3株猪源乙型脑炎病毒的分离鉴定及增值特性

Isolation and identification of three strsins of Japanese encephalitis virus and their proliferative characteristics

2012年从四川省多个地区的猪场采集流产死胎10份,采用BHK-21细胞培养方法分离出3株乙型脑炎病毒,分别命名为JEV-SC-1、JEV-SC-2与JEV-SC-3株,E基因与现用疫苗株SA-14-14-2的核苷酸相似性分别为99.24%、99.05%及98.76%,基因分型研究显示3株病毒株均为基因Ⅲ型,分离株在BHK-21细胞中均能产生明显的细胞病变,其TCID50分别为10-5.81/0.025、10-5.12/0.025及10-4.42/0.025mL。以JEV-SC-1株分别感染BHK-21细胞和Vero细胞,观察病变情况并利用荧光定量PCR法绘制其在这2种细胞中的生长曲线,检测该毒株在BHK-21细胞上连续传代后不同代次的病毒含量。结果显示,JEV-SC-1株在这2种细胞中均能增殖,但CPE及病毒滴度有所差异:JEV-SC-1株感染Vero细胞的病毒滴度较BHK-21细胞高,但出现细胞病变时间较晚;JEV-SC-1株在BHK-21细胞上连续传代至20代,第1代至第10代病毒含量呈上升趋势,10代以后病毒含量趋于稳定,波动幅度较小。本试验为大规模生产疫苗提供理论支持,同时也为进一步研究JEV-SC-1株的生物特性奠定了基础。

In 2012,10 samples of abortion and stillbirth pig were collected from pig farms in Sichuan province,and three strains of Japanese encephalitis virus(JEV-SC-1、JEV-SC-2and JEV-SC-3)were isolated.The E gene of JEV-SC-1 strain,JEV-SC-2 strain and JEV-SC-3 strain shared 99.24%,99.05% and 98.76% nucleotide identity with SA14-14-2vaccine strain,respectively.All of the three strains belonged to genotype Ⅲ.The three strains could cause cytopathic effect in BHK-21cells with titers of 10-5.81 TCID50/0.025 mL,10-5.12 TCID50/0.025 mL and 10-4.42TCID50/0.025mL,respectively.The JEV-SC-1strain were used to infect BHK-21and Vero cells respectively,supernatant were collected at different times points post-infection.Cytopathic effect in different cells was observed and real-time fluorescent quantitative PCR(FQ-PCR)was introduced to analyze the quantity of JEV RNA in different samples,and the one-step growth curves in different cells were determined according to the results.The virus content in different passages of the strains subcultured in BHK-21cells was also detected by FQ-PCR.As a result,JEV-SC-1strain could proliferate in BHK-21and Vero cells and caused different CPE.The titer of JEV-SC-1strain harvested from Vero cells is higher,but the time of CPE is later;subcultured JEV-SC-1strain in BHK-21cells to the 20generation,the first generation to the tenth generation virus showed an upward trend,after the 10generation virus content tends to be stable.This research provided the support for the large scale production of vaccines,also laid the foundation for further study on the biological characteristics of JEV-SC-1strain.