狂犬病病毒诱导小鼠脑产生高水平I型干扰素的机制研究

Rabies is a zoonotic disease, the scourge that each year kills more than 70,000 people over the world. Around 3.9 billion people are at risk of rabies worldwide, and more than 150 countries are affected. Some 0.9 billion of Chinese people are at threat of rabies. In the past decade, rabies kills coverage of 2224 persons each year in China. We isolated a rabies virus GX074 from a clinically normal dog in Guangxi province and identified that the rabies virus GX074 exerts strong pathogenicity in the adult mouse host. Rabies virus glycoprotein (G) could induce high-level type I interferon (IFN-α/β) in mouse brain, and it was verified that the level of IFN-α/β in brain was related to clinical signs of mouse. To explore the high pathogenicity underlying GX074 infection in mice, chimeric rabies virus mutants were constructed using an infectious cDNA clone of the fixed RC-HL strain (Japan) by replacing the native RC-HL genes with GX074 genes using a reverse-genetics approach and the pathogenicity was evaluated in vivo using adult mice..To specific this phenomenon that may be possible to relative with pathogenic mechanism of rabies, in this study we use the techniques, such as immunohistochemistry assay, immune-fluorescent assay, reverse genetic approach, to construct infectious cDNA clone of GX074 glycoprotein, genetic knock-out of IFN-α/β receptors’ (IFNAR) genes, (1) to explore rabies virus GX074 induce which kind cells, neuroglia cells or neurons, to produce high-level IFN-α/β, location of IFN-α/β in cells, and how the IFN-α/β affects neurons; (2) to explore the molecular signal transduction pathway of RABV GX074 induces production of IFN-α/β in mouse brain, and the relationship between production of IFN-α/β in mouse brain and clinical signs and /or mouse lethality. Based on the above works, the research project will reach the aims as follows: determine whether the high-level IFN-α/β in mouse brain induced by GX074 impairs neurons of brain; the molecular regulation mechanism (signal transduction pathway) of the high-level IFN-α/β in mouse brain, and biological importance of the high-level IFN-α/β in mouse brain. These researching results will be of momentous scientific significance to further explain pathogenic and lethal mechanism of rabies virus.

狂犬病是一种灾难性的人畜共患病，全世界每年超过7万人死于该病，超过150个国家、39亿人口受到该病的威胁。我国9亿人受到威胁，近10年平均年死亡2224人。我们从广西健康犬分离的狂犬病病毒GX074株对小鼠致死，发现其G蛋白能诱导小鼠脑组织产生高水平I型干扰素(IFNα/β)，脑内IFNα/β水平与临床症状严重性呈正相关。针对该现象可能与狂犬病致病机理有关，本项目采用免疫组化、免疫荧光、反向遗传及基因敲除等技术，探索GX074诱导小鼠脑产生高水平IFNα/β的细胞类型、细胞内定位及其对神经元细胞的影响，探索GX074诱导脑产生IFNα/β的分子调控通路及IFNα/β与临床症状、致死性的相关性。研究目标：确定GX074诱导小鼠脑产生高水平IFNα/β对神经元细胞的影响，阐明GX074诱导小鼠脑产生高水平IFNα/β的分子调控机制及其生物学意义，对进一步解析该病的发病机理具有重要科学意义。

本项目从广西健康犬分离的狂犬病病毒GX074株能引起小鼠死亡，证实其G蛋白可以诱导小鼠脑组织产生高水平I型干扰素(IFNα/β)，脑内IFNα/β水平与临床症状严重性呈显著正相关。因此，推测高水平细胞因子与狂犬病致病机理之间存在着紧密的联系。通过反向遗传技术成功拯救狂犬病病毒不同基因重组突变体证明G蛋白与 GX074 株的致病性显著相关。随后构建G 蛋白242-288 位氨基酸变异的 Mu1~Mu21共21个突变株，经动物实验确定 242~273 aa位点是与致病性相关的功能区。在此基础上，将第333 位精氨酸（Arg333）突变为谷氨酰胺（Glu333），证明了242~273 aa 区域与 Arg 333 是协同构成 G 蛋白的致病性。感染CVS-24、GX074、rRC-HLΔG、rRC-HLΔG242-288的小鼠在濒死期均检测到高水平的IFN-α、IFN-β、TNF-α、IFN-γ、IL-1β、IL-6、IL-12和MCP-1，说明狂犬病病毒可诱发脑内细胞因子风暴，小鼠脑中TNF-α、IFN-γ、IL-1β、IL-6产生水平与小鼠发病症状以及病毒毒力呈正相关，表明脑内过量的细胞因子与狂犬病致病性密切相关。I型干扰素受体缺失（IFNAR-/-）小鼠被感染后，其发病及死亡时间比正常小鼠提前。疫苗重组毒株rRC-HL和20%致死率的rRC-HL△G/R333Q均能导致IFNAR-/-小鼠发生100%死亡，并可引起基因缺失鼠更高水平干扰素的产生。IFNAR-/-小鼠感染狂犬病病毒可引起小鼠脑小胶质细胞和星形胶质细胞增生相对于正常小鼠明显减少，表明I型干扰素受体与小胶质细胞增生和星形胶质细胞增生有一定的相关性。重组弱毒株 rRC-HL感染 MyD88 基因敲除（MyD88-/-）小鼠后期，维持较高水平的 IFN-α和 IFN-β，同时在感染野生型小鼠早期产生 IL-6，而 MyD88-/-小鼠则相反。本项目系统的阐明了GX074诱导小鼠脑产生高水平IFNα/β的分子调控机制及其生物学意义，对进一步解析该病的发病机理具有重要科学意义。