南宁地区犬细小病毒VP2基因的克隆及遗传进化分析

Cloning and Phylogenetic Analysis of VP2 Gene of Canine Parvovirus in Nanning Region

为了解广西南宁地区犬细小病毒(CPV)的流行现状与病毒变异情况,本试验对初步确诊为犬细小病毒病的12份阳性病料提取基因组DNA,以提取的基因组DNA为模板,通过PCR扩增VP2基因序列并测序,将12个阳性毒株样本VP2基因测序结果与GenBank中登录的17株国内外CPV分离株VP2基因进行同源性比对,采用Mega 7.0软件绘制遗传进化树,分析其病毒亚型和遗传进化情况。结果显示,成功扩增得到12个毒株样本的VP2基因片段,大小约1 755bp,12株阳性样本毒株的VP2基因同源性在99.2%～100.0%之间,其中NN01与NN07、NN02与NN06同源性最高,为100.0%;阳性样本毒株与国内其他分离株VP2基因的同源性为97.6%～100.0%,其中NN08、NN10及NN04与CPV-ZJ1579同源性最高,均为100.0%,属于CPV-2a亚型;阳性样本毒株与国外代表性毒株同源性在98.1%～99.8%之间。遗传进化树分析表明,12个样本毒株中有3株属于CPV-2a亚型,3株属于CPV-2b亚型,6株属于CPV-2c亚型。这是继2018年初广西分离到CPV-2c型CPV后,首次发现南宁地区大规模流行CPV-2c亚型病毒,预示着CPV-2c亚型CPV在国内的流行正在增加。综上所述,广西南宁地区CPV-2a、CPV-2b与CPV-2c亚型并存,但CPV-2c亚型的比重比其他地区大,这也给该地区提供了新的防治信息,在实际CPV防控工作中除了对CPV-2a、CPV-2b等传统流行亚型的关注之外,更应该重视CPV-2c亚型CPV的防控。

In order to investigate the prevalence and mutation of canine parvovirus(CPV)in Nanning,Guangxi,this study extracted the viral genomic DNA of12positive samples that were initially diagnosed as CPV.Using the extracted samples DNA as templates,the entire VP2gene se-quence was amplified and sequenced.In this experiment,the VP2gene of all12positive strains were compared with the VP2gene of17domestic and foreign CPV isolates registered in Gen-Bank.The results showed that the VP2gene fragments of12strains were successfully amplified with the size about1755bp,and the homology between VP2gene of12positive strains was between99.2%and100.0%,among which NN01and NN07,NN02and NN06were the highest,accounting for100.0%.The homology of VP2gene between the positive sample strain and other domestic isolates was97.6%to100.0%,among which NN08,NN10and NN04were the highest with CPV-ZJ1579,all of which were100.0%,belonging to CPV-2a subtype.The homology between positive sample strains and foreign representative strains was98.1%to99.8%.Phylogenetic tree analysis showed that among the12strains,3strains were CPV-2a subtype,3strains were CPV-2b subtype,and6strains were CPV-2c subtype.This was the first time that CPV-2c subtype CPV had been found in Nanning area since the isolation of CPV-2c subtype CPV in Guan-gxi in early2018,which indicated that the epidemic of CPV-2c subtype CPV was slowly increasing in China.This experiment indicated that CPV-2a,CPV-2b and CPV-2c coexisted in Nanning area of Guangxi,but the proportion of CPV-2c subtype was larger than that of other areas,which also provided new prevention and control information for this area.In addition to paying attention to the traditional epidemic subtypes such as CPV-2a and CPV-2b,the prevention and control of CPV-2c subtype CPV should be paid more attention to in the actual prevention and control work of CPV.