【摘要】 目的　研究乙脑病毒变异性状与NS3区基因序列的关系。方法　应用乙脑病毒野生株及一种人肝癌细胞KN73建立持续感染模型 ,采用标准胰蛋白酶消化技术进行细胞传代 ,经反复冻融法收集细胞内变异病毒。采用BHK细胞空斑实验方法进行病毒滴定 ,利用NS3区特异引物进行逆转录 多聚酶链反应以获NS3区基因片段 ,应用ABI PRSMTM310测序系统进行序列分析。结果　在早期 (感染后 2 4～ 36h)细胞培养液中病毒量为 10 6PFU/ml,在后期 (感染后 3年 )细胞培养液中病毒量为10 3～ 4 PFU/ml,细胞内病毒含量一直维持在 10 2～ 3PFU/ml水平。NS3区测序结果可见 :位于核苷酸碱基序列上第 6 6位C→U ,第 72位A→C ,第 2 94位U→C ,第 30 6位A→G及第 342位A→G ,但相应编码的氨基酸未发生变异。结论　变异株存在性状变异 ,即增殖性低于野生株 ;NS3区编码蛋白对野生株及变异株均至关重要 ,亦提示病毒性状变异的原因可能由其它区域蛋白变异或病毒与宿主间蛋白相互作用所致更多还原

【Abstract】 Objective To study on the relationship between the characterization and NS3 sequence analysis of mutant strains during persistent infection of KN73 cells with Japanese encephalitis viruses. Methods Persistent infection model was established by the standard strains of Japanese encephalitis viruses and human hepatoma cells line. Cells were subcultured weekly by standard trypsinization techniques. Cell associated viruses of persistently infected cells were collected by the method of freezing and melting alternate. Viral titers were examined by plaque methods using BHK cells NS3 region of mutant virus strain from infected cells was detected by reverse transcription polymerase chain reaction (RT PCR) using specific primers. PCR products were purified using agarose gel, and then directly sequenced by ABI PRSN TM 310 sequencing system. Results In the early phase (24 36hr) post infection, virus titers of culture fluids in KN73 cells infected with JaGAr 01 were 10 6 PFU/ml. They were 10 3 4 PFU/ml in the late phase (3 years) post infection. The cell associated viruses in KN73 cells infected with JaGAr 01 were 10 2 3 PFU/ml. Sequence analysis of NS3 region from mutant viruses was found that the amino acids did not change though some based encoding them changed. Conclusions The reproduction of the mutant viruses were lower than that of the standard virus JaGAr 01. The protein encoded by NS3 region was very important for both the standard and the mutant viruses JaGAr 01. It was pointed out that the reason of virus mutation was resulted from protein encoded by other nucleotides sequences, or from the cooperation between virus and host protein.更多还原