Molecular Mechanisms of Eastern Equine Encephalitis Virus Pathogenesis

描述(申请人提供)：节肢动物传播的甲型病毒-东部马脑炎病毒(EEEV)是美国地方性病毒中毒性最强的病毒之一，在很高比例的被感染的人和马中导致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，并有可能用作生物武器，它被归类为选择代理病毒和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来对抗这种病毒，而且对它的研究严重不足。病毒与受体的相互作用代表了抗病毒治疗的靶点，可用于疫苗载体的合理设计。硫酸乙酰肝素(HS)是一种硫化多糖，被认为是多种甲病毒和黄病毒实验室毒株的附着受体，但其与病毒的相关性在自然界中受到质疑，因为它 用作受体可能是一种适应体外生长的人工制品。为了解决这个问题，我们比较了HS结合的EEEV实验室毒株和60多个EEEV毒株的E2附着蛋白氨基酸序列，包括直接从动物组织的未扩增现场样本中测序的病毒。这一分析证实了HS是自然循环EEEV的受体。通过对消除HS结合表型的EEEV附着蛋白的突变，我们确定HS结合是脊椎动物EEEV疾病的多个独特方面的原因，包括极端的神经毒力、有限的脾复制以及参与先天性和获得性免疫反应的细胞因子/趋化因子的抑制。这表明HS结合促进了EEEV在体内的复制适合性。本应用中的实验将研究HS结合在EEEV疾病中的作用以及脊椎动物和蚊子宿主中识别虫媒病毒复制/传播周期中易受治疗干预的点的组织靶向，为合理设计抗EEEV疫苗提供依据。

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.