四株猪伪狂犬病毒株的分离鉴定与主要毒力基因分析

Isolation and identification of 4 strains of porcine pseudorabies virus and analysis of main virulence genes

猪伪狂犬病毒(PRV)的流行对中国养猪业造成巨大损失,而2011年后许多已免疫PRV疫苗的猪场频繁出现gE抗体转为阳性现象,感染猪出现PRV的临床症状,并出现所谓的“流产风暴”,学者们怀疑PRV的重新流行与病毒毒力增强和基因变异有关。为了解PRV变异情况,从各地疑似PRV阳性病料中,通过PK-15细胞分离出4个毒株,对毒株传代培养,进行TCID50与LD50测定,对主要毒力基因gB、gC、gE和TK进行扩增测序后分析,确定该4株病毒为PRV株,分别命名为FJ01株、FJ03株、YK株和MS2018株,滴度分别为10-6.63、10-7.08、10-8.10、10-7.18TCID50s·0.1mL^-1,对Balb/c小鼠的LD50分别为102.17、102.72、103.44、103.51TCID50s,可见FJ01株的毒力最强。对4个毒株的毒力基因与其他PRV毒株进行同源性比对并建立进化树,FJ01株、FJ03株、MS2018株与中国近几年流行的变异毒株如HNX株、HNB株、JS-2012株等在一个大进化分支上,亲缘性较近,而与疫苗株Bartha-K61、SA215等,国际经典毒株Becker、Kaplan等亲缘性较远,变异较大。YK株与国际毒株亲缘性更近,其原因有待进一步探索。

Pseudorabies virus(PRV)’s prevalence brings huge economic losses to the domestic pig industry,however, many large-scale immunized pig herds broke out pseudorabies,and gE antybodies turned from negative to positive since2011,causing PRV clinical symptoms infected pigs,and then resulting in “abortion storm”. Most scholars believed that national outbreak of PRV might be related to factors such as viral virulence and genetic variation. In this study,four pseudorabies viruses were successfully isolated from suspected PRV infected pigs by PK-15 cells,moreover,their TCID 50 and LD 50 were determined. Their main immunogenic genes and virulence-related genes gB,gC,gE and TK were amplified,sequenced and analyzed. The 4 pseudorabies viruses were affirmed,named as FJ01,FJ03,YK and MS2018 strain,respectively,whose titers were10 -6.63,10 -7.08,10 -8.10,10 -7.18 TCID 50s ·0.1mL -1, respectively,and the results of virulence test in Balb/c mice showed as102.17,102.72,103.44,103.51 TCID 50s, respectively. The virulence of FJ01 strain was the strongest. Their nucleotide and amino acid homologies were analyzed and evolutionary trees were established. The results showed that the genes of FJ01,FJ03 and MS2018 strains had higher homology with the mutants such as HNX,HNB and JS-2012 strains which were isolated in recent years. In evolutionary relationship,they belonged to a large branch,however,they belonged to different evolutionary branches from vaccine Bartha-K61,SA215,and foreign strains Becker and Kaplan with evident variations. YK had the closer affinity with foreign strains in evolutionary relationship,but the reason remained to be further explored.