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）引起的高致死性、全系统性出血热是非洲、欧洲和美洲最可怕的疾病之一，直到20世纪30年代开发出17 D减毒活疫苗。将YFV的天然Asibi分离物在原代培养细胞中凭经验传代以衍生17 D病毒。基因组序列比较发现48个核苷酸和20个氨基酸的取代发生一致的衰减17 D。尽管17 D被认为是一种原型减毒活病毒疫苗，但由于缺乏小动物疾病模型，其减毒和免疫原性的分子基础的阐明受到极大阻碍。许多致病性病毒的毒力依赖于干扰素（IFN）的逃避和/或拮抗作用。通过物种特异性机制的反应，我们研究了IFN-γ的作用？保护小鼠免受嗜内脏的YFV感染。我们发现，野生型Asibi病毒和减毒活病毒17 D在缺乏I型IFN受体（IFNAR 1-/-）的小鼠中的发病率、死亡率和发病机制的基础上很容易区分。在这项提议中，将利用这种YFV疾病模型来鉴定和表征YFV减毒的决定因素，并揭示控制病毒/宿主相互作用的分子机制。在目标1中，我们将描述野生型Asibi和减毒17 D在IFNAR 1-/-小鼠中的发病机制，以确定17 D复制和传播相对于Asibi受阻的步骤。目前虫媒病毒发病机制的模型表明，皮肤中的树突状细胞（DC）和巨噬细胞的感染是一个关键的早期事件，其中病毒利用活化的DC的迁移特性来影响病毒血症传播。由于Asibi和17 D在早期病毒血症潜力方面存在很大差异，我们认为巨噬细胞/DC感染的差异可能会改变发病机制。在目的2中，我们将使用源自Asibi和17 D的cDNA克隆的嵌合病毒来系统地鉴定和映射17 D的减毒表型：i）结构或非结构基因区域; ii）特异性基因;以及最后iii）核苷酸/氨基酸突变的单个或组合。一组广泛的嵌合病毒已经可用。这些研究将开始阐明YFV减毒和毒力的分子机制，并将为“合理”设计用于保护免受其他黄病毒的减毒活疫苗提供框架。 公共卫生相关性：由蚊子传播的黄热病病毒（YFV）引起的高致死性病毒性出血热是非洲、欧洲和美洲最可怕的疾病之一，直到20世纪30年代开发出减毒活17 D疫苗。即使在今天，每年仍有20多万西非人感染YF，造成数万人死亡。通过在培养细胞中重复生长野生型YFV分离株（Asibi株）来获得减毒的17 D疫苗株。尽管17 D被认为是迄今为止开发的最有效的减毒活病毒疫苗之一，但控制这种减毒活疫苗减毒的分子机制仍然是一个谜。我们的长期目标是确定宿主如何能够控制17 D感染，并阐明病毒基因组中积累的哪些突变是导致减毒的原因。为了实现这一目标，Ryman博士和Barrett博士提出了一项联盟协议，其中Barrett博士的实验室将向Ryman博士的实验室提供病毒，在那里将使用新开发的YFV发病机制和疾病模型评估其毒力。我们对宿主-病原体相互作用的理解已经增加到足以允许活减毒病毒株的合理设计，并且存在在基因工程载体系统中引入和测试突变的技术。预期我们的发现将提高YFV疫苗的安全性和有效性，并且另外促进其他减毒活病毒疫苗的合理设计，特别是针对其他致病性黄病毒（例如，西尼罗河病毒和登革热病毒）和密切相关的甲病毒（例如，东部马脑炎病毒），其中大多数是新出现的传染病和生物恐怖主义/生物战的病原体。

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.