Comparative analysis of multiple consensus genomes of the same strain of Marek's disease virus reveals intrastrain variation.

Current strategies to understand the molecular basis of Marek's disease virus (MDV) virulence primarily consist of cataloging divergent nucleotides between strains with different phenotypes. However, most comparative genomic studies of MDV rely on previously published consensus genomes despite the confirmed existence of MDV strains as mixed viral populations. To assess the reliability of interstrain genomic comparisons relying on published consensus genomes of MDV, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates. In conjunction with the published genomes of CVI988 and Md5, this allowed us to perform three-way comparisons between multiple consensus genomes of the same strain. We found that consensus genomes of CVI988 can vary in as many as 236 positions involving 13 open reading frames (ORFs). By contrast, we found that Md5 genomes varied only in 11 positions involving a single ORF. Notably, we were able to identify 3 single-nucleotide polymorphisms (SNPs) in the unique long region and 16 SNPs in the unique short (US) region of CVI988GenBank.BAC that were not present in either CVI988Pirbright.lab or CVI988USDA.PA.field. Recombination analyses of field strains previously described as natural recombinants of CVI988 yielded no evidence of crossover events in the US region when either CVI988Pirbright.lab or CVI988USDA.PA.field were used to represent CVI988 instead of CVI988GenBank.BAC. We were also able to confirm that both CVI988 and Md5 populations were mixed, exhibiting a total of 29 and 27 high-confidence minor variant positions, respectively. However, we did not find any evidence of minor variants in the positions corresponding to the 19 SNPs in the unique regions of CVI988GenBank.BAC. Taken together, our findings suggest that continued reliance on the same published consensus genome of CVI988 may have led to an overestimation of genomic divergence between CVI988 and virulent strains and that multiple consensus genomes per strain may be necessary to ensure the accuracy of interstrain genomic comparisons.

Comparative Analysis of Multiple Consensus Genomes of the Same Strain of Marek's Disease Virus Reveals Intrastrain Variation.

Current strategies to understand the molecular basis of Marek's disease virus (MDV) virulence primarily consist of cataloguing divergent nucleotides between strains with different phenotypes. However, each MDV strain is typically represented by a single consensus genome despite the confirmed existence of mixed viral populations. To assess the reliability of single-consensus interstrain genomic comparisons, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates. In conjunction with the published genomes of CVI988 and Md5, this allowed us to perform 3-way comparisons between consensus genomes of the same strain. We found that consensus genomes of CVI988 can vary in as many as 236 positions involving 13 open reading frames (ORFs). In contrast, we found that Md5 genomes varied only in 11 positions involving a single ORF. Phylogenomic analyses showed all three Md5 consensus genomes clustered closely together, while also showing that CVI988 GenBank.BAC diverged from CVI988 Pirbright.lab and CVI988 USDA.PA.field . Comparison of CVI988 consensus genomes revealed 19 SNPs in the unique regions of CVI988 GenBank.BAC that were not present in either CVI988 Pirbright.lab or CVI988 USDA.PA.field . Finally, we evaluated the genomic heterogeneity of CVI988 and Md5 populations by identifying positions with >2% read support for alternative alleles in two ultra-deeply sequenced samples. We were able to confirm that both populations of CVI988 and Md5 were mixed, exhibiting a total of 29 and 27 high-confidence minor variant positions, respectively. We did not find any evidence of minor variants in the positions corresponding to the 19 SNPs in the unique regions of CVI988 GenBank.BAC . Taken together, our findings confirm that consensus genomes of the same strain of MDV can vary and suggest that multiple consensus genomes per strain are needed in order to maximize the accuracy of interstrain genomic comparisons.

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.

Aerosolized Hydrogen Peroxide Decontamination of N95 Respirators, with Fit-Testing and Viral Inactivation, Demonstrates Feasibility for Reuse during the COVID-19 Pandemic.

