Aaron W. Kolb

Using HSV-1 Genome Phylogenetics to Track Past Human Migrations

We compared 31 complete and nearly complete globally derived HSV-1 genomic sequences using HSV-2 HG52 as an outgroup to investigate their phylogenetic relationships and look for evidence of recombination. The sequences were retrieved from NCBI and were then aligned using Clustal W. The generation of a maximum likelihood tree resulted in a six clade structure that corresponded with the timing and routes of past human migration. The East African derived viruses contained the greatest amount of genetic diversity and formed four of the six clades. The East Asian and European/North American derived viruses formed separate clades. HSV-1 strains E07, E22 and E03 were highly divergent and may each represent an individual clade. Possible recombination was analyzed by partitioning the alignment into 5 kb segments, performing individual phylogenetic analysis on each partition and generating a.phylogenetic network from the results. However most evidence for recombination spread at the base of the tree suggesting that recombination did not significantly disrupt the clade structure. Examination of previous estimates of HSV-1 mutation rates in conjunction with the phylogenetic data presented here, suggests that the substitution rate for HSV-1 is approximately 1.38 × 10(-7) subs/site/year. In conclusion, this study expands the previously described HSV-1 three clade phylogenetic structures to a minimum of six and shows that the clade structure also mirrors global human migrations. Given that HSV-1 has co-evolved with its host, sequencing HSV-1 isolated from various populations could serve as a surrogate biomarker to study human population structure and migration patterns.

Multiple Peptides Homologous to Herpes Simplex Virus Type 1 Glycoprotein B Inhibit Viral Infection

ABSTRACT The 773-residue ectodomain of the herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) has been resistant to the use of mutagenic strategies because the majority of the induced mutations result in defective proteins. As an alternative strategy for the identification of functionally important regions and novel inhibitors of infection, we prepared a library of overlapping peptides homologous to the ectodomain of gB and screened for the ability of the peptides to block infection. Seven of 138 15-mer peptides inhibited infection by more than 50% at a concentration of 100 μM. Three peptides (gB94, gB122, and gB131) with 50% effective concentrations (EC50s) below 20 μM were selected for further studies. The gB131 peptide (residues 681 to 695 in HSV-1 gB [gB-1]) was a specific entry inhibitor (EC50, ∼12 μM). The gB122 peptide (residues 636 to 650 in gB-1) blocked viral entry (EC50, ∼18 μM), protected cells from infection (EC50, ∼72 μM), and inactivated virions in solution (EC50, ∼138 μM). We were unable to discern the step or steps inhibited by the gB94 peptide, which is homologous to residues 496 to 510 in gB-1. Substitution of a tyrosine in the gB122 peptide (Y640 in full-length gB-1) reduced the antiviral activity eightfold, suggesting that this residue is critical for inhibition. This peptide-based strategy could lead to the identification of functionally important regions of gB or other membrane proteins and identify novel inhibitors of HSV-1 entry.

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.

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

ABSTRACT 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.

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.

Prospects for Lentiviral Vector Mediated Prostaglandin F Synthase Gene Delivery in Monkey Eyes In vivo

Abstract Currently, the most effective outflow drugs approved for clinical use are prostaglandin F2α analogues, but these require daily topical self-dosing and have various intraocular, ocular surface and extraocular side effects. Lentiviral vector-mediated delivery of the prostaglandin F synthase (PGFS) gene, resulting in long-term reduction of intraocular pressure (IOP), may eliminate off-target tissue effects and the need for daily topical PGF2α self-administration. Lentiviral vector-mediated delivery of the PGFS gene to the anterior segment has been achieved in cats and non-human primates. Although these results are encouraging, our studies have identified a number of challenges that need to be overcome for prostaglandin gene therapy to be translated into the clinic. Using examples from our work in non-human primates, where we were able to achieve a significant reduction in IOP (2 mm Hg) for 5 months after delivery of the cDNA for bovine PGF synthase, we identify and discuss these issues and consider several possible solutions.

Phylogenetic and recombination analysis of the herpesvirus genus varicellovirus

BackgroundThe 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.ResultsA 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.ConclusionTogether, the data contribute to a greater understanding of varicellovirus genomics, and we also suggest a new clade nomenclature scheme based on genetic distances.

Genomic, Recombinational and Phylogenetic Characterization of Global Feline Herpesvirus 1 Isolates

Feline herpes virus type 1 (FHV-1) is widely considered to be the leading cause of ocular disease in cats and has been implicated in upper respiratory tract infections. Little, however is known about interstrain phylogenetic relationships, and details of the genomic structure. For the present study, twenty-six FHV-1 isolates from different cats in animal shelters were collected from eight separate locations in the USA, and the genomes sequenced. Genomic characterization of these isolates includied short sequence repeat (SSR) detection, with fewer SSRs detected, compared to herpes simplex viruses type 1 and 2. For phylogenetic and recombination analysis, 27 previously sequenced isolates of FHV-1 were combined with the 26 strains sequenced for the present study. The overall genomic interstrain genetic distance between all available isolates was 0.093%. Phylogenetic analysis identified four main FHV-1 clades primarily corresponding to geographical collection site. Recombination analysis suggested that interclade recombination has occurred.

Toll-like receptors 4, 5, 6 and 7 are constitutively expressed in non-human primate retinal neurons

The purpose of this study was to characterize cell-specific expression patterns of Toll-like receptors (TLR) in non-human primate (NHP) neural retina tissue. TLR 4, 5, 6, and 7 proteins were detected by immunblotting of macaque retina tissue lysates and quantitative PCR (qPCR) demonstrated TLRs 4-7 mRNA expression. Immunofluorescence (IF) microscopy detected TLRs 4-7 in multiple cell types in macaque neural retina including Muller, retinal ganglion cells (RGC), amacrine, and bipolar cells. These results demonstrate that TLRs 4-7 are constitutively expressed by neurons in the NHP retina raising the possibility that these cells could be involved in retinal innate inflammatory responses.