三株犬细小病毒的分离鉴定及VP2基因序列分析

Isolation and identification of 3 strains of canine parvoviruses and sequence analysis of VP2 gene

为了解江苏省犬细小病毒(CPV)的流行变异情况,对疑似感染犬细小病毒的病例样品进行特异性PCR鉴定和F81细胞培养,并对病毒VP2基因及其推导氨基酸序列进行遗传变异分析。结果显示,成功分离到3株CPV,间接免疫荧光试验测定的病毒滴度分别为3.2×10^(3)TCID50/mL、3.5×10^(3)TCID50/mL、1.0×10^(4)TCID50/mL;3株CPV分离株VP2基因的核苷酸序列同源性为99.4%~99.8%,与30株来自不同国家和地区的CPV参考株同源性分别为98.1%~99.9%。VP2基因序列进化树显示,3株CPV分离株与CPV-2型(疫苗株)、CPV-2a型、CPV-2b型、NewCPV-2a型、NewCPV-2b型及国外CPV-2c型毒株的亲缘关系较远,与国内CPV-2c型毒株的亲缘关系较近;根据VP2蛋白第426位氨基酸位点的分子特征,证明这3株CPV分离株均为CPV-2c型毒株。该结果为近期江苏省CPV分子流行病学调查提供了依据。

To investigate the prevalence and variation of canine parvovirus(CPV)in Jiangsu province,suspected CPV positive samples were identified by specific PCR and F81 cell culture method.The genetic variation of CPV VP2 gene and amino acid sequence were also analyzed.In this study,3 CPV strains were successfully isolated and the virus titers were 3.2×10^(3)TCID50/mL,3.5×10^(3)TCID50/mL and 1.0×10^(4)TCID50/mL,respectively,which was proved through indirect immunofluorescence test.The nucleotide sequence homology of the VP2 gene among the 3 CPV strains was 99.4%~99.8%.The homology with 30 reference strains from different countries and regions was 98.1%~99.9%.The phylogenetic tree of VP2 gene sequence showed that 3 CPV strains were distant related to CPV-2(vaccine strain),CPV-2a,CPV-2b,New CPV-2a,New CPV-2b and foreign CPV-2c strains,and were closely related to domestic CPV-2c strains.According to the molecular characteristics of the 426 th amino acid site of VP2 protein,all the 3 CPV strains were CPV-2c strains.This outcome provides a basis for the recent molecular epidemiological investigation of CPV in Jiangsu Province.