犬源伪狂犬病病毒的分离鉴定及其主要毒力基因分子特征分析

Isolation,Identification and the Major Virulence Genes Characteristic Analysis of Pseudorabies Virus from Canine

对发生临床疑似伪狂犬病毒(PRV)感染的牧羊犬组织样品,进行研磨、细胞接种、传代培养、细胞病变效应(CPE)观察、PCR检测、效价测定、电镜观察、IFA检测、家兔接种以及基因测序分析等,结果成功分离到1株犬源PRV毒株,命名为XJ-14株。该毒株可致PK-15细胞产生变圆、破碎、逐渐呈拉网式漂浮等特征性细胞病变;PRV gE-PCR检测阳性,病毒效价达10^(-7.45)/0.1 mL;病毒粒子在电镜下呈圆形,有囊膜,直径约150 nm;PRV荧光抗体检测阳性,接种家兔后可致家兔奇痒、麻痹死亡。对该毒株gC、gE、TK基因进行全基因测序,发现gE糖蛋白的48、497位各插入了1个D,另有12个氨基酸位点发生点突变,其gC蛋白氨基酸序列64~70位出现了7个氨基酸(AASTPAA)插入。结果表明,此牧羊犬病例是由PRV感染所致。

Tissue samples from a sheepdog infected with clinically suspected Pseudorabies virus(PRV)were identified by grinding,cell culture,subculture,CPE observation,PCR detection,virus titer determination,electron microscope observation,IFA assay,rabbit inoculation test gene sequencing and analysis.As a result,one PRV strain was isolated from dog successfully,named XJ-14strain.The isolate was able to cause typical CPE in PK-15cells,characterized by round,broken,cell mesh and floating.The result of PRV gE-PCR was positive,with the virus titer of10-7.45/0.1mL.The virus particles were round,with envelope,diameter about150nm under the electron microscope.The reaction with PRV fluorescent antibody was positive.After challenging the XJ-14isolate,the rabbits appeared itching,paralysis and death.The major virulence genes,such as gE,TK and gC gene were sequenced and analyzed.The results showed that two Asp were inserted in the gE glycoprotein48and497amino acid site,respectively.And there were12amino acid sites mutation.For the gC glycoprotein sequence,there were7amino acids insertion(AASTPAA)in the64~70amino acid sites.All the results showed that the dog case was caused by PRV.