In response to the demand for N95 respirators by health care workers during the COVID-19 pandemic, we evaluated decontamination of N95 respirators using an aerosolized hydrogen peroxide (aHP) system. This system is designed to dispense a consistent atomized spray of aerosolized, 7% hydrogen peroxide (H2O2) solution over a treatment cycle. Multiple N95 respirator models were subjected to 10 or more cycles of respirator decontamination, with a select number periodically assessed for qualitative and quantitative fit testing. In parallel, we assessed the ability of aHP treatment to inactivate multiple viruses absorbed onto respirators, including phi6 bacteriophage, herpes simplex virus 1 (HSV-1), coxsackievirus B3 (CVB3), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For pathogens transmitted via respiratory droplets and aerosols, it is critical to address respirator safety for reuse. This study provided experimental validation of an aHP treatment process that decontaminates the respirators while maintaining N95 function. External National Institute for Occupational Safety & Health (NIOSH) certification verified respirator structural integrity and filtration efficiency after 10 rounds of aHP treatment. Virus inactivation by aHP was comparable to the decontamination of commercial spore-based biological indicators. These data demonstrate that the aHP process is effective, with successful fit-testing of respirators after multiple aHP cycles, effective decontamination of multiple virus species, including SARS-CoV-2, successful decontamination of bacterial spores, and filtration efficiency maintained at or greater than 95%. While this study did not include extended or clinical use of N95 respirators between aHP cycles, these data provide proof of concept for aHP decontamination of N95 respirators before reuse in a crisis-capacity scenario. IMPORTANCE The COVID-19 pandemic led to unprecedented pressure on health care and research facilities to provide personal protective equipment. The respiratory nature of the SARS-CoV2 pathogen makes respirator facepieces a critical protective measure to limit inhalation of this virus. While respirator facepieces were designed for single use and disposal, the pandemic increased overall demand for N95 respirators, and corresponding manufacturing and supply chain limitations necessitated the safe reuse of respirators when necessary. In this study, we repurposed an aerosolized hydrogen peroxide (aHP) system that is regularly utilized to decontaminate materials in a biosafety level 3 (BSL3) facility, to develop a method for decontamination of N95 respirators. Results from viral inactivation, biological indicators, respirator fit testing, and filtration efficiency testing all indicated that the process was effective at rendering N95 respirators safe for reuse. This proof-of-concept study establishes baseline data for future testing of aHP in crisis-capacity respirator-reuse scenarios.

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.

Dermatitis during Spaceflight Associated with HSV-1 Reactivation.

Human alpha herpesviruses herpes simplex virus (HSV-1) and varicella zoster virus (VZV) establish latency in various cranial nerve ganglia and often reactivate in response to stress-associated immune system dysregulation. Reactivation of Epstein Barr virus (EBV), VZV, HSV-1, and cytomegalovirus (CMV) is typically asymptomatic during spaceflight, though live/infectious virus has been recovered and the shedding rate increases with mission duration. The risk of clinical disease, therefore, may increase for astronauts assigned to extended missions (>180 days). Here, we report, for the first time, a case of HSV-1 skin rash (dermatitis) occurring during long-duration spaceflight. The astronaut reported persistent dermatitis during flight, which was treated onboard with oral antihistamines and topical/oral steroids. No HSV-1 DNA was detected in 6-month pre-mission saliva samples, but on flight day 82, a saliva and rash swab both yielded 4.8 copies/ng DNA and 5.3 × 104 copies/ng DNA, respectively. Post-mission saliva samples continued to have a high infectious HSV-1 load (1.67 × 107 copies/ng DNA). HSV-1 from both rash and saliva samples had 99.9% genotype homology. Additional physiological monitoring, including stress biomarkers (cortisol, dehydroepiandrosterone (DHEA), and salivary amylase), immune markers (adaptive regulatory and inflammatory plasma cytokines), and biochemical profile markers, including vitamin/mineral status and bone metabolism, are also presented for this case. These data highlight an atypical presentation of HSV-1 during spaceflight and underscore the importance of viral screening during clinical evaluations of in-flight dermatitis to determine viral etiology and guide treatment.

Herpes Simplex Virus-2 Variation Contributes to Neurovirulence During Neonatal Infection.

Herpes simplex virus (HSV) infection of the neonatal brain causes severe encephalitis and permanent neurologic deficits. However, infants infected with HSV at the time of birth follow varied clinical courses, with approximately half of infants experiencing only external infection of the skin rather than invasive neurologic disease. Understanding the cause of these divergent outcomes is essential to developing neuroprotective strategies. To directly assess the contribution of viral variation to neurovirulence, independent of human host factors, we evaluated clinical HSV isolates from neonates with different neurologic outcomes in neurologically relevant in vitro and in vivo models. We found that isolates taken from neonates with encephalitis are more neurovirulent in human neuronal culture and mouse models of HSV encephalitis, as compared to isolates collected from neonates with skin-limited disease. These findings suggest that inherent characteristics of the infecting HSV strain contribute to disease outcome following neonatal infection.

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.

A holistic perspective on herpes simplex virus (HSV) ecology and evolution.

Herpes simplex viruses (HSV) cause chronic infection in humans that are characterized by periodic episodes of mucosal shedding and ulcerative disease. HSV causes millions of infections world-wide, with lifelong bouts of viral reactivation from latency in neuronal ganglia. Infected individuals experience different levels of disease severity and frequency of reactivation. There are two distantly related HSV species, with HSV-1 infections historically found most often in the oral niche and HSV-2 infections in the genital niche. Over the last two decades, HSV-1 has emerged as the leading cause of first-episode genital herpes in multiple countries. While HSV-1 has the highest level of genetic diversity among human alpha-herpesviruses, it is not yet known how quickly the HSV-1 viral population in a human host adapts over time, or if there are population bottlenecks associated with viral reactivation and/or transmission. It is also unknown how the ecological environments in which HSV infections occur influence their evolutionary trajectory, or that of co-occurring viruses and microbes. In this review, we explore how HSV accrues genetic diversity within each new infection, and yet maintains its ability to successfully infect most of the human population. A holistic examination of the ecological context of natural human infections can expand our awareness of how HSV adapts as it moves within and between human hosts, and reveal the complexity of these lifelong human-virus interactions. These insights may in turn suggest new areas of exploration for other chronic pathogens that successfully evolve and persist among their hosts.

