犬细小病毒CPV-BJ03/17株分离鉴定及VP2基因遗传进化分析

Isolation and Identification of Canine Parvovirus CPV-BJ03/17 Strain and Genetic Evolution Analysis of VP2 Gene

为了研究当前犬细小病毒(canine parvovirus,CPV)国内流行株的遗传变异情况,试验从北京某宠物基地采集疑似CPV感染死亡犬的肠道组织,无菌处理病料后,用F81细胞进行病毒培养,通过电镜形态学观察、血清学、分子生物学和攻毒试验进行鉴定。结果表明,病毒在F81细胞上出现明显的细胞病变(CPE),电镜观察可见直径20nm左右的病毒粒子,血凝效价1∶256,PCR鉴定在570bp处有特异性条带,证明分离出1株CPV,命名为CPV-BJ03/17。全基因组测序分析表明,病毒基因组全长4 620bp,提交GenBank,登录号为:MF134808;该毒株与广东(SC02/2011)、深圳(CPV-s5)和甘肃(CPV-LZ1)等地的分离株亲缘关系较近,核苷酸同源性均为99.7%。VP2氨基酸序列分析表明,CPV-BJ03/17分离株确定为New CPV-2a亚型,主要氨基酸位点与近期分离株BJ15-1等相比无明显变化。动物回归试验表明,CPV-BJ03/17为CPV强毒株。本研究分离出1株CPV强毒株,通过比较CPV的流行情况和遗传变异规律,为CPV的疾病治疗及疫苗研究提供参考。

In order to study the genetic variation of current domestic canine parvovirus（CPV）,the intestinal tissue were collected from a suspected CPV infected canine in Beijing.After sample tissues were processed by asepsis work,virus was inoculated in F81 cells and identified by electron microscopy,serology,molecular biology and challenge test.The results indicated that the virus produced typical CPE on F81 cells and the HA blood coagulation rate was 1∶256.The diameter of virus particle was about 20 nm observed by electron microscopy.The specific band was at570 bp in PCR identification which proved that the virus isolated from the samples was CPV,and named CPV-BJ03/17.The results of whole-genome sequencing and analysis indicated that the full length virus genome was 4 620 bp,GenBank accession No.：MF134808,and this virus strain hadacloser relationship with virus isolated from Guangdong（SC02/2011）,Shenzhen（CPV-s5）and Gansu（CPV-LZ1）whose nucleotide identities were all 99.7%.The analysis of VP2 amino acid sequence suggested that CPV-BJ03/17 belonged to a New CPV-2 asubtype.The major amino acid sites of CPV-BJ03/17 showed no significant changes compared with the recent isolated stain BJ15-1.Animal experiments showed that CPV-BJ03/17 was a high CPV virulent strain.In conclusion,a high CPV virulent strain was isolated in this study,according to the comparison of CPV prevalence and genetic variation,this results also provided a reference for CPV disease treatment and vaccine research.