Multi-phenotype analysis for enhanced classification of 11 herpes simplex virus 1 strains.

Herpes simplex virus 1 (HSV1) is best known for causing oral lesions and mild clinical symptoms, but it can produce a significant range of disease severities and rates of reactivation. To better understand this phenotypic variation, we characterized 11 HSV1 strains that were isolated from individuals with diverse infection outcomes. We provide new data on genomic and in vitro plaque phenotype analysis for these isolates and compare these data to previously reported quantitation of the disease phenotype of each strain in a murine animal model. We show that integration of these three types of data permitted clustering of these HSV1 strains into four groups that were not distinguishable by any single dataset alone, highlighting the benefits of combinatorial multi-parameter phenotyping. Two strains (group 1) produced a partially or largely syncytial plaque phenotype and attenuated disease phenotypes in mice. Three strains of intermediate plaque size, causing severe disease in mice, were genetically clustered to a second group (group 2). Six strains with the smallest average plaque sizes were separated into two subgroups (groups 3 and 4) based on their different genetic clustering and disease severity in mice. Comparative genomics and network graph analysis suggested a separation of HSV1 isolates with attenuated vs. virulent phenotypes. These observations imply that virulence phenotypes of these strains may be traceable to genetic variation within the HSV1 population.

Comparison of herpes simplex virus 1 genomic diversity between adult sexual transmission partners with genital infection.

Herpes simplex virus (HSV) causes chronic infection in the human host, characterized by self-limited episodes of mucosal shedding and lesional disease, with latent infection of neuronal ganglia. The epidemiology of genital herpes has undergone a significant transformation over the past two decades, with the emergence of HSV-1 as a leading cause of first-episode genital herpes in many countries. Though dsDNA viruses are not expected to mutate quickly, it is not yet known to what degree the HSV-1 viral population in a natural host adapts over time, or how often viral population variants are transmitted between hosts. This study provides a comparative genomics analysis for 33 temporally-sampled oral and genital HSV-1 genomes derived from five adult sexual transmission pairs. We found that transmission pairs harbored consensus-level viral genomes with near-complete conservation of nucleotide identity. Examination of within-host minor variants in the viral population revealed both shared and unique patterns of genetic diversity between partners, and between anatomical niches. Additionally, genetic drift was detected from spatiotemporally separated samples in as little as three days. These data expand our prior understanding of the complex interaction between HSV-1 genomics and population dynamics after transmission to new infected persons.

In vitro evolution of herpes simplex virus 1 (HSV-1) reveals selection for syncytia and other minor variants in cell culture.

The large dsDNA virus herpes simplex virus 1 (HSV-1) is considered to be genetically stable, yet it can rapidly evolve in response to strong selective pressures such as antiviral treatment. Deep sequencing has revealed that clinical and laboratory isolates of this virus exist as populations that contain a mixture of minor alleles or variants, similar to many RNA viruses. The classic virology approach of plaque purifying virus creates a genetically homogenous population, but it is not clear how closely this represents the mixed virus populations found in nature. We sought to study the evolution of mixed versus highly purified HSV-1 populations in controlled cell culture conditions, to examine the impact of this genetic diversity on evolution. We found that a mixed population of HSV-1 acquired more genetic diversity and underwent a more dramatic phenotypic shift than a plaque-purified population, producing a viral population that was almost entirely syncytial after just ten passages. At the genomic level, adaptation and genetic diversification occurred at the level of minor alleles or variants in the viral population. Certain genetic variants in the mixed viral population appeared to be positively selected in cell culture, and this shift was also observed in clinical samples during their first passages in vitro. In contrast, the plaque-purified viral population did not appear to change substantially in phenotype or overall quantity of minor allele diversity. These data indicate that HSV-1 is capable of evolving rapidly in a given environment, and that this evolution is facilitated by diversity in the viral population.

Genotypic and Phenotypic Diversity of Herpes Simplex Virus 2 within the Infected Neonatal Population.

More than 14,000 neonates are infected with herpes simplex virus (HSV) annually. Approximately half display manifestations limited to the skin, eyes, or mouth (SEM disease). The rest develop invasive infections that spread to the central nervous system (CNS disease or encephalitis) or throughout the infected neonate (disseminated disease). Invasive HSV disease is associated with significant morbidity and mortality, but the viral and host factors that predispose neonates to these forms are unknown. To define viral diversity within the infected neonatal population, we evaluated 10 HSV-2 isolates from newborns with a range of clinical presentations. To assess viral fitness independently of host immune factors, we measured viral growth characteristics in cultured cells and found diverse in vitro phenotypes. Isolates from neonates with CNS disease were associated with larger plaque size and enhanced spread, with the isolates from cerebrospinal fluid (CSF) exhibiting the most robust growth. We sequenced complete viral genomes of all 10 neonatal viruses, providing new insights into HSV-2 genomic diversity in this clinical setting. We found extensive interhost and intrahost genomic diversity throughout the viral genome, including amino acid differences in more than 90% of the viral proteome. The genes encoding glycoprotein G (gG; US4), glycoprotein I (gI; US7), and glycoprotein K (gK; UL53) and viral proteins UL8, UL20, UL24, and US2 contained variants that were found in association with CNS isolates. Many of these viral proteins are known to contribute to cell spread and neurovirulence in mouse models of CNS disease. This report represents the first application of comparative pathogen genomics to neonatal HSV disease.IMPORTANCE Herpes simplex virus (HSV) causes invasive disease in half of infected neonates, resulting in significant mortality and permanent cognitive morbidity. The factors that contribute to invasive disease are not understood. This study revealed diversity among HSV isolates from infected neonates and detected the first associations between viral genetic variations and clinical disease manifestations. We found that viruses isolated from newborns with encephalitis showed enhanced spread in culture. These viruses contained protein-coding variations not found in viruses causing noninvasive disease. Many of these variations were found in proteins known to impact neurovirulence and viral spread between cells. This work advances our understanding of HSV diversity in the neonatal population and how it may impact disease outcome.

