Irkut蝙蝠狂犬病毒感染后形成免疫抑制的分子基础

Rabies is a zoonosis caused by lyssavirus with typical symptom of acute progressive encephalitis，one of the characteristics of lyssavirus is its capacity to invade the central nervous system (CNS). CNS infection with wild-type (wt) lyssavirus is always fatal in humans, representing a serious public health problem worldwide. Rabies virus (RABV) is the major causative agent to humans and animals, however, can be effectively protected by preexposure prophylaxis (PrEP) or postexposure prophylaxis (PEP). In our previous study, a lyssavirus was isolated from a greater tube-nosed bat (Murina leucogaster) in Jilin Province, China. It was identified as Irkut virus (IRKV) (isolate IRKV-THChina12). In the PrEP or PEP experiments, vaccine or human RABV immunoglobution (HRIG) at the challenge site did not protected mice against IRKV infection. Besides, different neurological lesions in brain and chemokines response in serum suggested differences in the pathogenesis of the infection by RABV and IRKV. We are designed to use the reverse genetic technology platform to reverse serially chimeric lyssaviuses based on IRKV replaced with RABV structural protein, and we are planning to infect the different cells in vitro and the mice in vivo, to detect the infection, replication, migration, dissemination, and the molecular basis for immunosuppression in IRKV infection. On the other hand, the yeast two-hybrid system will be used to identify the interactions of the key structural protein with host proteins, and the binding properties of the host proteins will be analyzed by co-immunoprecipitation combined with confocal laser technology. We will also investigate whether the host protein influences IRKV replication and pathogenicity by the over-expression and knockdown technology. Clarifying the biologics and the molecular basis for immunosuppression in IRKV infection is the theoretical basis for the IRKV vaccine development and pathogenesis research.

狂犬病因其病原一旦侵入中枢神经系统后，感染者几乎100%死亡而引起社会的高度恐慌。本研究前期从我国蝙蝠体内分离到Irkut蝙蝠狂犬病毒（IRKV），研究发现不论是暴露前免疫市售狂犬病疫苗还是暴露后治疗，均不能100%保护IRKV的感染，而且感染IRKV的动物脑组织病理损伤及血清中炎性因子应答较感染狂犬病病毒（RABV）有明显差异。本项目拟拯救基于IRKV为骨架，结构蛋白依次被替换为RABV结构蛋白的系列嵌合病毒，通过体内、体外感染实验，揭示IRKV在感染、复制、移行及分布的生物学特性和造成免疫抑制的病毒结构蛋白；随后采用酵母双杂交实验以造成免疫抑制的关键结构蛋白为饵，钓取与其相互作用的宿主蛋白，并对宿主蛋白进行过表达及沉默，研究其对IRKV感染与复制的影响。阐明IRKV感染的生物学特性及免疫抑制的分子基础，为IRKV疫苗制剂研发及致病的分子机制研究提供理论前提。

狂犬病因其病原一旦侵入中枢神经系统后，感染者几乎100%死亡而引起社会的高度恐慌。实验室前期从我国蝙蝠体内分离到Irkut蝙蝠狂犬病毒（IRKV），研究发现不论是暴露前免疫市售狂犬病疫苗还是暴露后治疗，均不能100%保护IRKV的感染，而且感染IRKV的动物脑组织病理损伤及血清中炎性因子应答较感染狂犬病病毒（RABV）有明显差异。本研究建立了针对IRKV核酸检测的等温扩增结合荧光实时/试纸条读取的检测方法，灵敏度均可达100拷贝数/反应，反应在常温37℃ 20分钟内，不依靠复杂仪器设备，可作为现场快速诊断方法使用。驯化完成了适应细胞增殖的IRKV，滴度可达107.25TCID50/ml以上，对其进行了研究，表明IRKV P蛋白第441位氨基酸对于IRKV适应细胞增殖起着重要的调节作用，细胞适应株相较于分离株IRKV毒力降低；建立了狂犬病毒反向遗传学操作平台，已成功拯救出鼬獾源狂犬病病毒适应株及标准攻击毒株，拯救出的病毒株与母本毒株相比较，细胞增殖性无明显差异；这些对于后续IRKV拯救提供参考与基础。构建了IRKV感染性克隆质粒系统（全长感染性克隆及辅助质粒体系），通过瞬时转染采用相应抗体验证构建的辅助质粒均能较好表达。实现了脑组织类器官的诱导分化培养并成功建立了其感染狂犬病毒（含IRKV）的类器官感染模型，尚属首次报道，对于免疫机制及抗病毒药物研发提供模型支持及基础。..