摘要
OBJECTIVE: To study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression. METHODS: Persistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins. RESULTS: In the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed. CONCLUSIONS: The virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.
目的 研究乙脑病毒变异的特性以及变异株性状与E蛋白表达的关系。方法 通过应用乙脑病毒的野生株以及人肝癌细胞KN73建立持续感染模型。采用胰蛋白酶消化技术每周进行细胞传代。经反复冻融收集持续感染细胞内病毒。采用BHK细胞空斑实验方法进行病毒滴定。采用间接免疫荧光方法检测E和NS3蛋白抗原。采用Western印迹杂交检测E和NS3蛋白的表达。结果 感染早期 (2 4小时— 36小时 )野生株感染的KN73细胞的培养液中病毒滴度为 10 6 PFU/ml。在感染后期 (3年 )滴度为 10 3 4 PFU/ml。病毒重叠感染实验发现在急性重叠感染野生株的持续感染KN73细胞中培养液的病毒滴度比在同一时相急性感染的正常KN73细胞培养液的滴度要低得多。间接免疫荧光检测表明 ,在持续感染的KN73细胞中病毒抗原的存在低于急性感染的KN73细胞。Western印迹杂交检测表明 ,E、NS3蛋白的分子量分别为 5 3KD和 73KD。在持续感染的KN73细胞中NS3蛋白的表达很稳定 ,但E蛋白的表达却明显受抑。结论 从持续感染的KN73细胞中获得的病毒具有缺陷干扰病毒的一些特性且参与持续感染 ,其毒力和增殖均低于亲本病毒。
