Molecular Mechanisms of Eastern Equine Encephalitis Virus Pathogenesis

描述（由申请方提供）：节肢动物介导的甲病毒东部马脑炎病毒（EEEV）是美国（US）最致命的地方性病毒之一，在高比例的受感染人类和马中引起毁灭性疾病和死亡。由于其极端的神经毒性，在美国东部广泛分布，并有可能用作生物武器，它被归类为选择代理病毒和NIH类别B优先病原体。然而，没有抗病毒药物或获得许可的人类疫苗可用于对抗这种病毒，并且它的研究严重不足。病毒-受体相互作用是抗病毒治疗的靶点，可用于疫苗载体的合理设计。硫酸乙酰肝素（HS）是一种硫酸化多糖，被鉴定为多种甲病毒和黄病毒实验室毒株的附着受体，但其与自然界中病毒的相关性存在疑问，因为其 用作受体可能是适应体外生长的人工产物。为了解决这个问题，我们比较了E2附着蛋白的氨基酸序列之间的HS结合EEEV实验室株和超过60个EEEV株，包括病毒直接从未扩增的现场样品的动物组织测序。该分析证实HS是自然循环EEEV的受体。通过EEEV附着蛋白的诱变消除HS结合表型，我们确定HS结合是导致脊椎动物EEEV疾病的多个独特方面的原因，包括极端神经毒力、有限的脾脏复制和抑制参与先天性和适应性免疫应答的细胞因子/趋化因子。这表明HS结合促进EEEV在体内的复制适应性。本申请中的实验将研究HS结合在EEEV疾病中的作用以及脊椎动物和蚊子宿主中的组织靶向，鉴定虫媒病毒复制/传播周期中易受治疗干预的点，并为抗EEEV疫苗的合理设计提供基础。

DESCRIPTION (provided by applicant): The arthropod-vectored alphavirus eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to the United States (US), causing devastating disease and mortality in a high percentage of infected humans and equines. Due to its extreme neurovirulence, widespread distribution in the eastern US and potential for use as a bioweapon, it is categorized as a Select Agent virus and NIH Category B Priority Pathogen. Yet, no antiviral drugs or licensed human vaccines are available to combat this virus and it is critically understudied. The virus-receptor interaction represents a target fo antiviral therapeutics and can be used in rational design of vaccine vectors. Heparan sulfate (HS) is a sulfated polysaccharide identified as an attachment receptor for multiple alphavirus and flavivirus laboratory strains but its relevance to viruses in nature is in question because its use as a receptor is possibly an artifact of adaptation to in vitro growth. To address this question, we compared the E2 attachment protein amino acid sequence between a HS binding EEEV laboratory strain and over 60 EEEV strains, including viruses sequenced directly from unamplified field samples of animal tissues. This analysis confirmed that HS is a receptor for naturally circulating EEEV. By mutagenesis of the EEEV attachment protein eliminating the HS binding phenotype, we determined that HS binding was responsible for multiple unique aspects of EEEV disease in vertebrates including extreme neurovirulence, limited spleen replication and suppression of cytokines/chemokines involved in innate and adaptive immune responses. This suggests that HS binding promotes EEEV replicative fitness in vivo. The experiments in this application will investigate the role of HS binding in EEEV disease and tissue targeting in vertebrate and mosquito hosts identifying points in the arbovirus replication/transmission cycle vulnerable to therapeutic intervention and provide the basis for the rational design of anti-EEEV vaccines.