甘肃省猪香港病毒的巢式PCR检测及VP1基因序列的系统发育分析

Detection of Porcine Hokovirus(PHoV)by Nested-PCR in Domestic Pigs in Gansu Province and Phylogenetic Analysis Based on VP1 Gene Sequences

猪香港病毒(PHoV)于2008年在我国香港首次发现,属细小病毒新成员。本研究自甘肃省兰州、张掖、天水市养猪场和屠宰场采集血液样品147份,针对PHoV的VP1基因保守区(GenBank No.EU200677)设计2对引物,用巢式PCR(nPCR)检测PHoV。结果,甘肃省生猪PHoV总阳性率为38.10%(56/147)。其中,12周龄以上猪的阳性率(55.56%)显著高于6周龄以下猪(25.00%)(P<0.05);三地区间的阳性率差异不显著(P>0.05)。从56份阳性样本中挑选具地域代表性的12份,对VP1扩增片段测序(GenBank注册号:JQ177084~JQ177095),并和GenBank中的所有VP1序列一起进行系统发育分析。结果表明,12个VP1片段序列之间的相似性为95.2%~99.0%。其中,JQ177093、JQ177085和JQ177089构成一个独立的分支;其余9株和来自GenBank的12株参考序列均处于另一分支。9株中,JQ177095、JQ177087和JQ177091构成一个亚分支,紧邻罗马尼亚野猪株(JF738366);JQ177086、JQ177090和JQ177094构成第二个亚分支,紧邻香港株HK3;JQ177088、JQ177092和JQ177084构成第三个亚分支,紧邻香港株HK5。12株PHoV的VP1基因片段序列与香港株HK1~HK6的总体相似性为96.7%~99.7%,与罗马尼亚野猪株为95.2%~99.0%。本研究结果表明,源于罗马尼亚野猪株和香港株的PHoV均在甘肃省猪群中高水平流行。

Porcine Hokovirus(PHoV)is a new parvovirus of pigs which was recently discovered in Hong Kong,China in 2008.In present study,a total of 147 blood samples were collected from pig farms and slaughter houses in Lanzhou,Zhangye and Tianshui city,Gansu province.Two pairs of primers were designed based on VP1 regions of PHoV genome(GenBank accession No.EU200677).One hundred and forty seven samples were detected by nested PCR(nPCR)assay for PHoV.The nPCR showed an overall prevalence of PHoV as high as 38.10%(56/147).Of which,>12-week-old pigs showed a significantly higher positive rate(55.56%)than <6-week-old piglets(25.00%)(P<0.05),while there was no significant differences among three cities(P>0.05).Out of the 56 positives,12were selected in terms of geographical distribution for sequencing(GenBank accession No.:JQ177084-JQ177095).Phylogenetic analysis together with 11refer-ence VP1 sequences of PHoV from GenBank revealed a homology of 95.2%-99.0% among the present 12 VP1 sequences.Of which,JQ177093,JQ177085 and JQ177089 formed a separate branch in the phylogenetic tree,and other 9together with 12 reference sequences from GenBank formed the second branch.Of the later 9,JQ177095,JQ177087 and JQ177091formed a sub-branch closely related to JF738366 from wild boar in Romania,whereas JQ177086,JQ177090 and JQ177094formed another sub-branch just next to HK3 from Hong-kong pigs,and the third sub-branch of JQ177088,JQ177092 and JQ177084closely related to HK5.The present 12PHoV-VP1 sequences shared a homogeneity of 95.2%-99.0% with Romania wild boar isolate(JF738366),and 96.7%-99.7% with Hong Kong-originated HK1-HK6.The present findings confirmed a high prevalence of PHoVs both evolved from Romania wild boar isolate and Hong Kong pig isolates in Gansu province.