乙脑病毒持续感染模型建立及初步应用研究

Establishment and Initial Application of Persistent Infection of KN_(73)Cells With Japanese Encephalitis Virus

目的 :建立流行性乙型脑炎病毒持续感染模型 ,为抗病毒药物筛选提供实验模型体系。方法 :采用流行性乙型脑炎病毒标准株及人肝癌细胞株KN73 建立持续感染模型 ,应用标准胰蛋白酶消化技术进行细胞传代 ,经反复冻融方法收集细胞内病毒 ,采用BHK细胞空斑实验进行病毒滴定。选用Suramine进行病毒抑制实验。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 ～10 4PFU/ml,细胞内病毒含量一直维持在 10 2 ～ 10 3 PFU/ml水平 ;在细胞传代后第 2d ,80 μg/ml的Suramine可使病毒量由 2 .3× 10 3 PFU/ml降至 2 .3× 10 2 PFU/ml。结论

Objective: Our aims were to establish a feasible model of the persistent infection of KN73 cells with Japanese encephalitis viruses screening effective antivirus drugs. Methods: Persistent infection model was established by using the standard strains of Japanese encephalitis viruses, and the KN 73 cells which were derived from human liver cancer cells. Cells were subcultured weekly using standard trypsinization techniques. Cell associated viruses of persistently infected cells were collected with the method of freezing and melting alternate. Viral titers were determined with plaque methods by using BHK cells. Suramine regarded as antiviral effect was used to antiviral drugs. Results: In the early stage (24 to 36 hours after infection), virus titers of culture fluids in KN 73 cells infected with JaGAr 01 were 10 6 PFU/ml. They were 10 3- 10 4 PFU/ml in the late stage (3 years after infection). The cell associated viruses in KN 73 cells infected with JaGAr 01 were 10 2-10 3 PFU/ml. The inhibitory experiment of viruses was found that the amount of the viruses was decreased from 2.3×10 3 PFU/ml to 2.3×10 2 PFU/ml at the second day after cell passage when Suramin concentration which was not poisonous to cells was 80 μg/ml. Conclusion: The reproduction of the mutant viruses was lower than that of the standard virus JaGAr 01. The model of the persistent infection of KN 73 cells with Japanese encephalitis viruses was helpful to the study of antiviral drugs.