犬细小病毒昆明流行株的分离鉴定与VP2基因序列分析

为了解昆明市犬细小病毒(canine parvovirus,CPV)流行及抗原变异情况,有针对性地筛选疫苗及研发新疫苗,本研究从昆明市部分宠物医院收集6份疑似CPV粪便样品进行病毒分离培养,对获得的疑似病毒液进行PCR、免疫荧光试验(IFA)、血凝效价测定、TCID50测定及VP2基因序列分析。结果显示,分离的1株病毒能使F81猫肾细胞产生明显的细胞病变(CPE),PCR扩增产物大小为164bp,IFA鉴定感染的F81细胞出现绿色荧光,血凝效价为1∶512,病毒TCID50为10-4.375/0.1mL,VP2基因PCR扩增条带大小为1 755bp。对分离株VP2结构蛋白进行测序分析,判定该分离株为CPV-2a亚型,与标准毒株存在5个变异氨基酸位点,属于变异株。将测序结果与商品化疫苗株及国内参考株序列进行比对分析,结果显示与弱毒苗Intervet/vaccine/06和Pfizer/vaccine/06核苷酸同源性均为98.7%;与国内参考毒株序列同源性为98.2%～99.5%,与ANTU-1和WH02/06株同源性最高;与ANTU-1株、WH02/06株及BJ01株亲缘关系最近,位于进化树的同一簇。本研究对监测CPV的遗传变异趋势及疫苗的研制具有重要意义。

Isolation and Identification of Kunming Popular Strain of Canine Parvovirus and Sequence Analysis of VP2 Gene

In order to understand the prevalence and antigenic variation of canine parvovirus (CPV) in Kunming,and select and develop vaccines pertinently,six suspected CPV feces were collected from some pet hospitals in Kunming for virus isolation and culture in this study.The obtained suspected virus solution was subjected to PCR,IFA,hemagglutination titer measurement,TCID₅₀ measurement,and VP2 gene sequence analysis.The results showed that the isolated virus could produce obvious cytopathic effect (CPE) in F81 cat kidney cells.The length of PCR product was 164 bp.The F81 cat kidney cells infected by IFA showed green fluorescence and the blood coagulation titer was 1:512.The TCID₅₀ of the virus was 10^-4.375/0.1 mL,and the length of VP2 gene was 1 755 bp.The VP2 structural protein of the isolate was sequenced and analyzed,and the isolate was determined to be CPV-2a subtype,and there were five variant amino acid sites compared with the standard strain,which belonged to the mutant strain.The sequencing results were compared with the commercial vaccine strains and the domestic reference strains.The results showed that the nucleotide homology with the attenuated vaccine Intervet/vaccine/06 and Pfizer/vaccine/06 were both 98.7%;The sequence homology was 98.2% to 99.5%,which had the highest homology with ANTU-1 and WH02/06 strains.It was closely related to ANTU-1 strain,WH02/06 strain and BJ01 strain,and was located in the same cluster of evolutionary tree.This study was of great significance for monitoring the genetic variation trend of CPV and the development of vaccines.