Establishment of methodology for clinicopathological investigation of viral encephalitis occurring in the South-East Asia

在这项研究中，我们调查了中枢神经系统病毒感染的方法和发病机制。主要目标是狂犬病病毒，包括狂犬病病毒、属于弹状病毒科（大分病毒）的昆虫蝙蝠分离株、尼帕病毒和肠道病毒71。我们初步研究了在福尔马林固定的人和动物组织的石蜡切片中鉴定这些病毒的方法。我们通过在大肠杆菌中表达的重组蛋白和变性病毒颗粒免疫家兔，建立了检测这些核蛋白或衣壳蛋白的抗体。在Western印迹分析中证实这些兔抗血清与病毒蛋白反应后，我们对通过接种病毒制备的动物组织进行免疫组织化学检查。用狂犬病病毒核蛋白免疫制备的抗体能识别石蜡切片上的各种狂犬病病毒，并能检测70年代输入性狂犬病病例的抗原。针对尼帕病毒核蛋白的抗体在马来西亚尸检病例中可以检测到该抗原，但敏感性不太好。关于大分病毒，我们测定了核衣壳基因，揭示了弹状病毒科病毒的独特的发生位置。通过食蟹猴静脉注射建立肠道病毒71型动物模型，揭示肠道病毒71型与脊髓灰质炎病毒致病性的异同。这些结果对进一步研究病毒性脑炎，特别是东南亚地区的病毒性脑炎有重要意义。

In this study we investigated the methodology and pathogenesis of viral infections of the central nervous system. The main targets were lyssaviruses including rabies viruses, the isolate from an insect bat belonging Rhabdoviridae (Oita virus), nipah virus, and enterovirus 71. We initially studied the methods for identification of these viruses in paraffin sections of formalin-fixed human and animal tissues. We established the antibodies to detect these nucleoproteins or capsid proteins by immunization of recombinant proteins expressed in E.coli and degenerated viral particles to rabbits. After confirmation of these rabbit antiserum reacting with viral proteins in western blot analysis, we performed immunohistochemical examinations of animal tissues which were prepared by inoculation of the viruses. The antibody prepared by immunization of nucleoprotein of rabies virus could recognize the various lyssaviruses in paraffin sections and also was capable to detect the antigens in the imported rabies cases in 1970s. The antibody against the nucleoprotein of Nipah virus could detect the antigen in a human autopsy case in Malaysia but the sensitivity was not so good. Regarding Oita virus we sequenced of the nucleocapsid gene and disclosed the unique position of phylogeny of Rhabdoviridae. We established the animal model of enterovirus 71 using the cynomolgus monkeys through the intravenous way and disclosed the similarity and difference with and from the pathogenicity of poliovirus. These results are valuable for further studies on viral encephaltis, especially occurring in the South-east Asia.