猪细小病毒河南地方株HP104的分离与生物学特性鉴定

Isolation and identification of a porcine parvovirus HP104 strain in Henan province

采用PCR方法对河南采集的疑似PPV病料进行PCR检测，PCR检测为阳性的病料接种猪睾丸细胞，分离到1株病毒，命名为HP104。通过观察细胞病变（CPE）、病毒滴度TCID50和血凝效价的测定、理化特性试验等研究病毒的特性，利用电镜观察病毒的形态，NSI基因PCR扩增及测序分析。该病毒在sT细胞上连续盲传10代后，细胞出现了较稳定的CPE，传代至第15代时病毒血凝效价基本稳定为2，到第20代时TcID50达到10TCID／0．1mL左右。该毒株对热、pH3．0酸、氯仿、胰蛋白酶有抗性。在电镜下可观察到近似球形的病毒粒子，大小约23～26nm。PCR扩增产物与预期一致，与其他PPV毒株NSI基因序列同源性高达99％以上。该病毒特征与猪细小病毒特征基本相符，初步证实分离的病毒为猪细小病毒。

Polymerase chain reaction （PCR） was used to detect the DNA of suspected porcine par- vovirus （PPV） pathologic materials from Henan province,a PPV strain HP104 was successfully i solated by inoculating the positive pathologic material on the swine testicular （ST） cells. Biological characteristics of the virus was studied by observing the physiochemical characteristics of the vi rus,including the lesions of ST cell, detecting TCIDs0 and HA titer of the virus, electron micro scope was used for observing the morphology of virus. Specific gene fragment NS1 was amplified by PCR,NS1 gene was detected and the sequence of NS1 was analysed. The cells infected by the virus appeared stable CPE after 10 passages. Hemagglutination （HA） titer of the virus reached 21%at the 15th passage and the virus reached 10-8%TCIDs0/0.1 mL at the 20th passage,the virus was resistant to chloroform,heat,acid and trypsin. The virus was observed to be visible round with a diameter of 23 -26 nm by electron microscopy. PCR amplification product was consistent with ex pectation. Compared with other PPV strains,the homology of gene sequence of NS1 is above 99%. The virus characteristics were consistent with PPV,it was confirmed that the isolated virus was porcine parvovirus.