犬细小病毒VP2基因序列分析及BJ-1株的分离鉴定

Sequence Analysis of Canine Parvovirus VP2 Gene and Isolationand and Identification of BJ-1 Strain

为分离犬细小病毒毒株,对临床经胶体金试纸条检测CPV阳性的8份犬粪便样品进行VP2全长编码片段扩增与测序。VP2片段序列分析结果显示,8株临床样品的VP2基因全长均为1755 bp,氨基酸的同源性为98.5%~100%。遗传进化分析显示,所有分离的毒株均与所参考的中国分离株在一个分支上。其中1株属于New CPV-2b,其余7株均为New CPV-2a。在序列分析基础上,选择北京地区的1份犬粪便样品,接种F81细胞进行病毒分离。粪便样品接种F81细胞盲传5代后出现典型的病变。对该毒株进行电镜观察、免疫荧光试验以及蛋白电泳鉴定。电镜观察显示病毒粒子的外形结构为圆形或六边形,直径约25 nm,可见实心和空心病毒粒子。间接免疫荧光结果显示,接种病毒液的细胞出现特异性亮绿色荧光信号,对照细胞无荧光信号。纯化后的病毒蛋白电泳显示两条清晰的条带分别为VP1(76 ku)、VP2(64 ku)。结果表明,成功分离到1株New CPV-2a亚型毒株,并将其命名为CPV BJ-1株。

In order to isolate Canine parvovirus strains,in this study,the VP2 coding fragments of eight cases of canine fecal samples,which were CPV positive by colloidal gold test strip,were amplified and sequenced.The VP2 fragment sequence analysis showed that the VP2 gene of 8 clinical samples were all 1755 bp in length and the amino acid homology was between 98.5%and 100%.Phylogenetic analysis showed that the eight clinical samples were clustered with the Chinese isolates.One of them belongs to New CPV-2b,and the other seven are New CPV-2a.On the basis of sequence analysis,one fecal sample from Beijing was selected and inoculated into F81 cells for virus isolation.After five blind passages,typical cytopathic effects were observed.The strain was then subjected to electron microscopic observation,immunofluorescence assay and protein electrophoresis.Electron microscopic observation showed that the virion had a round and hexagonal shape with a diameter of about 25 nm,both solid and hollow virions can be seen.The results of indirect immunofluorescence showed that the cells inoculated with the virus showed a specific bright green fluorescent signal,and the mocked cells had no fluorescent signal.The purified virus showed two distinct bands by protein electrophoresis,VP1(76 ku)and VP2(64 ku).The above results indicated that we successfully isolated a New CPV-2a subtype strain and named it CPV BJ-1 strain.