregates. On top, analysis of viral samples by flow cytometry was confounded by swarm detection, as multiple objects are measured simultaneously and interpreted as a single object. Despite these technical difficulties, comparison of crude supernatant to gradient purified HCMV revealed that infectious virus is a minor proportion of the particles released from infected cells. Our data stress the need for a set of standardized controls and protocols when applying FACS to biological nanoparticles and highlights technical challenges that need to be solved before flow virometry can achieve its full potential.

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.

The Effect of Ultraviolet C Radiation Against SARS-CoV-2 Inoculated N95 Respirators

Since March 31st, 2020, during the height of the pandemic, we have decontaminated thousands of 3M 1860 respirators with Ultraviolet C (UVC) for our frontline workers. There is no published peer-reviewed data regarding the dose required to effectively disinfect SARS-CoV-2 on N95 filtering facepiece respirators (FFRs). Four different locations (facepiece and strap) on 5 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). UVC 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. This dose is an appropriate decontamination method to facilitate reuse of respirators for healthcare personnel when applied to certain models/materials. Increasing the dose may improve decontamination for the other models and straps; however, UVC radiation can degrade certain polymers in a dose dependent manner, and the effects may vary greatly between different models. Therefore, fit-testing of UVC decontaminated respirators must be performed each time a new model and/or dose is introduced into the healthcare system.

In Vitro Virucidal Effect of Intranasally Delivered Chlorpheniramine Maleate Compound Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Background. The initial global outbreak of the novel coronavirus disease 2019 (COVID-2019) pandemic, responsible for the severe acute respiratory syndrome 2 (SARS-CoV-2), was first reported in Wuhan, China, at the end of December 2019. COVID-19 shares similarities with the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and it 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 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 chlorpheniramine maleate (CPM) in a nasal spray composition currently in development as an anti-allergy medication.Methods. The virucidal activity of chlorpheniramine maleate was tested using viral stock of SARS-CoV-2, USA-WA1/2020 strain in Vero 76 infected cells. The end-point titer (CCID50) values were calculated with 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 by one-way ANOVA with Dunnett’s multiple comparison test in GraphPad Prism (version 8) software. Results. After 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 reduction of 2.5 log10 CCID50.Conclusions. This study demonstrates the strong virucidal effect against SARS-CoV-2 of a nasal spray containing chlorpheniramine maleate. Given that CPM has broad antiviral effects against influenza, virucidal effect against SARS-CoV-2, and coadjuvant effects with hydroxychloroquine in treating multidrug-resistant malaria with minimal side effects. 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 chloroquine alone, in hospitalized patients with SARS-CoV-2.

Differential Small RNA Responses against Co-Infecting Insect-Specific Viruses in Aedes albopictus Mosquitoes

The mosquito antiviral response has mainly been studied in the context of arthropod-borne virus (arbovirus) infection in female mosquitoes. However, in nature, both female and male mosquitoes are frequently infected with insect-specific viruses (ISVs). ISVs are capable of infecting the reproductive organs of both sexes and are primarily maintained by vertical transmission. Since the RNA interference (RNAi)-mediated antiviral response plays an important antiviral role in mosquitoes, ISVs constitute a relevant model to study sex-dependent antiviral responses. Using a naturally generated viral stock containing three distinct ISVs, Aedes flavivirus (AEFV), Menghai rhabdovirus (MERV), and Shinobi tetra virus (SHTV), we infected adult Aedes albopictus females and males and generated small RNA libraries from ovaries, testes, and the remainder of the body. Overall, both female and male mosquitoes showed unique small RNA profiles to each co-infecting ISV regardless of the sex or tissue tested. While all three ISVs generated virus-derived siRNAs, only MERV generated virus-derived piRNAs. We also studied the expression of PIWI genes in reproductive tissues and carcasses. In contrast to Piwi5-9, Piwi1-4 were abundantly expressed in ovaries and testes, suggesting that Piwi5-9 are involved in exogenous viral piRNA production. Together, our results show that ISV-infected Aedes albopictus produce viral small RNAs in a virus-specific manner and that male mosquitoes mount a similar small RNA-mediated antiviral response to that of females.

Differential small RNA responses against co-infecting insect-specific viruses in Aedes albopictus mosquitoes

The mosquito antiviral response has been mainly studied in the case of arthropod-borne virus (arbovirus) infection in female mosquitoes. However, in nature, both female and male mosquitoes are abundantly infected with insect-specific viruses (ISVs). ISVs are capable of infecting the reproductive organs of both sexes and are maintained primarily by vertical transmission. Since the RNA interference (RNAi)-mediated antiviral response plays an important antiviral role in mosquitoes, ISVs constitute a relevant model to study sex-dependent antiviral responses. Using a naturally generated viral stock containing three distinct ISVs, Aedes flavivirus (AEFV), Menghai Rhabdovirus (MERV) and Shinobi tetra virus (SHTV), we infected adult Aedes albopictus females and males and generated small RNA libraries from ovaries, testes, and the remainder of the body. Overall, both female and male mosquitoes showed unique small RNA profiles to each co-infecting ISV regardless the sex or tissue tested. While all three ISVs generated virus-derived siRNAs, only MERV generated virus-derived piRNAs. We also studied the expression of PIWI genes in reproductive tissues and carcasses. Piwi1-4 were abundantly expressed in ovaries and testes in contrast to Piwi5-9, suggesting that Piwi 5-9 are involved in exogenous viral piRNA production. Together, our results show that ISV-infected Aedes albopictus produce viral small RNAs in a virus-specific manner and that male mosquitoes mount a similar small RNA-mediated antiviral response to that of females.

