Elucidating molecular mechanisms of yellow fever virus virulence

描述（由申请人提供）：由蚊媒黄热病病毒（YFV）引起的高致命性全系统出血热是非洲、欧洲和美洲最可怕的疾病之一，直到20世纪30年代开发出17D减毒活疫苗。将自然分离的亚洲猪分离株在原代培养细胞中传代，得到17D病毒。基因组序列比较显示，随着17D的衰减，发生了48个核苷酸和20个氨基酸的替换。虽然17D被认为是一种原型减毒活疫苗，但由于缺乏小动物疾病模型，对其减毒和免疫原性的分子基础的阐明受到很大阻碍。许多致病病毒的毒力取决于逃避和/或对抗干扰素(IFN)-？通过物种特异性机制，我们研究了IFN-？保护小鼠免受嗜内脏型YFV感染。我们发现，在缺乏I型IFN受体（IFNAR1-/-）的小鼠中，根据其发病率、死亡率和发病机制，野生型Asibi病毒和减毒17D病毒很容易区分。在本研究中，该YFV疾病模型将被用于鉴定和表征YFV衰减的决定因素，并揭示控制病毒/宿主相互作用的分子机制。在Aim 1中，我们将在IFNAR1-/-小鼠中描述野生型Asibi和减毒17D的发病机制，以确定17D的复制和传播相对于Asibi受到阻碍的步骤。目前的虫媒病毒发病机制模型表明，皮肤中树突状细胞（dc）和巨噬细胞的感染是一个至关重要的早期事件，在这个过程中，病毒利用活化树突状细胞的迁移特性来影响病毒血症的传播。由于Asibi和17D在早期病毒毒潜能方面存在很大差异，我们认为巨噬细胞/DC感染的差异可能会改变发病机制。在目标2中，我们将使用来自Asibi和17D cDNA克隆的嵌合病毒系统地鉴定和绘制17D的减毒表型：1)到结构或非结构基因区域；Ii)特异基因；最后iii)单个或组合核苷酸/氨基酸突变。大量嵌合病毒已经可用。这些研究将开始阐明YFV衰减和毒力的分子机制，并将为“合理”设计抗其他黄病毒的减毒活疫苗提供框架。

DESCRIPTION (provided by applicant): The highly-lethal, pansystemic hemorrhagic fever caused by the mosquito-borne yellow fever virus (YFV) was one of the most feared diseases in Africa, Europe and the Americas, until the live-attenuated 17D vaccine was developed in the 1930's. The natural Asibi isolate of YFV was empirically passaged in primary cultured cells to derive the 17D virus. Genomic sequence comparisons revealed 48 nucleotide and 20 amino acid substitutions that occurred coincident with attenuation of 17D. Although 17D is considered a prototypic live- attenuated virus vaccine, elucidation of the molecular basis for its attenuation and immunogenicity has been greatly impeded by the lack of a small animal disease model. Reasoning that virulence of many pathogenic viruses depends upon evasion and/or antagonism of interferon (IFN)-??? responses by species-specific mechanisms, we investigated the role of IFN-??? in protection of mice from visceroptropic YFV infection. We discovered that wild-type Asibi virus and live-attenuated 17D were readily distinguishable on the basis of their morbidity, mortality and pathogenesis in mice lacking the type I IFN receptor (IFNAR1-/-). In this proposal, this model of YFV disease will be exploited to identify and characterize determinants of YFV attenuation and expose molecular mechanisms that control the virus/host interaction. In Aim 1, we will characterize the pathogenesis of wild-type Asibi and attenuated 17D in IFNAR1-/- mice to pinpoint the step(s) at which 17D replication and dissemination is impeded relative to Asibi. Current models of arbovirus pathogenesis indicate that infection of dendritic cells (DCs) and macrophages in the skin is a crucial early event, in which the viruses exploit migratory properties of activated DCs to effect viremic dissemination. Since Asibi and 17D differ greatly in early viremic potential, we propose that differences in macrophage/DC infection may alter pathogenesis. In Aim 2, we will use chimeric viruses derived from cDNA clones of Asibi and 17D to systematically identify and map the attenuated phenotype of 17D: i) to structural or non-structural gene regions; ii) to specific genes; and finally iii) to single or combinations of nucleotide/amino acid mutations. An extensive panel of chimeric viruses is already available. These studies will begin to elucidate the molecular mechanisms of YFV attenuation and virulence, and will provide a framework for "rational" design of live-attenuated vaccines for protection against other flaviviruses. PUBLIC HEALTH RELEVANCE: The highly-lethal viral hemorrhagic fever caused by the mosquito-borne yellow fever virus (YFV) was one of the most feared diseases in Africa, Europe and the Americas until the live-attenuated 17D vaccine was developed in the 1930's. Even today, over 200,000 West Africans contract YF annually, with tens of thousands of fatalities. The attenuated 17D vaccine strain was derived by repeatedly growing a wild-type YFV isolate (strain Asibi) in cultured cells. Although 17D is considered to be one of the most effective live-attenuated virus vaccines ever developed, the molecular mechanisms that control the attenuation of this live-attenuated vaccine remain a mystery. Our long-term goal is to determine how the host is able to control the 17D infection and elucidate which of the mutations accumulated in the virus genome are responsible for the attenuation. To achieve this goal, Drs. Ryman and Barrett have proposed a consortium agreement in which Dr. Barrett's laboratory will provide viruses to Dr. Ryman's laboratory, where their virulence will be assessed using a newly developed model of YFV pathogenesis and disease. Our understanding of host-pathogen interactions has increased sufficiently to allow rational design of live-attenuated virus strains and the technology exists to introduce and test mutations in genetically-engineered vector systems. It is anticipated that our findings will improve the safety and efficacy of the YFV vaccine, and additionally facilitate the rational design of other live-attenuated virus vaccines, particularly against other pathogenic flaviviruses (e.g., West Nile and dengue viruses) and the closely- related alphaviruses (e.g., eastern equine encephalitis virus), most of which are agents of both emerging infectious disease and bioterrorism/biowarfare.