猪伪狂犬病病毒鄂A株的分离鉴定

STUDY ON THE ISOLATION AND IDENTIFICATION OF THE EA STRAIN OF PSEUDORABIES VIRUS

从湖北省某些猪场大批死亡的新生仔猪的脑及内脏中分离到多株病毒，并用其中一代表毒株进行了全面鉴定。该病毒株在仓鼠肾传代细胞上连传１５代均出现典型细胞病变，用适应ＢＨＫ－２１细胞的病毒接种鸡胚成纤维细胞、Ｈｅｌａ细胞、Ｖｅｒｏ细胞、犊牛睾丸原代细胞、猪肾传代细胞ＩＢＲＳ－２均出现典型细胞病变。在电镜下可见到典型的伪狂犬病病毒粒子。病毒对５－碘脱氧尿核苷、氯仿、胰蛋白酶、乙醚敏感，在ｐＨ５．０～９．０下稳定，５６℃３０ｍｉｎ灭活，病毒能被伪狂犬病病毒标准阳性血清中和。病毒接种家兔和小白鼠均出现典型伪狂犬病症状。进而用所分离病毒的ＤＮＡ作为模板，应用特异性引物，通过聚合酶链式反应扩增出伪狂犬病病毒ｇｐ５０基因中４３３－６５１之间２１７ｂｐ长的特异性片段，用地高辛标记的伪狂犬病病毒ＤＮＡＢａｍＨＩ－７片段作探针与所分离病毒的ＤＮＡ进行斑点杂交，出现特异性杂交斑点。结果表明所分离病毒为伪狂犬病病毒，并将该毒株命名为猪伪狂犬病病毒鄂Ａ株。

オn recent years,the disease that makes lots of piglets die had outbroken on many farms all over China,we have isolated several virus strains from the samples of brain and visceral organs of dead piglets from some farms in Hubei.One strain of them had been identified.The virus was continually inoculated 15 generations onto hamster kindey cell (BHK21) and induced typical cytopathogenic effect(CPE).Culturing the virus on chicken embryonic fibroblast cell,bovine testis cell,pig kindey cell line IBRS2,Vero cell and Hela cell,the typical CPE could be seen,and the typical pseudorabies virus particles could be found under electromicroscope.The isolated virus was sensitive to 5iodo2deoxyuridine,chloroform,ethy1 ether as well as trypsin.It remained alive from pH5.0 to pH9.0,and it was inactivated for 30 minutes at 56℃.The isolates could be neutralized by the positive serum to pseudorabies virus.Two rabbits and four Balb/c mice were injected with the isolates and the typical clinical syndrome could be observed.The DNA extracted from the isolated virus was subjected to polymerase chain reaction(PCR)analysis,utilizing a set of oligonucleotide primers,and the sequence consisting of 217 base pairs lying in the gene encoding the glycoprotein gp50 was on hand.Dot hybridization was performed to detect the genomic DNA of the isolate by means of the Digoxin labelled PRV DNA BamHI7 fragment and the result was proved to be positive.All of the evidences indicated that the isolated virus was pseudorabies virus(PRV),and it was named EA strain of pseudorabies virus.