General epidemiological study of wildlife and domestic animal rabies in Brazil

对巴西野生动物和家畜分离的狂犬病毒（RV）核蛋白基因进行了分子流行病学分析。家畜狂犬病分离株定义为吸血蝙蝠相关病毒（VRRV），表明吸血蝙蝠主要将该病毒传播给家畜。果食性蝙蝠存在VRRV，而食虫性蝙蝠的大部分分离株与VRRV组无关。巴西蝙蝠保持着物种特异性的RV变异和复杂的传染周期。糖蛋白氨基酸比对显示狗相关狂犬病毒（DRRV）和VRRV在抗原位点和表位位置上存在差异，提示DRRV和VRRV可以通过抗原性差异进行区分。在分子流行病学研究的基础上，建立RT-PCR结合限制性片段长度多态性（RFLP）分析和多重RT-PCR方法，对不同动物野外分离的RV病毒库进行鉴别和鉴定。多重RT-PCR将野外分离物分为狐、犬、食虫蝙蝠和吸血/食腐蝙蝠4类。该方法对巴西感染周期特别复杂的包括人在内的多种宿主的狂犬病传染源分类具有实用价值。为了研制有效的野生源性狂犬病活疫苗，利用缺失全部P基因的疫苗株cDNA进行反向遗传，获得了RV缺陷P基因。缺陷病毒诱导的小鼠对致命的狂犬病感染具有保护性免疫，而没有任何致病作用。由于该基因缺陷病毒可以通过重组田间分离株的糖蛋白基因进行有用的遗传修饰，因此它将适用于基于各流行地区田间分离株抗原性的新型RV疫苗策略。

Molecular epidemiological analysis of rabies virus (RV) nucleoprotein gene isolated from Brazilian wildlife and domestic animals was performed. Livestock rabies isolates were defined as the vampire bat-related virus(VRRV), showing that vampire bat mainly transmitted these viruses to the livestock. While frugivorous bat had the VRRV, most isolates from insectivorous bat were independent of VRRV group. Brazilian bats maintain species-specific RV variants and complicated jnfectious cycle each other. Amino acid alignment of glycoprotein revealed differences between dog-related rabies virus(DRRV) and VRRV at the positions of antigenic sites and epitopes, suggesting that DRRV and VRRV can be distinguished by differences of antigenicity.Based on the molecular epidemiological studies, RT-PCR along with restriction fragment length polymorphism (RFLP) analysis and multiplex RT-PCR method were established to discriminate and identify the RV reservoirs of field isolates from various animals. Multiplex RT-PCR classified the examined field isolates into four groups, such as fox, dog, insectivorous bat, and vampire/furugivorous bat. This method is useful for the classification of a source of rabies infection in the variety host including human in Brazil which has especially complicated infectious cycle.To develop effective live vaccine against wildlife-derived rabies, which has recently increased, RV deficient P gene was generated by reverse genetics from cDNA of a vaccine strain lacking the entire P gene. The defective virus induced conferred protective immunity against a lethal rabies infection without any pathogenic effects in mice. Since this genetic defective virus can be applied to useful genetic modification by recombining the glycoprotein gene of the field isolate stocks, it would be applicable to the novel RV vaccine strategy based on the antigenicity of field isolates in each prevalent area.