Genome Sequence of the Virulent Model Herpes Simplex Virus 1 Strain McKrae Demonstrates the Presence of at Least Two Widely Used Variant Strains.

Herpes simplex virus 1 (HSV-1) strain McKrae was isolated in 1965 and has been utilized by many laboratories. Three HSV-1 strain McKrae stocks have been sequenced previously, revealing discrepancies in key genes. We sequenced the genome of HSV-1 strain McKrae from the laboratory of James M. Hill to better understand the genetic differences between isolates.

Viral infection of human neurons triggers strain-specific differences in host neuronal and viral transcriptomes.

Infection with herpes simplex virus 1 (HSV-1) occurs in over half the global population, causing recurrent orofacial and/or genital lesions. Individual strains of HSV-1 demonstrate differences in neurovirulence in vivo, suggesting that viral genetic differences may impact phenotype. Here differentiated SH-SY5Y human neuronal cells were infected with one of three HSV-1 strains known to differ in neurovirulence in vivo. Host and viral RNA were sequenced simultaneously, revealing strain-specific differences in both viral and host transcription in infected neurons. Neuronal morphology and immunofluorescence data highlight the pathological changes in neuronal cytoarchitecture induced by HSV-1 infection, which may reflect host transcriptional changes in pathways associated with adherens junctions, integrin signaling, and others. Comparison of viral protein levels in neurons and epithelial cells demonstrated that a number of differences were neuron-specific, suggesting that strain-to-strain variations in host and virus transcription are cell type-dependent. Together, these data demonstrate the importance of studying virus strain- and cell-type-specific factors that may contribute to neurovirulence in vivo, and highlight the specificity of HSV-1-host interactions.

Alphaherpesvirus Genomics: Past, Present and Future.

Alphaherpesviruses, as large double-stranded DNA viruses, were long considered to be genetically stable and to exist in a homogeneous state. Recently, the proliferation of high-throughput sequencing (HTS) and bioinformatics analysis has expanded our understanding of herpesvirus genomes and the variations found therein. Recent data indicate that herpesviruses exist as diverse populations, both in culture and in vivo, in a manner reminiscent of RNA viruses. In this review, we discuss the past, present, and potential future of alphaherpesvirus genomics, including the technical challenges that face the field. We also review how recent data has enabled genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures, including those introduced by cell culture. While we focus on the human alphaherpesviruses, we draw key insights from related veterinary species and from the beta- and gamma-subfamilies of herpesviruses. Promising technologies and potential future directions for herpesvirus genomics are highlighted as well, including the potential to link viral genetic differences to phenotypic and disease outcomes.

Interferon-independent STING signaling promotes resistance to HSV-1 in vivo.

The Stimulator of Interferon Genes (STING) pathway initiates potent immune responses upon recognition of DNA. To initiate signaling, serine 365 (S365) in the C-terminal tail (CTT) of STING is phosphorylated, leading to induction of type I interferons (IFNs). Additionally, evolutionary conserved responses such as autophagy also occur downstream of STING, but their relative importance during in vivo infections remains unclear. Here we report that mice harboring a serine 365-to-alanine (S365A) mutation in STING are unexpectedly resistant to Herpes Simplex Virus (HSV)-1, despite lacking STING-induced type I IFN responses. By contrast, resistance to HSV-1 is abolished in mice lacking the STING CTT, suggesting that the STING CTT initiates protective responses against HSV-1, independently of type I IFNs. Interestingly, we find that STING-induced autophagy is a CTT- and TBK1-dependent but IRF3-independent process that is conserved in the STING S365A mice. Thus, interferon-independent functions of STING mediate STING-dependent antiviral responses in vivo.

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.

Oligonucleotide Enrichment of HSV-1 Genomic DNA from Clinical Specimens for Use in High-Throughput Sequencing.

