2株伪狂犬病毒株的分离鉴定及生物学特性分析

Isolation and identification of two pseudorabies virus isolates and analysis of their biological characteristics

为了解伪狂犬病病毒(PRV)变异情况,从山东和江苏两地的某发病猪场分别采集阳性病料接种BHK-21细胞,连续传代进行病毒分离,观察细胞病变;对细胞培养物进行间接免疫荧光与蛋白印迹鉴定,通过对病毒滴度的测定绘制了分离病毒株的一步生长曲线;进一步动物试验鉴定分离株的毒力,并对其gE基因进行遗传进化分析。结果显示:利用PCR成功扩增出PRV的gB、gC和gE特异性基因,病料接种BHK-21细胞后产生了特异性细胞病变;病毒感染细胞后表达了gE蛋白,并与抗PRV单抗特异性结合,显示成功分离到2株分离毒株,分别命名为SDWF2019和JSNT2019;通过序列分析,2个分离株与我国新流行的PRV毒株属于同一遗传进化分支。本研究为我国华东区猪伪狂犬病的疫情监测提供了参考。

In order to understand the mutation of the PRV gene,positive samples were collected from affected pig farms in the Shandong and Jiangsu areas,respectively,and were inoculated with BHK-21 cells.Then,viruses were isolated and identified by PCR,and were successively passaged to observe their cytopathic changes.The positive samples were identified by immunofluorescence and western blotting,and the one-step growth curve of the viruses was plotted by measuring their titer.In addition,mice were inoculated to calculate their mortality rates,to analyze the virulence of the strains,and to construct a phylogenetic tree for the gE genes.The results showed that the gB,gC,and gE specific genes of PRV were successfully amplified by PCR,and the viruses produced specific cytopathic lesions after inoculation with BHK-21 cells.The viruses expressed gE protein and specifically bound to the FITC-labeled anti-PRV monoclonal antibodies after infection with the cells.The virulent strains were named as SDWF2019 and JSNT2019,respectively,and sequencing and sequence analysis showed that they belonged to the same genetic evolutionary branch as that of the newly prevalent PRV strains in China.This study provided a scientific basis for further control of pseudorabies epidemics in China.