Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

The RLR intrinsic antiviral system is expressed in neural retina and restricts lentiviral transduction of human Mueller cells.

The retinoic acid-inducible gene I (RIG)-I-like receptor (RLR) family of RNA sensor proteins plays a key role in the innate immune response to viral nucleic acids, including viral gene delivery vectors, but little is known about the expression of RLR proteins in the retina. The purpose of this study was to characterize cell-specific expression patterns of RLR proteins in non-human primate (NHP) neural retina tissue and to examine if RLR pathway signaling restricts viral gene delivery transduction. Since RLR protein signaling converges at the mitochondrial antiviral signaling protein (MAVS), experiments were performed to determine if knockdown of MAVS affected FIVGFP transduction efficiency in the human Mueller cell line MIO-M1. Immunoblotting confirmed expression of RIG-I, melanoma differentiation-associated protein 5 (MDA5), laboratory of genetics and physiology 2 (LGP2), and MAVS proteins in MIO-M1 cells and NHP retina tissue. Double label immunofluorescence (IF) studies revealed RIG-I, LGP2, and MAVS were expressed in Mueller microglial cells in the NHP retina. In addition, LGP2 and MDA5 proteins were detected in cone and retinal ganglion cells (RGC). MDA5 was also present in a subset of calretinin positive amacrine cells, and in nuclei within the inner nuclear layer (INL). Knockdown of MAVS significantly increased the transduction efficiency of the lentiviral vector FIVGFP in MIO-M1 cells, compared to control cells. FIVGFP or AAVGFP challenge did not alter expression of the LGP2, MAVS, MDA5 or RIG-I genes in MIO-M1 cells or NHP retina tissue compared to media treated controls. Our data demonstrate that innate immune response proteins involved in viral RNA sensing, including MDA5, RIG-I, LGP2, and MAVS, are expressed in several cell types within the NHP neural retina. In addition, the MAVS protein restricts non-human lentiviral transduction efficiency in MIO-M1 cells.

ICTV Virus Taxonomy Profile: Herpesviridae 2021.

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125-241 kbp contain 70-170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.

Knockdown of TRIM5α or TRIM11 increases lentiviral vector transduction efficiency of human Muller cells.

The goal of this study was to determine the expression and distribution of the host restriction factors (RFs) TRIM5α and TRIM11 in non-human primate (NHP) neural retina tissue and the human Muller cell line MIO-M1. In addition, experiments were performed to determine the effect of TRIM5α and TRIM11 knockdown on FIVGFP transduction of MIO-M1 cells with the goal of devising strategies to increase the efficiency of lentiviral (LV) gene delivery. Immunofluorescence (IF) studies indicated that TRIM5α and TRIM11 were localized predominantly in nuclei within the outer nuclear layer (ONL) and inner nuclear layer (INL) of NHP retina tissue. Double label IF indicated that TRIM5α and TRIM11 were localized to some of the retinal Muller cell nuclei. MIO-M1 cells expressed TRIM5α predominantly in the nucleus and TRIM11 primarily in the cytosol. FIVGFP transduction efficiency was significantly increased, at 4 and 7 days post transduction, in TRIM5α and TRIM11 knockdown clones (KD) compared to WT MIO-M1 cells. In addition, pretreatment with the proteasome inhibitor MG132 increased the transduction efficiency of FIVGFP in WT MIO-M1 cells. The nuclear translocation of NF-κB (p65), at 72 h post FIVGFP transduction, was enhanced in TRIM5α and TRIM11 KD clones. The expression of TRIM5α and TRIM11 in macaque neural retina tissue and MIO-M1 cells indicate the presence of these RFs in NHP retina and human Muller cells. Our data indicate that even partial knockdown of TRIM5α or TRIM11, or a short proteasome inhibitor pretreatment, can increase the transduction efficiency of a LV vector.

Genomic nucleotide-based distance analysis for delimiting old world monkey derived herpes simplex virus species.

