猪伪狂犬病病毒ZY-2014株的分离与鉴定

Isolation and Identification of Variant Strain ZY-2014 of Pseudorabies Virus

关键词:  猪伪狂犬病毒  分离  鉴定

Key words:  pseudorabies virus  isolation  identification

摘要:

从河南省某规模化猪场的死亡仔猪中分离到1株疑似猪伪狂犬病病毒，命名为ZY-2014株，并对其进行了PCR鉴定、gE基因序列分析、动物感染试验等鉴定。病毒经vero细胞培养可产生典型细胞病变，用Reed-Muench法测定病毒的滴度为107.4TCID50/100 μL。分离病毒基因组DNA经过gD、gE、gG、三对特异性检测引物扩增后，均出现伪狂犬病毒特异性目的条带，分别为217 bp、636 bp、1440 bp。测序结果与预期结果相符。将ZY-2014毒株gE基因测序结果与10个新分离毒株、14个经典毒株进行同源性比对分析，其核苷酸及推导的氨基酸序列与国内2012年以来新分离毒株的同源性分别为99.6%～100%和99.1%~100%，与2012年以前分离毒株核苷酸及推导的氨基酸序列的同源性分别为97.7%～99.6% 和95.7%～99.3%。遗传进化树结果显示，所分离的ZY-2014毒株与2012年以来新分离毒株同属一分支，与经典毒株分属不同进化分支。动物攻毒试验中，家兔和小鼠均出现明显的伪狂犬病临床症状。鉴定试验表明，分离株ZY-2014是一株猪伪狂犬变异毒株。

Abstract:

A suspected pseudorabies virus variant, named ZY-2014, was isolated from dead piglets in a large-scale pig farms of Henan province and determined by PCR identification,gE gene sequence analysis and animal infection tests. The virus grown on vero cell culture can produced typical cytopathic effect. The virus titer was 107.4 TCID50/100 μL confirmed by the Reed-Muench methods.Three specific pseudorabies virus target bands of 217 bp, 636 bp, 1440 bp were detected using gD, gE, gG specific PCR respectively. Sequencing results were consistent with the expected results.The homologies of gE nucleotide and amino acid sequence were analyzed with other reference strains, including 10 viral strain that had been isolated since 2012 and 14 classical strains. The gE gene of ZY-2014 strain shared 99.6% to 100% nucleotide homology and 99.1% to 100% amino acid homology with newly isolated PRV strains respectively, and shared 97.7% to 99.6% homologies in nucleotide sequence and 95.7% to 99.3% in amino acid sequence with classical PRV strain respectively. Based on the phylogenetic analysis, ZY-2014 strain was clustered to a relatively independent branch together with newly isolated strain in recent three year, and far from the classical strains. The typical pseudorabies clinical symptoms were presented in rabbits and mice inoculated with ZY-2014 strain.These results demonstrated that a new wild pseudorabies virus had been isolated.