Genetic determinants of HSV-1 virulence in neurons

我的职业目标是建立一个学术研究实验室，致力于了解病原体如何影响神经系统，以及神经元的哪些特征可以提供有用的治疗靶点来对抗这些感染。基于我在神经生物学研究生培训和病毒学博士后培训的坚实基础，我已经做好了充分的准备，为我们对嗜神经性病原体的理解做出重大贡献。这个奖项将促进我的职业发展，让我获得生物信息学和免疫学方面的培训，并提供资金和时间来生成数据和出版物，以促进未来的R 01应用。这项研究计划的总体目标是了解病原体如何与宿主神经系统相互作用，特别是掌握哪些遗传特征可以使嗜神经性病原体更具或更少毒性。单纯疱疹病毒1（HSV-1）是一种广泛存在的人类病原体，在人类外周神经元中建立潜伏期，并在患者的一生中反复重新激活。HSV-1是美国传染性失明的主要原因，也是散发性致命脑炎的主要原因。目前尚不清楚为什么某些HSV-1感染进展到脑炎的水平，但几乎可以肯定它受到病毒和宿主因素的影响。这种疾病的小鼠模型已经证明，病毒株的毒力和引起脑炎的能力不同。然而，由于缺乏关于这种病毒的比较基因组信息，将这些表型差异映射到特定病毒基因的能力受到限制-在过去二十年中，只有一种野生型HSV-1基因组已知。我最近证明了高通量测序（HTS）和生物信息学组装可用于获得更多的HSV-1基因组。我建议使用HTS来检验这一假设：即富含神经毒力菌株的遗传位点，以及缺乏不会引起小鼠脑炎的菌株的遗传位点，极有可能与神经毒力表型有因果关系。为了实现这一目标，我的具体目标是：（1）使用HTS，基因组组装和生物信息学比较HSV-1的不同临床分离株，以找到在高神经毒力菌株与非毒性菌株中富集的基因座;（2）通过添加或去除推定的神经毒力基因座来产生病毒重组体，并使用病毒性脑炎的体内模型测试表型的相关变化。这些目标将导致未来的研究方向，使用标准的分子和细胞生物学方法来探索这些神经毒力位点如何影响神经元功能和病毒感染周期。这一提议将阐明这种普遍存在的人类病毒的神经毒力机制，并强调在其他嗜神经性病原体中考虑的毒力方面。 简述：单纯疱疹病毒1型（HSV-1）是一种广泛传播的人类病毒。这项研究将调查如何HSV-1病毒分离出一个病人可能不同于另一个。这将有助于我们理解为什么有些患者比其他人遭受更严重的HSV-1感染症状。

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.