BACKGROUND: Herpes simplex viruses form a genus within the alphaherpesvirus subfamily, with three identified viral species isolated from Old World monkeys (OWM); Macacine alphaherpesvirus 1 (McHV-1; herpes B), Cercopithecine alphaherpesvirus 2 (SA8), and Papiine alphaherpesvirus 2 (PaHV-2; herpes papio). Herpes B is endemic to macaques, while PaHV-2 and SA8 appear endemic to baboons. All three viruses are genetically and antigenically similar, with SA8 and PaHV-2 thought to be avirulent in humans, while herpes B is a biosafety level 4 pathogen. Recently, next-generation sequencing (NGS) has resulted in an increased number of published OWM herpes simplex genomes, allowing an encompassing phylogenetic analysis. RESULTS: In this study, phylogenetic networks, in conjunction with a genome-based genetic distance cutoff method were used to examine 27 OWM monkey herpes simplex isolates. Genome-based genetic distances were calculated, resulting in distances between lion and pig-tailed simplex viruses themselves, and versus herpes B core strains that were higher than those between PaHV-2 and SA8 (approximately 14 and 10% respectively). The species distance cutoff was determined to be 8.94%, with the method recovering separate species status for PaHV-2 and SA8 and showed that lion and pig-tailed simplex viruses (vs core herpes B strains) were well over the distance species cutoff. CONCLUSIONS: We propose designating lion and pig-tailed simplex viruses as separate, individual viral species, and that this may be the first identification of viral cryptic species.

Quantitative Trait Locus Based Virulence Determinant Mapping of the HSV-1 Genome in Murine Ocular Infection: Genes Involved in Viral Regulatory and Innate Immune Networks Contribute to Virulence.

Herpes simplex virus type 1 causes mucocutaneous lesions, and is the leading cause of infectious blindness in the United States. Animal studies have shown that the severity of HSV-1 ocular disease is influenced by three main factors; innate immunity, host immune response and viral strain. We previously showed that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) resulted in recombinants that exhibit a range of disease phenotypes from severe to avirulent, suggesting epistatic interactions were involved. The goal of this study was to develop a quantitative trait locus (QTL) analysis of HSV-1 ocular virulence determinants and to identify virulence associated SNPs. Blepharitis and stromal keratitis quantitative scores were characterized for 40 OD4:CJ994 recombinants. Viral titers in the eye were also measured. Virulence quantitative trait locus mapping (vQTLmap) was performed using the Lasso, Random Forest, and Ridge regression methods to identify significant phenotypically meaningful regions for each ocular disease parameter. The most predictive Ridge regression model identified several phenotypically meaningful SNPs for blepharitis and stromal keratitis. Notably, phenotypically meaningful nonsynonymous variations were detected in the UL24, UL29 (ICP8), UL41 (VHS), UL53 (gK), UL54 (ICP27), UL56, ICP4, US1 (ICP22), US3 and gG genes. Network analysis revealed that many of these variations were in HSV-1 regulatory networks and viral genes that affect innate immunity. Several genes previously implicated in virulence were identified, validating this approach, while other genes were novel. Several novel polymorphisms were also identified in these genes. This approach provides a framework that will be useful for identifying virulence genes in other pathogenic viruses, as well as epistatic effects that affect HSV-1 ocular virulence.

Phylogenetic and recombination analysis of the herpesvirus genus varicellovirus.

