猪细小病毒潍坊株的分离鉴定及VP2基因遗传进化分析  

Isolation and Identification of Porcine Parvovirus Weifang Strain and Genetic Evolutionary Analysis of VP2 Gene

作　　者：孙卓 张洪亮[1] 秦志华[1] 单虎[1] 杨瑞梅[1] SUN Zhuo;ZHANG Hongliang;QIN Zhihua;SHAN Hu;YANG Ruimei(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109

出　　处：《青岛农业大学学报（自然科学版）》2023年第2期112-117,共6页Journal of Qingdao Agricultural University(Natural Science)

基　　金：山东省农业重大技术协同推广计划(SDNYXTTG-2022-02);山东省农业重大应用技术创新项目(SD2019XM003);山东省生猪产业技术体系疫病控制岗位专家(SDAIT-08-07)。

摘　　要：为了分析猪细小病毒(porcine parvovirus virus,PPV)VP2基因遗传情况,从山东省潍坊地区某猪场疑似猪细小病毒病死胎的淋巴结、肝脏中分离到一株病毒,将病料处理后接种PK-15细胞,观察细胞病变,采用血凝和血凝抑制检测、电镜观察及对病毒VP2基因进行PCR扩增并进行遗传进化分析。结果显示:病毒接种到PK-15细胞后产生明显的圆缩、拉网、灶性脱落等病变;分离毒可凝集豚鼠红细胞,能被已知PPV阳性血清中和;电镜观察病毒为近似圆形、无囊膜、大小不一的病毒粒子,直径约20 nm;对病毒的VP2基因用PCR方法扩增后测序并进行遗传进化分析,测序后确定分离株为PPV,将其命名为PPV SD-21。VP2基因进化树发现,PPV SD-21株与Kresse株和NADL-2株等分离株处于同一分支,遗传距离较近,属于PPV基因Ⅰ型,PPV SD-21株与GenBank中PPV基因Ⅰ型中的10株PPV参考毒株的同源性为97.5%~99.8%,其中与NADL-2株的同源性为99.8%。PPV SD-21株的VP2基因序列只是个别碱基发生变化,所编码氨基酸未发生变异。上述实验结果说明该分离病毒为猪细小病毒基因Ⅰ型弱毒株,该毒株的分离鉴定可为猪细小病毒的诊断和流行病学研究奠定基础。In order to analyze the VP2 gene inheritance of porcine parvovirus virus(PPV)in this study,a strain of the virus was isolated from lymph nodes and liver of a stillborn fetus with suspected porcine parvovirus(PPV)disease from a pig farm in Weifang,Shandong Province.After treatment,the diseased substances were inoculated into PK-15 cells,and the pathological situation of the diseased cells was observed.The virus was detected by hemagglutination and hemagglutination inhibition,electron microscopy,and the VP2 gene of the virus was amplified by PCR for genetic evolution analysis.The results showed that the isolated virus produced obvious lesions such as circular shrinkage,retraction,and focal shedding after inoculation into PK-15 cells;the isolated virus agglutinated guinea pig erythrocytes and was neutralized by known PPV-positive sera;the isolated virus morphology was observed by electron microscopy as nearly circular virus particles of different sizes without vesicles,with a diameter of about 20 nm;the VP2 gene of the virus was amplified by PCR and then sequenced and analyzed for genetic evolution,and the isolated strain was identified as PPV SD-21 after sequencing.The phylogenetic tree of VP2 revealed that the PPV SD-21 strain was in the same branch and genetically close to isolates such as the Kresse and NADL-2 strains of PPV genotype I.Therefore,PPV SD-21 belonged to PPV genotype I.Its homology with the 10 PPV reference strains of PPV genotype I in GenBank was 97.5%-99.8%,among which the homology with NADL-2 strain was 99.8%.The sequence of the VP2 gene of the PPV SD-21 strain was only changed in some bases and encoded amino acid was no variation occurred.The above experimental results indicated that the isolated virus was a weak strain of PPV genotype I.The isolation and identification of this strain layed the foundation for the PPV diagnosis and epidemiological study.

关 键 词：猪细小病毒Ⅰ型 VP2基因 分离 鉴定 PK-15细胞 

分 类 号：S855.3[农业科学—临床兽医学]