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 Objective To study the relationship between the variation of JE virus and the sequence of NS3 gene. Methods The model of persistent infection was established by wild-type JE virus and a human hepatocellular carcinoma cell line KN73. The cells were passaged by standard trypsin digestion technique and the intracellular virus was collected by repeated freeze-thaw cycles. The BHK cell plaque assay was used to titrate the virus and reverse transcriptase polymerase chain reaction (PCR) with NS3 region specific primers to obtain NS3 gene fragments. Sequence analysis was performed using the ABI PRSMTM310 sequencing system. The results showed that the virus in the cell culture medium was 10 6 PFU / ml in the early stage (2-4 h after infection) and 10 3 ~ 4 PFU / ml in the late stage (3 years after infection). The intracellular virus content Has been maintained at 10 2 ~ 3PFU / ml level. NS3 region sequencing results can be seen: located in the nucleotide base sequence 6 6 C → U, 72 A → C, 94 94 U → C, 30 6 A → G and 342 A → G, but the corresponding encoded amino acids did not mutate. CONCLUSION: Variants present in the mutant strain are less proliferative than those in the wild-type strain. The protein encoded by NS3 is critical to both the wild-type strain and the mutant strain. It also suggests that the variation of the virus may be caused by protein mutation in other regions or interaction between the virus and the host protein Due to the role