BACKGROUND: The varicelloviruses comprise a genus within the alphaherpesvirus subfamily, and infect both humans and other mammals. Recently, next-generation sequencing has been used to generate genomic sequences of several members of the Varicellovirus genus. Here, currently available varicellovirus genomic sequences were used for phylogenetic, recombination, and genetic distance analysis. RESULTS: A phylogenetic network including genomic sequences of individual species, was generated and suggested a potential restriction between the ungulate and non-ungulate viruses. Intraspecies genetic distances were higher in the ungulate viruses (pseudorabies virus (SuHV-1) 1.65%, bovine herpes virus type 1 (BHV-1) 0.81%, equine herpes virus type 1 (EHV-1) 0.79%, equine herpes virus type 4 (EHV-4) 0.16%) than non-ungulate viruses (feline herpes virus type 1 (FHV-1) 0.0089%, canine herpes virus type 1 (CHV-1) 0.005%, varicella-zoster virus (VZV) 0.136%). The G + C content of the ungulate viruses was also higher (SuHV-1 73.6%, BHV-1 72.6%, EHV-1 56.6%, EHV-4 50.5%) compared to the non-ungulate viruses (FHV-1 45.8%, CHV-1 31.6%, VZV 45.8%), which suggests a possible link between G + C content and intraspecies genetic diversity. Varicellovirus clade nomenclature is variable across different species, and we propose a standardization based on genomic genetic distance. A recent study reported no recombination between sequenced FHV-1 strains, however in the present study, both splitstree, bootscan, and PHI analysis indicated recombination. We also found that the recently sequenced Brazilian CHV-1 strain BTU-1 may contain a genetic signal in the UL50 gene from an unknown varicellovirus. CONCLUSION: Together, the data contribute to a greater understanding of varicellovirus genomics, and we also suggest a new clade nomenclature scheme based on genetic distances.

Mapping Murine Corneal Neovascularization and Weight Loss Virulence Determinants in the Herpes Simplex Virus 1 Genome and the Detection of an Epistatic Interaction between the UL and IRS/US Regions.

UNLABELLED: Herpes simplex virus 1 (HSV-1) most commonly causes recrudescent labial ulcers; however, it is also the leading cause of infectious blindness in developed countries. Previous research in animal models has demonstrated that the severity of HSV-1 ocular disease is influenced by three main factors: host innate immunity, host immune response, and viral strain. We have previously shown that mixed infection with two avirulent HSV-1 strains (OD4 and CJ994) results in recombinants with a wide range of ocular disease phenotype severity. Recently, we developed a quantitative trait locus (QTL)-based computational approach (vQTLmap) to identify viral single nucleotide polymorphisms (SNPs) predicted to influence the severity of the ocular disease phenotypes. We have now applied vQTLmap to identify HSV-1 SNPs associated with corneal neovascularization and mean peak percentage weight loss (MPWL) using 65 HSV-1 OD4-CJ994 recombinants. The vQTLmap analysis using Random Forest for neovascularization identified phenotypically meaningful nonsynonymous SNPs in the ICP4, UL41 (VHS), UL42, UL46 (VP11/12), UL47 (VP13/14), UL48 (VP22), US3, US4 (gG), US6 (gD), and US7 (gI) coding regions. The ICP4 gene was previously identified as a corneal neovascularization determinant, validating the vQTLmap method. Further analysis detected an epistatic interaction for neovascularization between a segment of the unique long (UL) region and a segment of the inverted repeat short (IRS)/unique short (US) region. Ridge regression was used to identify MPWL-associated nonsynonymous SNPs in the UL1 (gL), UL2, UL4, UL49 (VP22), UL50, and ICP4 coding regions. The data provide additional insights into virulence gene and epistatic interaction discovery in HSV-1. IMPORTANCE: Herpes simplex virus 1 (HSV-1) typically causes recurrent cold sores; however, it is also the leading source of infectious blindness in developed countries. Corneal neovascularization is critical for the progression of blinding ocular disease, and weight loss is a measure of infection severity. Previous HSV-1 animal virulence studies have shown that the severity of ocular disease is partially due to the viral strain. In the current study, we used a recently described computational quantitative trait locus (QTL) approach in conjunction with 65 HSV-1 recombinants to identify viral single nucleotide polymorphisms (SNPs) involved in neovascularization and weight loss. Neovascularization SNPs were identified in the ICP4, VHS, UL42, VP11/12, VP13/14, VP22, gG, US3, gD, and gI genes. Further analysis revealed an epistatic interaction between the UL and US regions. MPWL-associated SNPs were detected in the UL1 (gL), UL2, UL4, VP22, UL50, and ICP4 genes. This approach will facilitate future HSV virulence studies.

