바이러스 분자유전학 기술을 이용한 차세대 신증후출혈열 백신주 (株)의 개발

초록

Hantaan (HTN) virus which is major causative agent of HFRS is prototype virus of genus hantavirus established newly in family Bunyaviridae. Because there are no effective treatment drugs, vaccines against HFRS, the only applicable method is control and prevention of the disease. Therefore, development of effective vaccine is the most valuable means to control this disease. We could expect more effective vaccine can be achieved via genetic engineering technology. For the major character of bunyaviruses, Hantaan virus has 3 segmented negative sense single stranded RNA molecules, respectively L, M, S, as genome. Because of difficulty to treat RNA, genetic engineering technique of this virus has not been achieved until now. To solve this problem and develop a vaccine strain which has effectiveness and safety, we would like to construct two systems; the one is the super infection method and the other is reverse genetic system for reassortant Hantaan virus. From HTN and Prospect Hill virus (PHV) super-infected cell and medium, we tried to screen the presence of reassortants or recombinants using multiplex PCR. As a result, we could find the reassortant/recombinant viruses in the cell. The pathogenesis of those viruses was tested by inoculation into mouse brain the viruses. From this study, it was evident that those established strains through superinfection are less (or non) pathogenic phenotypes. On the other hands, reverse genetic system also constructed for more advanced study. To serve viral or antiviral RNA in the host cell, we constructed T7 RNA polymerase stable cell line derived from Vero E-6, well known host cell line for hantavirus. And all list of viral plasmid was constructed by RT-PCR and cloning. Using this system, PHV was infected into T7 RNA polymerase stable cell line, and plasmid which has Hantaan S genome as + or - sense was transfected into the cell line subsequently. From this cell line we could detect and isolate recombinant/reassortant viruses which have mixed genome with each other. And also, this recombination and reassortment phenomena occurred only in case of that transfected RNA is positive sense. We expect that those established recombinant/reassortant viruses will offer more diverse chances and be a new candidate for hantaviral vaccine. And also, the methods to obtain recombinant/reassortant virus will offer powerful tools to study viral genetics.

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