Comparison of Herpes Simplex Virus 1 Strains Circulating in Finland Demonstrates the Uncoupling of Whole-Genome Relatedness and Phenotypic Outcomes of Viral Infection.

A majority of adults in Finland are seropositive carriers of herpes simplex viruses (HSV). Infection occurs at epithelial or mucosal surfaces, after which virions enter innervating nerve endings, eventually establishing lifelong infection in neurons of the sensory or autonomic nervous system. Recent data have highlighted the genetic diversity of HSV-1 strains and demonstrated apparent geographic patterns in strain similarity. Though multiple HSV-1 genomes have been sequenced from Europe to date, there is a lack of sequenced genomes from the Nordic countries. Finland's history includes at least two major waves of human migration, suggesting the potential for diverse viruses to persist in the population. Here, we used HSV-1 clinical isolates from Finland to test the relationship between viral phylogeny, genetic variation, and phenotypic characteristics. We found that Finnish HSV-1 isolates separated into two distinct phylogenetic groups, potentially reflecting historical waves of human (and viral) migration into Finland. Each HSV-1 isolate harbored a distinct set of phenotypes in cell culture, including differences in the amount of virus production, extracellular virus release, and cell-type-specific fitness. Importantly, the phylogenetic clusters were not predictive of any detectable pattern in phenotypic differences, demonstrating that whole-genome relatedness is not a proxy for overall viral phenotype. Instead, we highlight specific gene-level differences that may contribute to observed phenotypic differences, and we note that strains from different phylogenetic groups can contain the same genetic variations.IMPORTANCE Herpes simplex viruses (HSV) infect a majority of adults. Recent data have highlighted the genetic diversity of HSV-1 strains and demonstrated apparent genomic relatedness between strains from the same geographic regions. We used HSV-1 clinical isolates from Finland to test the relationship between viral genomic and geographic relationships, differences in specific genes, and characteristics of viral infection. We found that viral isolates from Finland separated into two distinct groups of genomic and geographic relatedness, potentially reflecting historical patterns of human and viral migration into Finland. These Finnish HSV-1 isolates had distinct infection characteristics in multiple cell types tested, which were specific to each isolate and did not group according to genomic and geographic relatedness. This demonstrates that HSV-1 strain differences in specific characteristics of infection are set by a combination of host cell type and specific viral gene-level differences.

Viral forensic genomics reveals the relatedness of classic herpes simplex virus strains KOS, KOS63, and KOS79.

Herpes simplex virus 1 (HSV-1) is a widespread global pathogen, of which the strain KOS is one of the most extensively studied. Previous sequence studies revealed that KOS does not cluster with other strains of North American geographic origin, but instead clustered with Asian strains. We sequenced a historical isolate of the original KOS strain, called KOS63, along with a separately isolated strain attributed to the same source individual, termed KOS79. Genomic analyses revealed that KOS63 closely resembled other recently sequenced isolates of KOS and was of Asian origin, but that KOS79 was a genetically unrelated strain that clustered in genetic distance analyses with HSV-1 strains of North American/European origin. These data suggest that the human source of KOS63 and KOS79 could have been infected with two genetically unrelated strains of disparate geographic origins. A PCR RFLP test was developed for rapid identification of these strains.

Rapid genome assembly and comparison decode intrastrain variation in human alphaherpesviruses.

UNLABELLED: Herpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. Here we developed and applied a streamlined new approach for assembly and comparison of large DNA viral genomes such as HSV-1. This viral genome assembly (VirGA) workflow incorporates a combination of de novo assembly, alignment, and annotation strategies to automate the generation of draft genomes for large viruses. We applied this approach to quantify the amount of variation between clonal derivatives of a common parental virus stock. In addition, we examined the genetic basis for syncytial plaque phenotypes displayed by a subset of these strains. In each of the syncytial strains, we found an identical DNA change, affecting one residue in the gB (UL27) fusion protein. Since these identical mutations could have appeared after extensive in vitro passaging, we applied the VirGA sequencing and comparison approach to two clinical HSV-1 strains isolated from the same patient. One of these strains was syncytial upon first culturing; its sequence revealed the same gB mutation. These data provide insight into the extent and origin of genome-wide intrastrain HSV-1 variation and present useful methods for expansion to in vivo patient infection studies. IMPORTANCE: Herpes simplex virus (HSV) infects more than 70% of adults worldwide, causing epithelial lesions and recurrent disease that manifests over a lifetime. Prior work has demonstrated that HSV strains vary from country to country and between individuals. However, the amount of variation within one strain has not been well studied. To address this, we developed a new approach for viral genome assembly (VirGA) and analysis. We used this approach to quantify the amount of variation between sister clones of a common parental virus stock and to determine the basis of a unique fusion phenotype displayed by several variants. These data revealed that while sister clones of one HSV stock are more than 98% identical, these variants harbor enough genetic differences to change their observed characteristics. Comparative genomics approaches will allow us to explore the impacts of viral inter- and intrastrain diversity on drug and vaccine efficacy.