Diacylglycerol acyltransferase-2 and monoacylglycerol acyltransferase-2 are ubiquitinated proteins that are degraded by the 26S proteasome.

Acyl-CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is one of the two DGAT enzymes that catalyzes the synthesis of triacylglycerol, which is an important form of stored energy for eukaryotic organisms. There is currently limited information available regarding how DGAT2 and triacylglycerol synthesis are regulated. Recent studies have indicated that DGAT2 can be regulated by changes in gene expression. How DGAT2 is regulated post-transcriptionally remains less clear. In this study, we demonstrated that DGAT2 is a very unstable protein and is rapidly degraded in an ubiquitin-dependent manner via the proteasome. Many of the 25 lysines present in DGAT2 appeared to be involved in promoting its degradation. However, the six C-terminal lysines were the most important in regulating stability. We also demonstrated that acyl-CoA:monoacylglycerol acyltransferase (MGAT)-2, an enzyme with extensive sequence homology to DGAT2 that catalyzes the synthesis of diacylglycerol, was also ubiquitinated. However, MGAT2 was found to be much more stable than DGAT2. Interestingly, when co-expressed, MGAT2 appeared to stabilize DGAT2. Finally, we found that both DGAT2 and MGAT2 are substrates of the endoplasmic reticulum-associated degradation pathway.

Biochemical characterization of human acyl coenzyme A: 2-monoacylglycerol acyltransferase-3 (MGAT3).

BACKGROUND: MGAT3 catalyzes the synthesis of 1,2-diacylglycerol from 2-monoacylglycerol in an acyl CoA-dependent reaction. Although initially identified as an MGAT enzyme, MGAT3 is more closely related to DGAT2 than to MGAT1 and MGAT2. Furthermore, MGAT3 possesses both DGAT and MGAT activities, in vitro. MGAT3 is almost exclusively expressed in the small intestine in humans, suggesting that it has a role in dietary fat absorption. Although identified many years ago, little information is available regarding the contribution of MGAT3 to triacylglycerol biosynthesis. RESULTS: This study confirmed the initial observations that MGAT3 possessed both MGAT and DGAT activities. When expressed in cells in culture, MGAT3 stimulated lipid droplet growth, but unlike DGAT2, does not become concentrated around the lipid droplet surface. We also characterized the MGAT activity of an MGAT3 mutant in which a conserved cysteine was changed to a tyrosine residue. Lastly, although they share significant sequence identity, MGAT3 is a much more stable protein than DGAT2, yet they are both polyubiquitinated and degraded through ER-associated degradation by the proteasome. CONCLUSION: Our findings provide additional evidence that MGAT3 likely functions as a TG synthase in cells.

Genomic, phylogenetic, and recombinational characterization of herpes simplex virus 2 strains.

UNLABELLED: Herpes simplex virus 2 (HSV-2) is a major global pathogen, infecting 16% of people 15 to 49 years old worldwide and causing recurrent genital ulcers. Little is known about viral factors contributing to virulence, and there are currently only two genomic sequences available. In this study, we determined nearly complete genomic sequences of six additional HSV-2 isolates, using Illumina MiSeq. We report that HSV-2 has a genomic overall mean distance of 0.2355%, which is less than that of HSV-1. There were approximately 100 amino-acid-encoding and indels per genome. Microsatellite mapping found a bias toward intergenic regions in the nonconserved microsatellites and a genic bias in all detected tandem repeats. Extensive recombination between the HSV-2 strains was also strongly implied. This was the first study to analyze multiple HSV-2 sequences, and the data will be valuable in future evolutionary, virulence, and structure-function studies. IMPORTANCE: HSV-2 is a significant worldwide pathogen, causing recurrent genital ulcers. Here we present six nearly complete HSV-2 genomic sequences, and, with the addition of two previously sequenced strains, for the first time genomic, phylogenetic, and recombination analysis was performed on multiple HSV-2 genomes. Our results show that microsatellite mapping found a bias toward intergenic regions in the nonconserved microsatellites and a genic bias in all detected tandem repeats and confirm that chimpanzee herpesvirus 1 (ChHV-1) is a separate species and that each of the HSV-2 strains is a genomic mosaic.

