Inna V. Larsen

Genomic, Phylogenetic, and Recombinational Characterization of Herpes Simplex Virus 2 Strains

ABSTRACT 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.

Ocular Distribution, Spectrum of Activity, and In Vivo Viral Neutralization of a Fully Humanized Anti-Herpes Simplex Virus IgG Fab Fragment following Topical Application

ABSTRACT 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 (MEC50 and MEC90, 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.

In Vivo Anti-Herpes Simplex Virus Activity of a Sulfated Derivative of Agaricus brasiliensis Mycelial Polysaccharide

ABSTRACT Agaricus brasiliensis (syn. A. subrufescens), a basidiomycete fungus native to the Atlantic forest in Brazil, contains cell walls rich in glucomannan polysaccharides. The β-(1→2)-gluco-β-(1→3)-mannan was isolated from A. brasiliensis mycelium, chemically modified by sulfation, and named MI-S. MI-S has multiple mechanisms of action, including inhibition of herpes simplex virus (HSV) attachment, entry, and cell-to-cell spread (F. T. G. S. Cardozo, C. M. Camelini, A. Mascarello, M. J. Rossi, R. J. Nunes, C. R. Barardi, M. M. de Mendonça, and C. M. O. Simões, Antiviral Res. 92:108–114, 2011). The antiherpetic efficacy of MI-S was assessed in murine ocular, cutaneous, and genital infection models of HSV. Groups of 10 mice were infected with HSV-1 (strain KOS) or HSV-2 (strain 333). MI-S was given either topically or by oral gavage under various pre- and posttreatment regimens, and the severity of disease and viral titers in ocular and vaginal samples were determined. No toxicity was observed in the uninfected groups treated with MI-S. The topical and oral treatments with MI-S were not effective in reducing ocular disease. Topical application of MI-S on skin lesions was also not effective, but cutaneously infected mice treated orally with MI-S had significantly reduced disease scores (P < 0.05) after day 9, suggesting that healing was accelerated. Vaginal administration of MI-S 20 min before viral challenge reduced the mean disease scores on days 5 to 9 (P < 0.05), viral titers on day 1 (P < 0.05), and mortality (P < 0.0001) in comparison to the control groups (untreated and vehicle treated). These results show that MI-S may be useful as an oral agent to reduce the severity of HSV cutaneous and mucosal lesions and, more importantly, as a microbicide to block sexual transmission of HSV-2 genital infections.

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.

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.

ABSTRACT 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.

A cationic TAT peptide inhibits Herpes simplex virus type 1 infection of human corneal epithelial cells.

UNLABELLED Abstract Purpose: To determine if a peptide, TAT-Cd(0), inhibits Herpes simplex virus type 1 infection of human corneal epithelial cells. METHODS TAT-Cd(0) and a control peptide, E(50,51)TAT-Cd(0), were added at various times throughout infection with the lacz-expressing hrR3 virus, and viral replication was measured by β-galactosidase activity. Toxicity was assessed using a dye reduction assay. RESULTS The CC(50) value for TAT-Cd(0) was ∼100 μM. In assays with peptide present at all times, TAT-Cd(0) was 150-fold more active than E(50,51)TAT-Cd(0) (EC(50) 0.2 vs. 30.0 μM). The EC(50) values of TAT-Cd(0) for entry inhibition, cell protection, virus inactivation, and inhibition of attachment were 0.1, 0.4, 9.5, and 3.0 μM, respectively. TAT-Cd(0) was less effective when added 1 h postinfection (EC(50) = 30.0 μM). CONCLUSIONS TAT-Cd(0) is an effective inhibitor of Herpes simplex virus type 1 infection in human corneal epithelial cells and affects multiple steps before, or very early, in infection. The peptide has potential as an antiviral and further studies are warranted.

Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork.

Purpose To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector–mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS). Methods TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 μM MG132 and transduced with FIV.GFP (green fluorescent protein)– or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 μM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA. Results In the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 μM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus. Conclusions Proteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.

Both CD8+ and CD4+ T Cells Contribute to Corneal Clouding and Viral Clearance following Vaccinia Virus Infection in C57BL/6 Mice

ABSTRACT Vaccinia virus (VACV) keratitis is a serious complication following smallpox vaccination and can lead to blindness. The pathological mechanisms involved in ocular VACV infection are poorly understood. Previous studies have used rabbits, but the lack of immune reagents and transgenic or knockout animals makes them less suitable for mechanistic studies. We report that infection of C57BL/6 mice with 1 × 107 PFU of vaccinia virus strain WR results in blepharitis, corneal neovascularization, and stromal keratitis. The DryVax strain of VACV was completely attenuated. Infection required corneal scarification and replication-competent virus, and the severity of ocular disease was similar in 4- to 6-week-old and 1-year-old mice. Viral titers peaked at approximately 1 × 106 PFU on day 5 postinfection, and virus had not cleared by day 13 postinfection. Neutrophils were found in the peripheral cornea on day 1 after infection and then declined, followed by infiltration of both CD4+ and CD8+ T cells, which remained peripheral throughout the infection. Blood vessel growth extended 2 to 5 mm into the cornea from the limbus. Infection of CD4−/−, CD8−/−, or antibody-depleted mice resulted in similar disease severity and corneal clouding, indicating that both T-cell subsets were involved in the immunopathological response. Depletion of both CD4+ and CD8+ T cells resulted in significantly more severe disease and failure to clear the virus. On the basis of our results, the pathology of VACV keratitis is significantly different from that of herpes simplex virus keratitis. Further studies are likely to reveal novel information regarding virulence and immune responses to viral ocular infection. IMPORTANCE Potentially blinding eye infections can occur after vaccination for smallpox. Very little is known about the pathological mechanisms that are involved, and the information that is available was generated using rabbit models. The lack of immunological reagents for rabbits makes such studies difficult. We characterized a mouse model of vaccinia virus ocular disease using C57BL/6 mice and strain WR and show that both CD4+ and CD8+ T-cell subsets play a role in the blinding eye disease and in controlling virus replication. On the basis of these results, vaccinia virus keratitis is significantly different from herpes simplex virus keratitis, and further studies using this model should generate novel insights into immunopathological responses to viral ocular infection.