To date more than 400 genomes of herpes simplex virus 1 (HSV-1) and the distantly related HSV-2 have been examined using deep sequencing techniques. This powerful approach has been especially useful for revealing the global genetic diversity that exists within and between strains of each virus species. However, most early methods for high-throughput sequencing required the input of abundant viral genomic DNA to enable the successful production of sequencing libraries, and the generation of sufficient short-read sequencing data for de novo genome assembly and similar applications. Therefore, the majority of sequenced HSV strains have been cultured and expanded in vitro prior to genomic analysis, to facilitate isolation of sufficient viral DNA for sequencing-library preparation. Here, we describe an in-solution targeted enrichment procedure for isolating, enriching, and sequencing HSV genomic DNA directly from clinical specimens. When this enrichment technique is combined with traditional sequencing-library preparation procedures, the need for in vitro culturing, expansion, and purification of viral DNA is eliminated. Furthermore, enrichment reduces the large amount of nonviral DNA that is typically present in specimens obtained directly from natural infections, thereby increasing the likelihood of successful viral genome sequencing and assembly. We have used this approach to prepare viral DNA libraries from clinical specimens derived from skin swabs, saliva, blood, and similar sources. We then use these libraries for deep sequencing and successful de novo assembly of the ~152 kb viral genomes, at coverage depths exceeding 100-1000×, for both HSV-1 and HSV-2.

Persistent Infection with Herpes Simplex Virus 1 and Alzheimer's Disease-A Call to Study How Variability in Both Virus and Host may Impact Disease.

Increasing attention has focused on the contributions of persistent microbial infections with the manifestation of disease later in life, including neurodegenerative conditions such as Alzheimer's disease (AD). Current data has shown the presence of herpes simplex virus 1 (HSV-1) in regions of the brain that are impacted by AD in elderly individuals. Additionally, neuronal infection with HSV-1 triggers the accumulation of amyloid beta deposits and hyperphosphorylated tau, and results in oxidative stress and synaptic dysfunction. All of these factors are implicated in the development of AD. These data highlight the fact that persistent viral infection is likely a contributing factor, rather than a sole cause of disease. Details of the correlations between HSV-1 infection and AD development are still just beginning to emerge. Future research should investigate the relative impacts of virus strain- and host-specific factors on the induction of neurodegenerative processes over time, using models such as infected neurons in vitro, and animal models in vivo, to begin to understand their relationship with cognitive dysfunction.

Personalized viral genomic investigation of herpes simplex virus 1 perinatal viremic transmission with dual fatality.

Here we present a personalized viral genomics approach to investigating a rare case of perinatal herpes simplex virus 1 (HSV-1) transmission that ended in death of both mother and neonate. We sought to determine whether the virus involved in this rare case had any unusual features that may have contributed to the dire patient outcome. A pregnant woman with negative HerpeSelect antibody test underwent cesarean section at 30 wk gestation and died the same day. The premature newborn died 5 d later. Both individuals were found postmortem to have positive blood HSV-1 PCR tests. Using oligonucleotide enrichment and deep sequencing, we determined that viral transmission from mother to infant was nearly perfect at the consensus genome level. At the virus population level, 77% of minor variants (MVs) in the mother's blood also appeared on the neonate's skin, of which more than half were disseminated into the neonate's blood. We also detected nonmaternal MVs that arose de novo in the neonate's viral populations. Of note, one de novo MV in the neonate's skin virus induced a nonsynonymous mutation in the UL6 protein, which is a component of the portal that allows DNA entry into new progeny capsids. This case suggests that perinatal viremic HSV-1 transmission includes the majority of genetic diversity from the maternal virus population and that new, nonsynonymous mutations can occur after relatively few rounds of replication. This report expands our understanding of viral transmission in humans and may lead to improved diagnostic strategies for neonatal HSV-1 acquisition.

Molecular epidemiology of Marek's disease virus in central Pennsylvania, USA.

The evolution of Marek's disease virus (MDV, Gallid herpesvirus 2) has threatened the sustainability of poultry farming in the past and its continued evolution remains a concern. Genetic diversity is key to understanding evolution, yet little is known about the diversity of MDV in the poultry industry. Here, we investigate the diversity of MDV on 19 Pennsylvanian poultry farms over a 3-year period. Using eight polymorphic markers, we found that at least twelve MDV haplotypes were co-circulating within a radius of 40 km. MDV diversity showed no obvious spatial clustering nor any apparent clustering by bird line: all of the virus haplotypes identified on the commercial farms could be found within a single, commonly reared bird line. On some farms, a single virus haplotype dominated for an extended period of time, while on other farms the observed haplotypes changed over time. In some instances, multiple haplotypes were found simultaneously on a farm, and even within a single dust sample. On one farm, co-occurring haplotypes clustered into phylogenetically distinct clades, putatively assigned as high and low virulence pathotypes. Although the vast majority of our samples came from commercial poultry farms, we found the most haplotype diversity on a noncommercial backyard farm experiencing an outbreak of clinical Marek's disease. Future work to explore the evolutionary potential of MDV might therefore direct efforts toward farms that harbor multiple virus haplotypes, including both backyard farms and farms experiencing clinical Marek's disease.

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.