Recombination Analysis of Herpes Simplex Virus 1 Reveals a Bias toward GC Content and the Inverted Repeat Regions.

UNLABELLED: Herpes simplex virus 1 (HSV-1) causes recurrent mucocutaneous ulcers and is the leading cause of infectious blindness and sporadic encephalitis in the United States. HSV-1 has been shown to be highly recombinogenic; however, to date, there has been no genome-wide analysis of recombination. To address this, we generated 40 HSV-1 recombinants derived from two parental strains, OD4 and CJ994. The 40 OD4-CJ994 HSV-1 recombinants were sequenced using the Illumina sequencing system, and recombination breakpoints were determined for each of the recombinants using the Bootscan program. Breakpoints occurring in the terminal inverted repeats were excluded from analysis to prevent double counting, resulting in a total of 272 breakpoints in the data set. By placing windows around the 272 breakpoints followed by Monte Carlo analysis comparing actual data to simulated data, we identified a recombination bias toward both high GC content and intergenic regions. A Monte Carlo analysis also suggested that recombination did not appear to be responsible for the generation of the spontaneous nucleotide mutations detected following sequencing. Additionally, kernel density estimation analysis across the genome found that the large, inverted repeats comprise a recombination hot spot. IMPORTANCE: Herpes simplex virus 1 (HSV-1) virus is the leading cause of sporadic encephalitis and blinding keratitis in developed countries. HSV-1 has been shown to be highly recombinogenic, and recombination itself appears to be a significant component of genome replication. To date, there has been no genome-wide analysis of recombination. Here we present the findings of the first genome-wide study of recombination performed by generating and sequencing 40 HSV-1 recombinants derived from the OD4 and CJ994 parental strains, followed by bioinformatics analysis. Recombination breakpoints were determined, yielding 272 breakpoints in the full data set. Kernel density analysis determined that the large inverted repeats constitute a recombination hot spot. Additionally, Monte Carlo analyses found biases toward high GC content and intergenic and repetitive regions.

Primate neural retina upregulates IL-6 and IL-10 in response to a herpes simplex vector suggesting the presence of a pro-/anti-inflammatory axis.

Injection of herpes simplex virus vectors into the vitreous of primate eyes induces an acute, transient uveitis. The purpose of this study was to characterize innate immune responses of macaque neural retina tissue to the herpes simplex virus type 1-based gene delivery vector hrR3. PCR array analysis demonstrated the induction of the pro-inflammatory cytokine IL-6, as well as the anti-inflammatory cytokine IL-10, following hrR3 exposure. Secretion of IL-6 was detected by ELISA and cone photoreceptors and Muller cells were the predominant IL-6 positive cell types. RNA in situ hybridization confirmed that IL-6 was expressed in photoreceptor and Muller cells. The IL-10 positive cells in the inner nuclear layer were identified as amacrine cells by immunofluorescence staining with calretinin antibody. hrR3 challenge resulted in activation of NFκB (p65) in Muller glial cells, but not in cone photoreceptors, suggesting a novel regulatory mechanism for IL-6 expression in cone cells. hrR3 replication was not required for IL-6 induction or NFκB (p65) activation. These data suggest a pro-inflammatory (IL-6)/anti-inflammatory (IL-10) axis exists in neural retina and the severity of acute posterior uveitis may be determined by this interaction. Further studies are needed to identify the trigger for IL-6 and IL-10 induction and the mechanism of IL-6 induction in cone cells.

Diacylglycerol acyltransferase-2 (DGAT2) and monoacylglycerol acyltransferase-2 (MGAT2) interact to promote triacylglycerol synthesis.

