Elucidating molecular mechanisms of yellow fever virus virulence

描述(申请人提供)：由蚊媒黄热病病毒(YFV)引起的高度致命的全身性出血热是非洲、欧洲和美洲最可怕的疾病之一，直到20世纪30年代S开发出17D减毒活疫苗。YFV天然亚西比分离株在原代培养细胞中经验性传代获得17D病毒。基因组序列比较发现，发生了48个核苷酸和20个氨基酸的替换，与17D的衰减一致。尽管17D被认为是一种典型的减毒活疫苗，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了极大的阻碍。推论许多致病病毒的毒力取决于对干扰素的逃避和/或拮抗-？通过物种特异性机制进行反应，我们研究了干扰素的作用。保护小鼠免受嗜脏性YFV感染。我们发现，在缺乏I型干扰素受体(IFNAR1-/-)的小鼠中，根据发病率、死亡率和致病机制，野生型Asibi病毒和减毒活17D很容易区分。在这项建议中，将利用YFV病的这种模型来识别和表征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和弱化17D在IFNAR1/-小鼠中的发病机制，以确定相对于ASIBI，17D复制和传播受阻的步骤(S)。目前的虫媒病毒致病模型表明，皮肤中树突状细胞(DC)和巨噬细胞的感染是一个关键的早期事件，病毒利用激活的DC的迁移特性来实现病毒的传播。由于ASIBI和17D在早期病毒潜势上有很大的不同，我们认为巨噬细胞/DC感染的不同可能改变了发病机制。在目标2中，我们将使用来自Asibi和17D克隆的嵌合病毒来系统地鉴定和定位17D的弱化表型：i)结构或非结构基因区域；ii)特定基因；以及最后iii)核苷酸/氨基酸突变的单个或组合。目前已有大量嵌合病毒问世。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”设计针对其他黄病毒的减毒活疫苗提供一个框架。 公共卫生意义：由蚊媒黄热病病毒引起的高致命性病毒性出血热一直是非洲、欧洲和美洲最可怕的疾病之一，直到20世纪30年代S开发出17D减毒活疫苗。即使在今天，每年仍有20多万西非人感染黄热病，导致数万人死亡。减毒17D疫苗株是通过在培养细胞中反复培养野生型YFV分离株(Asibi株)而产生的。尽管17D被认为是有史以来最有效的减毒活疫苗之一，但控制这种减毒活疫苗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17D感染，并阐明病毒基因组中积累的哪些突变对病毒的衰减负责。为了实现这一目标，莱曼博士和巴雷特博士提出了一项联盟协议，根据该协议，巴雷特博士的实验室将向莱曼博士的实验室提供病毒，在那里，将使用新开发的YFV致病机制和疾病模型来评估它们的毒力。我们对宿主-病原体相互作用的了解已经足够多，可以合理设计减毒活病毒株，并且存在在基因工程载体系统中引入和测试突变的技术。预计我们的发现将提高YFV疫苗的安全性和有效性，并进一步促进其他减毒活疫苗的合理设计，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)以及与之密切相关的甲型病毒(例如东部马脑炎病毒)，这些病毒大多既是新发传染病的病原体，也是生物恐怖主义/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.