Moriah L. Szpara - US grants

DESCRIPTION (provided by applicant): My career goal is to establish an academic research laboratory devoted to understanding how pathogens affect the nervous system, and what features of neurons could provide useful therapeutic targets to combat these infections. I am well-prepared to contribute significantly to our understanding of neurotropic pathogens, based on the strong foundations of my graduate training in neurobiology and postdoctoral training in virology. This award will facilitate my career development by allowing me to gain training in bioinformatics and immunology, and also by providing funding and time to generate data and publications to facilitate a future R01 application. The overall goal of this research proposal is to understand how pathogens interact with the host nervous system, specifically to grasp what genetic features can make a neurotropic pathogen more or less virulent. Herpes simplex virus 1 (HSV-1) is a widespread human pathogen that establishes latency in human peripheral neurons and reactivates repeatedly throughout a patient's lifetime. HSV-1 is a leading cause of infectious blindness in the United States, and the main cause of sporadic, fatal encephalitis. It is unknown why certain HSV-1 infections progress to the level of encephalitis, but it is almost certainly influenced by both viral and host factors. Mouse models of this disease have demonstrated that viral strains differ in their virulence and ability to cause encephalitis. However the ability to map these phenotypic differences to specific viral genes has been limited by the lack of comparative genomic information about this virus - only one wild type HSV-1 genome has been known for the last two decades. I recently demonstrated that high-throughput sequencing (HTS) and bioinformatic assembly can be used to obtain further HSV-1 genomes. I propose to use HTS to test this hypothesis: that genetic loci which are enriched in neurovirulent strains, and lacking in strains that do not cause encephalitis in mice, are highly likely to be causally associated with the neurovirulence phenotype. To achieve this, my specific aims are: (1) to use HTS, genome assembly, & bioinformatic comparison of diverse clinical isolates of HSV-1, to find loci that are enriched in highly neurovirulent strains versus non-virulent ones; and (2) to generate viral recombinants, by either adding or removing putative neurovirulence loci, and test for associated changes in phenotype using an in vivo model of viral encephalitis. These aims will lead to future research directions, using standard molecular and cell biological approaches to explore how these neurovirulence loci affect neuronal function and the viral infectious cycle. This proposal will shed light on mechanisms of neurovirulence in this pervasive human virus, and highlight aspects of virulence to consider in other neurotropic pathogens. NARRATIVE: Herpes simplex virus 1 (HSV-1) is a widespread human virus. This research will investigate how HSV-1 virus isolated from one patient may differ from that of another. This will help us understand why some patients suffer more severe symptoms from HSV-1 infection than others.

Project Summary Herpes simplex virus 1 (HSV-1) and HSV-2 cause millions of chronic infections. These viruses cause epithelial oral ?cold? sores and genital lesions, which recur lifelong due to reactivation from a latent viral reservoir in neurons. 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. The rise of genital HSV-1 coincides with a decline in seroprevalence of HSV-1, particularly among youth, and with changing sexual behaviors (13, 23). This shift raises the possibility that HSV-1 may face new selective pressures and undergo genetic adaptation as it moves from the oral to the genital niche. We propose to conduct a genome- wide analysis of HSV-1 variation in human infections, to observe how the virus adapts to the human genital niche during the first year of infection, and to discern how the virus is affected by the laboratory culture that is required for further studies in vitro, or in vivo in animal models. These data will reveal the degree to which HSV-1 undergoes bottlenecks or develops new variations in humans or in culture. The experiments in this exploratory R21 proposal provide the foundation for a future R01 proposal aimed at investigating how HSV-1 genetic variations impact clinical disease outcomes in patients. Insights on viral genomic variation will be obtained using Illumina sequencing of longitudinal HSV-1 genital and oral shedding swabs, which are being collected as part of an ongoing clinical study, P01AI030731. In Aim 1, we will use sensitive enrichment and viral genome sequencing techniques to assess HSV-1 genome diversity directly from human shedding. We will compare HSV-1 genomes from genital and oral swabs, collected at primary infection and during any recurrences over the course of a year. These will provide insights on viral genetic diversity between individuals, as well as within-host diversity over the first year of infection. Since P01AI030731 includes sub-studies on mucosal and humoral immune responses during the first year of infection, this proposal will yield additional data on how viral antigen variation correlates with the epitopes detected in each person's immune repertoire. In Aim 2, we will address whether HSV-1 strains that are cultured in vitro accurately reflect the viral genomic diversity shed by infected humans. We will do this by comparing viral genomes derived from cultured stocks to the genomes derived from the matched swab samples in Aim 1. This will establish the foundation for future studies that utilize cultured HSV-1 strains for in vitro and in vivo animal studies of viral pathogenesis, antiviral drug discovery, and vaccine development.