Acyl CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is an integral membrane protein that catalyzes triacylglycerol (TG) synthesis using diacylglycerol and fatty acyl CoA as substrates. DGAT2 resides in the endoplasmic reticulum (ER), but when cells are incubated with fatty acids, DGAT2 interacts with lipid droplets presumably to catalyze localized TG synthesis for lipid droplet expansion. Previous studies have shown that DGAT2 interacts with proteins that synthesize its fatty acyl CoA substrates. In this study, we provide additional evidence that DGAT2 is present in a protein complex. Using a chemical cross-linker, disuccinimidyl suberate (DSS), we demonstrated that DGAT2 formed a dimer and was also part of a protein complex of ∼ 650 kDa, both in membranes and on lipid droplets. Using co-immunoprecipitation experiments and an in situ proximity ligation assay, we found that DGAT2 interacted with monoacylglycerol acyltransferase (MGAT)-2, an enzyme that catalyzes the synthesis of diacylglycerol. Deletion mutagenesis showed that the interaction with MGAT2 was dependent on the two transmembrane domains of DGAT2. No significant interaction of DGAT2 with lipin1, another enzyme that synthesizes diacylglycerol, could be detected. When co-expressed in cells, DGAT2 and MGAT2 co-localized in the ER and on lipid droplets. Co-expression also resulted in increased TG storage compared with expression of DGAT2 or MGAT2 alone. Incubating McArdle rat hepatoma RH7777 cells with 2-monoacylglycerol caused DGAT2 to translocate to lipid droplets. This also led to the formation of large cytosolic lipid droplets, characteristic of DGAT2, but not DGAT1, and indicated that DGAT2 can utilize monoacylglycerol-derived diacylglycerol. These findings suggest that the interaction of DGAT2 and MGAT2 serves to channel lipid substrates efficiently for TG biosynthesis.

Phylogenetic and Recombination Analysis of Clinical Vitreous Humor-Derived Adenovirus Isolates Reveals Discordance Between Serotype and Phylogeny.

Purpose: To sequence, identify, and perform phylogenetic and recombination analysis on three clinical adenovirus samples taken from the vitreous humor at the Bascom Palmer Eye Institute. Methods: The PacBio Sequel II was used to sequence the genomes of the three clinical adenovirus isolates. To identify the isolates, a full genome-based multiple sequence alignment (MSA) of 722 mastadenoviruses was generated using multiple alignment using fast Fourier transform (MAFFT). MAFFT was also used to generate genome-based human adenovirus B (HAdV-B) MSAs, as well as HAdV-B fiber, hexon, and penton protein-based MSAs. To examine recombination within HAdV-B, RF-Net 2 and Bootscan software programs were used. Results: In the course of classifying three new atypical ocular adenovirus samples, taken from the vitreous humor, we found that all three isolates were HAdV-B species. The three Bascom Palmer HAdV-B genomes were then combined with over 300 HAdV-B genome sequences, including nine ocular HAdV-B genome sequences. Attempts to categorize the penton, hexon, and fiber serotypes using phylogeny of the three Bascom Palmer samples were inconclusive due to incongruence between serotype and phylogeny in the dataset. Recombination analysis using a subset of HAdV-B strains to generate a hybridization network detected recombination between nonhuman primate and human-derived strains, recombination between one HAdV-B strain and the HAdV-E outgroup, and limited recombination between the B1 and B2 clades. Conclusions: The discordance between serotype and phylogeny detected in this study suggests that the current classification system does not accurately describe the natural history and phylogenetic relationships among adenoviruses.

Disease parameters following ocular herpes simplex virus type 1 infection are similar in male and female BALB/C mice.

