河北部分地区猪圆环病毒2型分离鉴定及遗传变异分析  

Isolation,identification and phylogenetic analysis of porcine circovirus type 2 in Hebei Province

作　　者：赵云环 赵强[1] 刘莹[1] 张帅[1] 郭禹 左玉柱[1,2] 范京惠 ZHAO Yunhuan;ZHAO Qiang;LIU Ying;ZHANG Shuai;GUO Yu;ZUO Yuzhu;FAN Jing-hui(College of Veterinary Medicine,Hebei Agricultural University,Baoding,Hebei 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding,Hebei 071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001

出　　处：《中国兽医学报》2022年第5期853-861,共9页Chinese Journal of Veterinary Science

基　　金：河北省重点研发计划资助项目(19226622D);河北省农业产业技术体系生猪创新团队资助项目(HBCT2018110207)。

摘　　要：为了解河北省猪圆环病毒2型(porcine circovirus type 2,PCV2)基因型感染及遗传变异情况,对采自河北省6个不同地区的32份疑似PCV2感染的组织通过PCR进行初步鉴定,并将鉴定为阳性的组织通过接种PK-15细胞分离病毒,通过PCR、间接免疫荧光、透射电镜对分离的病毒进行鉴定,利用特异性引物对PCV2进行PCR全基因序列扩增,测序后进行全基因及ORF2基因核苷酸序列分析。结果显示,32份疑似样品中有13份PCV2阳性组织,阳性率为40.65%。对其中1份仅感染PCV2的样品进行病毒的细胞分离及后续鉴定,得到1株可稳定感染和增殖的PCV2毒株。将初步筛选的阳性组织进行PCV2全基因扩增并测序,得到8株PCV2全基因序列,基因组全长均为1768 bp;8株PCV2全基因同源性比对结果为95%~99.8%,ORF2同源性比对结果为94%~100%;遗传进化分析显示,8个分离株归属于3个基因亚型(PCV2a、PCV2b和PCV2d),同源性氨基酸序列分析显示主要变异区在47~63 aa抗原表位区。本试验结果为进一步研究该地区PCV2分子流行病学及相关疾病的免疫防控奠定了基础。In order to understand the genotype and genetic variation of porcine circovirus type 2(PCV2)infection in Hebei Province,32 suspected PCV2 tissues collected from six different areas were preliminarily identified by PCR.The virus was isolated by inoculating the identified positive tissues to PK-15 cells,then the isolated virus was identified by PCR,indirect immunofluorescence and transmission electron microscope observation.The full gene sequence of PCV2 was amplified by PCR with specific primers.After sequencing,nucleotide sequence homology of the whole gene and ORF2 gene was analyzed.The results showed that 13 of the 32 suspected samples were PCV2 positive tissues,with PCV2 positive rate of 40.65%.One of the samples infected with PCV2 only was subjected to cellular isolation and subsequent virus identification,and a PCV2 strain that could stably infect and proliferate was obtained.The PCV2 whole gene was amplified and sequenced from the initially screened positive tissues,and the whole genome of eight PCV2 strains were obtained,all with a full length of 1768 bp.The nucleotide and amino acid homology of the whole genome of PCV2 and ORF2 gene were 95%-99.8%and 94%-100%,respectively.Genetic evolutionary analysis showed that the eight isolates belonged to three subtypes(PCV2 a,PCV2 b and PCV2 d).The genetic variation analysis of ORF2 sequence showed that the main variation area located in the 47-63aa in the epitope region.The results of this study laid the foundation for further research on the molecular epidemiology of PCV2and the prevention and control of porcine circovirus related diseases in this area.

关 键 词：猪圆环病毒2型 分离鉴定 遗传进化分析 

分 类 号：S852.65[农业科学—基础兽医学]