Elucidating molecular mechanisms of yellow fever virus virulence

阐明黄热病病毒毒力的分子机制

• 批准号: 8067087
• 负责人: KATHERINE D RYMAN
• 金额: $ 22.42万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8067087
• 关键词: Affect; Africa; African; Agreement; Alphavirus; Americas; Amino Acid Substitution; Amino Acids; Animal Disease Models; Animals; Arboviruses; Attenuated; Attenuated Live Virus Vaccine; Biological Warfare; Bioterrorism; Bite; Complementary DNA; Contracts; Culicidae; Cultured Cells; Data; Dendritic Cells; Dengue Virus; Disease; Eastern Equine Encephalitis Virus; Emerging Communicable Diseases; Engineering; Europe; Evaluation; Event; Exploratory/Developmental Grant; Flavivirus; Fright; Genes; Genome; Genomics; Goals; Human; IFNAR1 gene; Immune response; Immunization; In Vitro; Individual; Infection; Interferon Type I; Interferons; Laboratories; Life; Liver; Maps; Modeling; Molecular; Morbidity - disease rate; Mus; Mutation; Nucleotides; Pathogenesis; Pathology; Phenotype; Primary Cell Cultures; Property; Relative (related person); Research Personnel; Resources; Role; Safety; Site; Skin; Spleen; Structural Genes; System; Technology; Testing; Vaccines; Viral Genes; Viral Hemorrhagic Fevers; Viral Pathogenesis; Virulence; Virulent; Virus; Virus Diseases; West Nile virus; Yellow Fever Virus Infection; Yellow fever virus; attenuation; base; cytokine; design; immunogenicity; improved; in vivo; lymph nodes; macrophage; mortality; pathogen; public health relevance; receptor; research study; response; subcutaneous; type I interferon receptor; vector; vector mosquito; virus host interaction; virus pathogenesis

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.

描述（由申请人提供）：由蚊媒黄热病病毒 (YFV) 引起的高致命性泛系统出血热是非洲、欧洲和美洲最令人恐惧的疾病之一，直到 17D 减毒活疫苗问世之前。发展于1930年代。根据经验，YFV 的天然 Asibi 分离株在原代培养细胞中传代以衍生 17D 病毒。基因组序列比较揭示了与 17D 减毒同时发生的 48 个核苷酸和 20 个氨基酸取代。尽管17D被认为是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。推断许多病原病毒的毒力取决于干扰素（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-??? 的作用保护小鼠免受内脏 YFV 感染。我们发现野生型 Asibi 病毒和减毒活病毒 17D 在缺乏 I 型 IFN 受体 (IFNAR1-/-) 的小鼠中的发病率、死亡率和发病机制很容易区分。在该提案中，将利用这种 YFV 疾病模型来识别和表征 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 IFNAR1-/- 小鼠中野生型 Asibi 和减毒 17D 的发病机制，以查明 17D 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，其中病毒利用活化 DC 的迁移特性来影响病毒血症传播。由于 Asibi 和 17D 在早期病毒血症潜力方面存在很大差异，因此我们认为巨噬细胞/DC 感染的差异可能会改变发病机制。在目标 2 中，我们将使用源自 Asibi 和 17D cDNA 克隆的嵌合病毒来系统地鉴定和定位 17D 的减毒表型： i) 结构或非结构基因区域； ii) 特定基因；最后iii)单个或核苷酸/氨基酸突变的组合。广泛的嵌合病毒组已经可用。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”设计用于预防其他黄病毒的减毒活疫苗提供框架。 公共卫生相关性：由蚊媒黄热病病毒 (YFV) 引起的高度致命的病毒性出血热是非洲、欧洲和美洲最令人恐惧的疾病之一，直到 1930 年代开发出减毒活疫苗 17D 为止。即使在今天，每年仍有超过 20 万西非人感染黄热病，造成数万人死亡。 17D 减毒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）而获得的。尽管17D被认为是有史以来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导致了减毒。为了实现这一目标，博士。莱曼和巴雷特提出了一项联合协议，巴雷特博士的实验室将向莱曼博士的实验室提供病毒，并使用新开发的 YFV 发病机制和疾病模型来评估病毒的毒力。我们对宿主-病原体相互作用的了解已经充分增加，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。预计我们的研究结果将提高 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）以及密切相关的疫苗。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。