Project Summary Herpes simplex virus 1 (HSV-1) and HSV-2 cause millions of chronic infections. These viruses cause epithelial oral ?cold? sores and genital lesions, which recur lifelong due to reactivation from a latent viral reservoir in neurons. Clinical outcomes from HSV-1 infection are highly diverse, ranging from surface lesions with quite different rates of recurrence, to asymptomatic shedding, to severe and potentially lethal encephalitis. This variation is thought to be caused by a combination of factors, including viral genetic differences, human genetic predisposition, and environmental variables. Animal models serve as the cornerstone of our molecular understanding of HSV-1 disease in vivo, and provide an opportunity to dissect viral genetic contributions to pathogenesis, while controlling for host genetic factors and environmental variables. We have recently sequenced a collection of low-passage clinical isolates of HSV-1 and categorized each isolate according to its reproducible virulence phenotype in a mouse ocular model of infection. We measured virulence in this model by the virus' ability to infect, replicate, and induce lethal disease, which involved the virus initiating infection at the ocular surface, migrating to the peripheral nervous system (trigeminal ganglia), and even penetrating into the central nervous system (CNS). Using multiple statistical approaches, we found two loci in the viral tegument protein VP22 (UL49) that predictably distinguish high-virulence clinical isolates from low-virulence isolates. VP22 is a viral tegument protein that is conserved among alpha-herpesviruses, which functions as a hub of viral and cellular protein interactions. VP22 functions in close concert with the viral transactivator protein VP16 (UL48), and the viral RNAse VHS (UL41). The variant loci detected in association with murine virulence exist at a frequency of approximately 50% in the clinical HSV-1 isolates we have surveyed to date, suggesting that knowledge gleaned from these experiments will provide data on viral variants of relevance to ongoing trials for antivirals and vaccines. With increased power from additional genotype-phenotype data, we will use a genome-wide association study (GWAS) to measure the association of VP22 and other candidate loci with virulence in HSV-1. We will make recombinant HSV-1 strains to test the role(s) of VP22 and other candidate virulence loci both in vivo and in vitro. We will test the hypothesis that virulent and non-virulent phenotypes in vivo result from distinct biochemical differences in VP22 between these isolates. Data from these experiments will enable us to test the robustness of our forward genetic predictions of virulence loci in HSV-1. If these data can be linked to human impacts in the future, the ability to gauge likely virulence level based on viral genotype would provide a powerful tool for future diagnostics and prediction of clinical outcomes.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) cause millions of lifelong, recurring infections. In adults, HSV infection typically leads to epithelial lesions, which recur whenever the virus reactivates from its latent reservoir in neurons. HSV infection of neonates can lead to more devastating outcomes than those seen in adults, including neurologic impairment or death. Around 1 in 3,200 births results in neonatal HSV disease, often from mothers who are not aware of their infection or of active viral shedding. Half of these neonates experience disseminated multi-organ or invasive disease in the central nervous system (CNS), while the remainder have more limited infections of the skin, eyes, or mouth. Rates of mortality and lifelong morbidity are significantly higher for the invasive CNS and disseminated forms of neonatal infection, than for the more limited surface infections. The contribution of HSV genetic variation to these different clinical outcomes is unknown. However, the genetic dissection of other viruses such as influenza and HIV has led to the identification of viral genetic factors that influence virulence and disease. We therefore propose to conduct a genomic and phenotypic analysis of HSV variation in neonatal infections. We will examine HSV genomic variation using isolates from published clinical studies of neonatal disease outcomes. The combination of viral comparative genomics, phenotypic analyses of cell-to-cell spread, and detailed clinical data will enable us to lay the foundation for a future genome-wide association study (GWAS) for neonatal HSV, which would test for correlations between viral genetic variation and clinical data such as invasive CNS vs. skin disease, severity of neurologic impairment, or response to antiviral chemotherapy. In Aim 1, we will use high-throughput sequencing and viral comparative genomics to dissect all of the viral genetic variations found in ?30 cases of neonatal HSV disease. This will enable us to detect single nucleotide variations (SNPs), insertions, and deletions that correlate with clinical measures of disease, or with in vitro data from Aim 2. In Aim 2 we will use a panel of cellular and biochemical assays to characterize spread phenotypes, protein expression, and antiviral drug resistance for each neonatal HSV isolate. This will reveal any strains with overt growth differences, and enable us to measure the expression and localization of viral proteins that vary in our genomic comparisons. Data on in vitro acyclovir resistance will be compared to clinical data on subsequent recurrences for each patient. These data will establish the foundation for future studies that test hypotheses about how viral genetic variations impact neonatal disease, using animal models of neonatal infection. The identification of viral genetic loci associated with invasive neonatal HSV disease would provide new insights on host-pathogen interactions and potential targets for therapeutic development, as well as informing treatment duration and the use of drugs in development.