一株猪伪狂犬病病毒变异株的分离鉴定

Isolation and Identification of a Mutant Strain of Pseudorabies Virus

为了解广东省猪伪狂犬病病毒(pseudorabies virus,PRV)野毒株的基因变异及遗传演化的情况,本试验对广东佛山疑似暴发伪狂犬病的猪场采集的病料(脑、肺脏、扁桃体、肝脏、脾脏)进行了PCR鉴定,初步鉴定为PRV毒株后,将阳性病料接种非洲绿猴肾细胞(Vero),进行毒株传代培养,对分离毒株进行PCR检测及小鼠感染试验,证实该病毒为PRV,并命名为PRV FS-2015株;并对该毒株进行细胞病变观察、病毒TCID50测定、毒株gC和TK基因扩增及序列分析。结果显示,PRV FS-2015株TCID50为10-7.5/0.1mL。PRV FS-2015株的gC和TK基因序列与国内外PRV参考毒株进行同源性比对分析发现,其核苷酸序列同源性分别为95.8%~100.0%和99.4%~100.0%,氨基酸同源性分别为92.3%~100.0%和98.7%~100.0%。遗传进化分析表明,PRV FS-2015株与国内近几年分离的PRV变异株GY、ZJ01、HB1201、HN1201、JS2012、BJ/YT和BP属于同一分支,同源性较高,亲缘关系更近;但与PRV经典株Kaplan、Becker、NIA3、Kolchis、Bartha、Yangsan、Min-A、SS和SL株的同源性较低,基因变异较大,表明PRV FS-2015毒株属于近几年流行的变异株。本研究结果可为广东省伪狂犬病的防控工作和疫苗株的选择提供科学依据。

The purpose of this study was to understand the genetic variation and evolution of pseudorabies virus（PRV）in Guangdong province.In this experiment,the PRV strain was primarily identified by PCR from samples（heart,lung,tonsil,liver and spleen）collected from sick pig in Foshan,Guangdong province,and the positive samples were inoculated Vero cells for subculture.And a strain was identifited as PRV by PCR and mice test,and named as PRV FS-2015.The cytopathic effect of the strain was observed and TCID50 was tested,the whole gene of TKand gCwere amplified and the sequence were analyzed.The results showed that the titer of TCID50 of isolated virus in Vero cells was 10-7.5/0.1 mL.The sequence analysis results showed that the nucleotide identities of the gCand TK genes of PRV FS-2015 strain with other PRV strains in GenBank were 95.8% to 100.0% and 99.4% to 100.0%,respectively,the homology of amino acid sequence were 92.3% to 100.0% and 98.7% to 100.0%,respectively.Phylogenetic tree analysis showed that the PRV FS-2015 stain had closer relationship with GY,ZJ01,HB1201,HN1201,JS2012,BJ/YT and BP stains,while had a distant relationship with Kaplan,Becker,NIA3,Kolchis,Bartha,Yangsan,Min-A,SS and SL stains.The experiment showed that PRV FS-2015 strain belonged to the variant popular in recent years,and the results could provide data for the prevention and control of pseudorabies and the selection of vaccine strains in Guangdong province.