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: 7896372
• 负责人: KATHERINE D RYMAN
• 金额: $ 20.18万
• 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 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7896372
• 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）：由蚊子 - 传播的黄热病病毒（YFV）引起的高度致命的，启发性的启示热是非洲，欧洲和美洲最令人恐惧的疾病之一，直到1930年代开发了17D疫苗。 YFV的天然ASIBI分离物在原代培养细胞中经验传递，以推导17D病毒。基因组序列比较揭示了48个核苷酸和20个氨基酸取代，这与17d的衰减相一致。尽管17D被认为是原型的活病毒疫苗，但由于缺乏小动物疾病模型，阐明了分子衰减和免疫原性的分子基础。推理许多病毒病毒的毒力取决于干扰素的逃避和/或拮抗作用（IFN） - ???通过物种特异性机制的响应，我们研究了IFN的作用 - ???在保护小鼠免受内脏YFV感染中。我们发现，根据缺乏I型IFN受体（IFNAR1 - / - ）的小鼠的发病率，死亡率和发病机理，野生型ASIBI病毒和活衰减的17D很容易区分。在此提案中，将利用这种YFV疾病模型来识别和表征YFV衰减的决定因素，并暴露控制病毒/宿主相互作用的分子机制。在AIM 1中，我们将表征野生型ASIBI的发病机理，并在IFNAR1 - / - 小鼠中减弱17D，以确定相对于ASIBI阻碍17D复制和传播的步骤。 arbovirus发病机理的当前模型表明，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，其中病毒利用活化的DC的迁移特性来影响病毒性传播。由于Asibi和17d在早期病毒潜力方面有很大差异，因此我们认为巨噬细胞/DC感染的差异可能会改变发病机理。在AIM 2中，我们将使用源自Asibi和17d的cDNA克隆的嵌合病毒来系统地识别并绘制17d的衰减表型：i）到结构或非结构性基因区域； ii）特定基因；最后是iii）到核苷酸/氨基酸突变的单一或组合。已经有大量的嵌合病毒。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”设计实时疫苗的设计框架，以保护其他黄病毒。 公共卫生相关性：由蚊子传播的黄热病病毒（YFV）引起的高致命病毒出血热是非洲，欧洲和美洲最令人恐惧的疾病之一，直到1930年代开发了现场直播的17D疫苗。即使在今天，每年有超过200,000西非人签订YF，数以万计的死亡人数。通过反复在培养细胞中生长野生型YFV分离株（菌株ASIBI）来得出减毒的17D疫苗菌株。尽管17D被认为是有史以来开发的最有效的活衰减病毒疫苗之一，但控制这种实时侵入疫苗的衰减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控制17D感染并阐明病毒基因组中累积的突变是导致衰减的原因。为了实现这一目标，博士。莱曼（Ryman）和巴雷特（Barrett）提出了一项财团协议，其中巴雷特（Barrett）博士的实验室将向莱曼（Ryman）博士的实验室提供病毒，在那里将使用新开发的YFV发病机理和疾病模型来评估其毒力。我们对宿主病原体相互作用的理解已经充分增加了，以允许对实时衰减的病毒菌株进行合理的设计，并且该技术的存在以引入和测试基因设计的载体系统中的突变。可以预料，我们的发现将提高YFV疫苗的安全性和功效，并促进其他活衰减的病毒疫苗的合理设计，尤其是针对其他致病性黄病毒（例如，西尼罗河和登革热病毒），以及密切相关的静脉疾病（例如，Easterter Emere Enceen sore emerion sore emerion sore emeriantion）和生物恐怖主义/生物贸易。