Sex related differences in the incidence or severity of infection have been described for multiple viruses. With herpes simplex viruses, the best example is HSV-2 genital infection where women have a higher incidence of infection and can have more severe infections than men. HSV-1 causes several types of infections including skin and mucosal ulcers, keratitis, and encephalitis in humans that do not appear to have a strong biological sex component. Given that mouse strains differ in their MHC loci it is important to determine if sex differences occur in multiple strains of mice. Our goal was to answer two questions: Are virus related sex differences present in BALB/C mice and does virulence of the viral strain have an effect? We generated a panel of recombinant HSV-1 viruses with differing virulence phenotypes and characterized multiple clinical correlates of ocular infection in BALB/c mice. We found no sex-specific differences in blepharitis, corneal clouding, neurovirulence, and viral titers in eye washes. Sex differences in neovascularization, weight loss and eyewash titers were observed for some recombinants, but these were not consistent across the phenotypes tested for any recombinant virus. Considering these findings, we conclude that there are no significant sex specific ocular pathologies in the parameters measured, regardless of the virulence phenotype following ocular infection in BALB/c mice, suggesting that the use of both sexes is not necessary for the bulk of ocular infection studies.

Phylogenetic and Genomic Characterization of Whole Genome Sequences of Ocular Herpes Simplex Virus Type 1 Isolates Identifies Possible Virulence Determinants in Humans.

Purpose: There are limited data on the prevalence and genetic diversity of herpes simplex virus type 1 (HSV-1) virulence genes in ocular isolates. Here, we sequenced 36 HSV-1 ocular isolates, collected by the Bascom Palmer Eye Institute, a university-based eye hospital, from three different ocular anatomical sites (conjunctiva, cornea, and eyelid) and carried out a genomic and phylogenetic analyses. Methods: The PacBio Sequel II long read platform was used for genome sequencing. Phylogenetic analysis and genomic analysis were performed to help better understand genetic variability among common virulence genes in ocular herpetic disease. Results: A phylogenetic network generated using the genome sequences of the 36 Bascom Palmer ocular isolates, plus 174 additional strains showed that ocular isolates do not group together phylogenetically. Analysis of the thymidine kinase and DNA polymerase protein sequences from the Bascom Palmer isolates showed multiple novel single nucleotide polymorphisms, but only one, BP-K14 encoded a known thymidine kinase acyclovir resistance mutation. An analysis of the multiple sequence alignment comprising the 51 total ocular isolates versus 159 nonocular strains detected several possible single nucleotide polymorphisms in HSV-1 genes that were found significantly more often in the ocular isolates. These genes included UL6, gM, VP19c, VHS, gC, VP11/12, and gG. Conclusions: There does not seem to be a specific genetic feature of viruses causing ocular infection. The identification of novel and common recurrent polymorphisms may help to understand the drivers of herpetic pathogenicity and specific factors that may influence the virulence of ocular disease.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Ocular distribution, spectrum of activity, and in vivo viral neutralization of a fully humanized anti-herpes simplex virus IgG Fab fragment following topical application.

Herpes simplex ocular infection is a major cause of corneal blindness. Local antiviral treatments exist but are associated with corneal toxicity, and resistance has become an issue. We evaluated the biodistribution and efficacy of a humanized anti-herpes simplex virus (anti-HSV) IgG FAb fragment (AC-8; 53 kDa) following repeated topical administration. AC-8 was found in the corneal epithelium, anterior stroma, subepithelial stromal cells, and retinal glial cells, with preferential entry through the ocular limbus. AC-8 was active against 13 different strains of HSV-1, with 50% and 90% mean effective concentrations (MEC(50) and MEC(90), respectively) ranging from 0.03 to 0.13 µg/ml, indicating broad-spectrum activity. The in vivo efficacy of AC-8 was evaluated in a mouse model of herpes-induced ocular disease. Treatment with low-dose AC-8 (1 mg/ml) slightly reduced the ocular disease scores. A greater reduction of the disease scores was observed in the 10-mg/ml AC-8-treated group, but not as much as with trifluridine (TFT). AC-8 treatment reduced viral titers but less than trifluridine. AC-8 did not display any toxicity to the cornea or other structures in the eye. In summary, topical instillation of an anti-HSV FAb can be used on both intact and ulcerated corneas. It is well tolerated and does not alter reepithelialization. Further studies to improve the antiviral effect are needed for AC-8 to be considered for therapeutic use.