Molecular Mechanisms of Eastern Equine Encephalitis Virus Pathogenesis

描述（由申请人提供）：以节肢动物为载体的甲病毒东部马脑炎病毒（EEEV）是美国（US）流行的毒性最强的病毒之一，在受感染的人和马中造成毁灭性的疾病和死亡。由于其极端的神经毒力、在美国东部广泛分布以及用作生物武器的潜力，它被归类为选择病原体病毒和 NIH B 类优先病原体。然而，目前还没有抗病毒药物或获得许可的人类疫苗可以对抗这种病毒，而且对其的研究也严重不足。病毒-受体相互作用代表了抗病毒治疗的目标，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，被确定为多种甲病毒和黄病毒实验室毒株的附着受体，但其与自然界病毒的相关性存在疑问，因为它 用作受体可能是适应体外生长的产物。为了解决这个问题，我们比较了 HS 结合 EEEV 实验室毒株和 60 多种 EEEV 毒株之间的 E2 附着蛋白氨基酸序列，其中包括直接从未扩增的动物组织现场样本中测序的病毒。该分析证实 HS 是自然循环 EEEV 的受体。通过对 EEEV 附着蛋白进行诱变，消除 HS 结合表型，我们确定 HS 结合导致脊椎动物 EEEV 疾病的多个独特方面，包括极端神经毒力、有限的脾脏复制以及抑制参与先天性和适应性免疫反应的细胞因子/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的组织靶向，识别虫媒病毒复制/传播周期中易受治疗干预的点，并为合理设计抗 EEEV 疫苗提供基础。

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.