IN VITRO CONSTRUCTION OF ATTENUATED VEE VIRUS MUTANTS

减毒 VEE 病毒突变体的体外构建

• 批准号: 6187208
• 负责人: ROBERT E. JOHNSTON
• 金额: $ 26.9万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-03-01 至 2001-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6187208
• 关键词: SCID mouse; Venezuelan equine encephalitis virus; apoptosis; attenuated microorganism; cell type; flow cytometry; histopathology; immunization; immunoglobulin A; in situ hybridization; infectious encephalomyelitis; laboratory mouse; lymphatic tissue; mucosal immunity; mutant; neurotropic virus; neutralizing antibody; synapses; tissue /cell culture; virulence; virus cytopathogenic effect; virus genetics; virus infection mechanism; virus load; virus replication

Venezuelan equine encephalitis virus (VEE) is a lymphotropic and neurotropic alphavirus, currently causing an explosive human epidemic in Colombia and Venezuela with over 45,000 cases to date. The long-term objectives of the proposed experiments are to define the molecular genetics of VEE induced disease and to use the VEE system to elucidate fundamental aspects of viral pathogenesis. The approach combines molecular genetics with classical histopathology and in situ hybridization. The experiments will utilize a mouse model which approximates VEE disease, molecularly cloned wild-type virus (V3000) which recapitulates the pathogenesis of natural virus isolates; and avirulent or highly attenuated site-directed mutants of V3000. Each of the mutations interdicts the VEE pathogenic sequence at a different stage and therefore links specific phenotypes in vivo with defined mutations. Vector systems have been developed which carry the gene for green fluorescent protein packaged in wild-type or mutant glycoprotein envelopes, facilitating the sensitive detection of ongoing virus replication in living cells or prior replication in preserved tissues. The proposed experiments take advantage of these powerful tools for further genetic dissection of VEE pathogenesis. The experiments are organized into four Specific Aims: 1) To define parameters of the lymphotrophic stage of VEE pathogenesis (e.g. elucidation of the earliest events in infection, identification of lymphoid target cells in vivo and in vitro, and the source of the viremia), 2) To determine the mechanism by which avirulent VEE mutants induce mucosal IgA following subcutaneous inoculation (e.g. characterization of viral spread to inductive sites of the mucosal immune system and correlation of mucosal IgA induction with virus mutation, mouse strain, and ability to spread to inductive sites), 3) To define parameters related to invasion of, dissemination within, and clearance of virus from the CNS (e.g. genetic characterization of CNS invasion, synaptic transmission, persistent infections in cell culture and animals, antibody curing of neuronal infections, and the mechanism of VEE induced neuronal death), and 4) To determine the in vivo and cell culture phenotypes of mutations at nucleotide 3 in the 5' untranslated region of the VEE genome.

委内瑞拉马脑炎病毒（VEE）是一种淋巴机， 神经性α病毒，目前引起爆炸性人类流行病 迄今为止，哥伦比亚和委内瑞拉有45,000多个案件。 长期 提出的实验的目标是定义分子遗传学 VEE诱导的疾病并使用VEE系统阐明基本 病毒发病机理的各个方面。 该方法结合了分子遗传学 具有经典的组织病理学和原位杂交。 实验 将利用近似VEE疾病的小鼠模型，分子 克隆的野生型病毒（V3000）概括了 天然病毒分离株；和无毒或高度衰减的现场定向 V3000的突变体。 每个突变都禁止VEE致病性 在不同阶段的序列，因此将特定表型连接起来 带有定义突变的体内。 已经开发了向量系统 携带包装在野生型或包装的绿色荧光蛋白的基因或 突变的糖蛋白信封，促进敏感检测 在活细胞中持续的病毒复制或保留的先验复制 组织。 拟议的实验利用了这些强大的工具 用于进一步的VEE发病机理的遗传解剖。 实验是 组织为四个特定目标：1）定义参数 VEE发病机理的淋巴营养阶段（例如最早阐明 感染事件，体内和体内淋巴靶细胞的鉴定 体外和病毒血症的来源），2）确定由 皮下后，哪种无毒的Vee突变体诱导粘膜Iga 接种（例如，病毒扩散表征到电感部位 粘膜免疫系统和粘膜IgA诱导与 病毒突变，小鼠应变和传播到电感位点的能力），3） 定义与入侵，在内部传播和 从CNS清除病毒（例如CNS的遗传表征 入侵，突触传播，细胞培养中的持续感染和 动物，神经元感染的抗体固化以及VEE的机制 诱导神经元死亡），4）确定体内和细胞培养 在5'未翻译区域的核苷酸3的突变表型 VEE基因组。