The Characterization of Immunoprotection Induced by a cDNA Clone Derived from the Attenuated Taiwan Porcine Epidemic Diarrhea Virus Pintung 52 Strain

The porcine epidemic diarrhea virus (PEDV) poses a great threat to the global swine industries and the unreliable protection induced by the currently available vaccines remains a major challenge. We previously generated a genogroup 2b (G2b) PEDV Taiwan Pintung 52 (PEDVPT) strain, PEDVPT-P96, and determined its promising host immune response against the virulent PEDVPT-P5 strain. To study the attenuation determinants of PEDVPT-P96 and establish a PEDVPT-P96-based recombinant vector as a vaccine platform for further antigenicity modification, iPEDVPT-P96, a full-length cDNA clone of PEDVPT-P96, was established. Comparing to the parental PEDVPT-P96 virus, the iPEDVPT-P96 virus showed efficient replication kinetics with a delayed decline of viral load and similar but much more uniform plaque sizes in Vero cells. In the 5-week-old piglet model, fecal viral shedding was observed in the PEDVPT-P96-inoculated piglets, whereas those inoculated with iPEDVPT-P96 showed neither detectable fecal viral shedding nor PEDV-associated clinical signs. Moreover, inoculation with iPEDVPT-P96 elicited comparable levels of anti-PEDV specific plasma IgG and fecal/salivary IgA, neutralizing antibody titers, and similar but less effective immunoprotection against the virulent PEDVPT-P5 challenge compared to the parental PEDVPT-P96. In the present study, an infectious cDNA clone of an attenuated G2b PEDV strain was successfully generated for the first time, and the in vitro and in vivo data indicate that iPEDVPT-P96 is further attenuated but remains immunogenic compared to its parental PEDVPT-P96 viral stock. The successful development of the iPEDVPT-P96 cDNA clone could allow for the manipulation of the viral genome to study viral pathogenesis and facilitate the rapid development of effective vaccines.

A Single Amino Acid Substitution within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes

ABSTRACTOne of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-β) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-β-inducing virus. IFN-β induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.IMPORTANCEThe type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-β induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity.

Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification

ABSTRACT Knowledge of influenza virus evolution at the point of transmission and at the intrahost level remains limited, particularly for human hosts. Here, we analyze a unique viral data set of next-generation sequencing (NGS) samples generated from a human influenza challenge study wherein 17 healthy subjects were inoculated with cell- and egg-passaged virus. Nasal wash samples collected from 7 of these subjects were successfully deep sequenced. From these, we characterized changes in the subjects' viral populations during infection and identified differences between the virus in these samples and the viral stock used to inoculate the subjects. We first calculated pairwise genetic distances between the subjects' nasal wash samples, the viral stock, and the influenza virus A/Wisconsin/67/2005 (H3N2) reference strain used to generate the stock virus. These distances revealed that considerable viral evolution occurred at various points in the human challenge study. Further quantitative analyses indicated that (i) the viral stock contained genetic variants that originated and likely were selected for during the passaging process, (ii) direct intranasal inoculation with the viral stock resulted in a selective bottleneck that reduced nonsynonymous genetic diversity in the viral hemagglutinin and nucleoprotein, and (iii) intrahost viral evolution continued over the course of infection. These intrahost evolutionary dynamics were dominated by purifying selection. Our findings indicate that rapid viral evolution can occur during acute influenza infection in otherwise healthy human hosts when the founding population size of the virus is large, as is the case with direct intranasal inoculation. IMPORTANCE Influenza viruses circulating among humans are known to rapidly evolve over time. However, little is known about how influenza virus evolves across single transmission events and over the course of a single infection. To address these issues, we analyze influenza virus sequences from a human challenge experiment that initiated infection with a cell- and egg-passaged viral stock, which appeared to have adapted during its preparation. We find that the subjects' viral populations differ genetically from the viral stock, with subjects' viral populations having lower representation of the amino-acid-changing variants that arose during viral preparation. We also find that most of the viral evolution occurring over single infections is characterized by further decreases in the frequencies of these amino-acid-changing variants and that only limited intrahost genetic diversification through new mutations is apparent. Our findings indicate that influenza virus populations can undergo rapid genetic changes during acute human infections.