山羊痘病毒的分离与鉴定

对贵州省2002年以来发生的疑似山羊痘病例样品进行了病毒的分离与鉴定。取山羊痘病羊的皮肤痘疹和水疱作待检病料,接种BHK-21传代细胞盲传3代后,出现了明显的、规律的细胞病变(CPE),病毒细胞培养物在F4代以后,能与山羊痘标准阳性血清在琼脂扩散试验中出现白色沉淀线,而与正常细胞的培养物及PBS不出现沉淀线;参照GenBank上山羊痘病毒P32基因序列,设计了1对特异性引物,对现场分离毒株进行PCR扩增,可扩增出963bp特异性的DNA条带。用待检病料感染的BHK-21细胞培养物接种9日龄鸡胚绒毛尿囊膜,随着传代次数的增加,痘斑病变的出现率从13.3%～20%上升至33.3%～40%;用山羊痘病变皮肤、鸡胚绒毛尿囊膜和感染细胞进行超薄切片,在电子显微镜下可以观察到典型的山羊痘病毒粒子。

Isolation and identification of goat poxvirus

Goat poxviruses were isolated from suspected goat pox cases in Guizhou province since 2002.Samples from skin and mucous membrane of affected goat were collected and inoculated BHK-21 cells.Typical cytopathic effect(CPE) was observed was observed in the F3 passage.The virus cultures reacted with immune positive serum of goat pox in agar gel precipitation,but no cross reaction was observed with normal cell cultures and PBS controls.A pair of primers was designed according to the goat pox virus P32 gene sequence in GenBank and a specific PCR fragment of 963 bp was amplified.Pox formation increased from 13.3 %-20 % to 33.3 %-40 % after passaging through 9 days old chicken embryo.Typical virus particles were observed under transmission electronic microscope in ultrathin sections of pathological tissues,allantochorion membrane of chicken embryos and affected cells.