猪伪狂犬病病毒JSSQ2013株的分离鉴定及重要功能基因序列分析

Isolation and Identification of Porcine pseudorabies virus (PRV) strain JSSQ2013 and Sequence Analysis of Important Functional Genes

关键词:  猪伪狂犬病毒  变异毒株  分离鉴定  遗传进化分析  致病性

Key words:  Pseudorabies virus (PRV)  Variant strains  Isolation and Identification  Phylogenetic analysis  Virulence

摘要:

为了解江苏省猪伪狂犬病病毒（Pseudorabies virus, PRV）野毒株的特点，本研究从2013年采自江苏省宿迁市的疑似PRV感染病料中分离纯化了一株PRV病毒，对其进行了PCR和间接免疫荧光法（IFA）鉴定，并进一步在Vero细胞上测定该分离株的病毒滴度TCID50和一步生长曲线，扩增其gB、gC、gD和gE基因进行序列比对及分子遗传进化分析，并将该分离株分别接种新西兰白兔和15日龄仔猪研究其致病性。结果显示，该病毒为一株PRV，命名为PRV JSSQ2013株，纯化后的病毒滴度为107.8 TCID50/ml；生长曲线测定显示在感染20h后病毒滴度即达到最高，为108.6 TCID50/ml。与我国近几年分离的PRV变异株序列相比，PRV JSSQ2013株的gB、gC、gD和gE基因核苷酸序列同源性分别为99.5~99.6%、99.5~99.6%、99.5~99.6%和98.7~99.7%，氨基酸序列同源性分别为98.9~99.0 %、99.5~99.7%、99.0~99.2%和98.1~99.3%，均高于其与经典毒株（Ea、Fa和SC株）和欧美毒株（Becker、Kaplan、Bartha、Kolchis和NIA3）的同源性；基于gB、gC、gD和gE基因的遗传进化树分析均显示PRV JSSQ2013株与国内近几年分离的PRV 变异株属同一分支。该病毒接种新西兰白兔后均出现典型的PR症状，如厌食、兴奋、啃咬或用爪挠接种部位等典型症状，且在48h内全部死亡；接种仔猪后第1天开始出现典型的PR症状，第5天全部死亡。以上结果证实，从江苏省宿迁市采集的疑似PRV感染病料中分离到一株强毒力的PRV变异株。本研究为了解江苏PRV分子流行特征、丰富我国PRV分子流行病学资料及新型疫苗的研制奠定了基础。

Abstract:

In order to investigate the characteristics of Porcine pseudorabies virus (PRV) in Jiangsu Province, a PRV field isolate JSSQ2013 was isolated from suspicious PRV samples collected in Suqian city, Jiangsu province in 2013 and identified by PCR and IFA. The titer and growth kinetic of PRV JSSQ2013 strain was determined on Vero cells and gB, gC, gD and gE genes were amplified and sequenced. In addition, the virulence of PRV JSSQ2013 strain was investigated by inoculating rabbits and pigs with the virus. The result showed that the titer of PRV JSSQ2013 strain stock was 107.8 TCID50/ml and the peak titer reached 108.6 TCID50/ml at 20h post infection. Sequence analysis showed that the nucleotide homologies of gB, gC, gD and gE of PRV JSSQ2013 strain were 99.5~99.6%, 99.5~99.6%, 99.5~99.6% and 98.7~99.7%, respectively, and amino acid homologies were 98.9~99.0%, 99.5~99.7%, 99.0~99.2% and 98.1~99.3%, respectively, when compared with Chinese variant strains, which was higher than classical and Euramerican strains. In addition, phylogenetic analysis showed that PRV JSSQ2013 strain shared close genetic relationship with Chinese variant strains and were clustered into the same branch. Furthermore, the typical pseudorabies clinical sign was observed in rabbits and pigs inoculated with PRV JSSQ201 strain. In summary, a PRV variant strain JSSQ2013 was isolated from Jiangsu Province and was highly virulent in rabbit and pigs. The result could provide data for molecular epidemiology and vaccine development for PRV in China.