The impact of viral genomic variation on neonatal disease outcomes

全球单纯疱疹病毒1型(HSV1)的负担约为37亿，而HSV2的全球负担约为4亿。在成年人中，这些终生感染通常会导致上皮损伤，每当病毒从其终生潜伏在神经元中的蓄水池重新激活时，上皮损伤就会复发。在新生儿中，结果更为可怕，大约一半的HSV感染会导致病毒侵袭性传播到中枢神经系统(CNS)，或病毒传播到肺或肝脏等器官。侵袭性中枢神经系统和播散型新生儿感染的死亡率和终生发病率明显高于仅限于体表的感染。目前尚不清楚HSV基因变异对这些不同临床结局的影响。在最近对新生儿HSV样本的初步分析中，我们发现了与侵袭性传播表型相关的病毒遗传变异模式。我们现在建议将我们对病毒变异的基因组和表型分析扩展到更大的新生儿数据集，并纳入毒力的体内模型。病毒比较基因组学、基于细胞的表型、体内致病模型和未识别的临床数据的结合将为未来新生儿单纯疱疹病毒的全基因组关联研究(GWAS)奠定基础。利用这些数据，我们将探索病毒基因变异与临床结果之间的联系，如侵袭性中枢神经系统与皮肤病、神经功能损害的严重程度以及抗病毒药物的反应。在目标1中，我们将使用高通量测序(HTSeq)和比较基因组学来剖析HSV1或HSV2新生儿HSV疾病患者之间和体内的病毒遗传变异。我们将在总体一致基因组的水平上量化宿主之间的差异，并通过检查来自身体不同利基的样本中的微小变异来量化宿主内的差异。在目标2中，我们将通过检测病毒复制率、细胞间传播、斑块形态以及病毒蛋白在包括神经元在内的一组细胞类型中的表达和定位，来确定每个培养的HSV分离株的体外表型特征。在目标3中，我们将使用播散性或中枢神经系统感染的小鼠模型来确定新生儿HSV1和HSV2分离株的体内传播和神经侵袭率。这些数据将使我们能够将病毒遗传学(来自AIM 1)和基于细胞的表型(来自AIM 2)的差异与在体内(AIM 3)观察到的神经侵袭和毒力水平或通过这些新生儿分离株的未鉴定临床数据联系起来。我们的最终目标是找到能够预测新感染新生儿高侵袭性疾病风险的措施，以便识别高危个体并针对其进行干预(S)，以限制病毒入侵并改善临床结果。

The global burden of herpes simplex virus 1 (HSV1) is ~3.7 billion, while HSV2 afflicts ~400 million. In adults, these lifelong infections typically cause epithelial lesions, which recur whenever the virus reactivates from its lifelong latent reservoir in neurons. In newborns the outcomes are more dire, with approximately half of all HSV infections leading to invasive viral spread into the central nervous system (CNS), or viral dissemination into organs such as the lungs or liver. Rates of mortality and lifelong morbidity are significantly higher for the invasive CNS and disseminated forms of neonatal infection, than for infections that remain limited to the body surface. The contribution of HSV genetic variation to these different clinical outcomes is as yet unknown. In a recent pilot analysis of neonatal HSV samples, we found patterns of viral genetic variation that correlated with invasive spread phenotypes. We now propose to extend our genomic and phenotypic analyses of viral variation to a larger neonatal dataset and to incorporate in vivo models of virulence. The combination of viral comparative genomics, cell-based phenotyping, in vivo models of pathogenesis, and de-identified clinical data will lay the foundation for a future genome-wide association study (GWAS) for neonatal HSV. Using these data, we will probe connections between viral genetic variation and clinical outcomes such as invasive CNS vs. skin disease, severity of neurologic impairment, and response to antivirals. In Aim 1, we will use high-throughput sequencing (HTSeq) and comparative genomics to dissect viral genetic variation between and within individuals with HSV1 or HSV2 neonatal HSV disease. We will quantify differences between-hosts at the level of the overall consensus genomes, and within-host by examining minor variants in samples from distinct niches in the body. In Aim 2 we will determine the in vitro phenotypic profile of each cultured HSV isolate, by examining rates of viral replication, cell-to-cell spread, plaque morphology, and viral protein expression and localization in a panel of cell types, including neurons. In Aim 3 we will determine the rate of spread and neuroinvasion in vivo for neonatal HSV1 and HSV2 isolates, using murine models of either disseminated or CNS infection. These data will allow us to link differences in viral genetics (from Aim 1) and cell-based phenotypes (from Aim 2) with levels of neuroinvasion and virulence observed in vivo (Aim 3) or via de-identified clinical data for these neonatal isolates. Our ultimate goal is to find measures than can predict the risk of highly-invasive disease for newly infected newborns, so that individuals at highest risk can be identified and targeted for intervention(s) to limit viral invasion and improve clinical outcomes.