羊口疮病毒内蒙古株的分离与初步鉴定

Isolation and Preliminary Identification of Inner Mongolia Strains of Orf Virus

[目的]确定内蒙古地区规模化养殖场发生的疑似羊口疮的病原。[方法]无菌采集疑似羊口疮病料7份,经剪碎、研磨、离心等处理后接种山羊皮肤成纤维细胞(goat skin fibroblasts,GSFs)培养;对分离到的病毒毒株进行电镜观察;利用B2L基因引物进行特异性PCR鉴定,对获得的B2L基因序列测序,构建系统发育树,并进行同源性分析。[结果]3份病料经GSFs培养48 h后出现明显病变,分离的病毒经负染后在电镜下观察呈卵圆形,病毒粒子长220~250 nm,宽125~200 nm,符合羊口疮病毒(orf virus,ORFV)粒子的形态特征,并将其命名为NM-ORFV-1株、NM-ORFV-2株、NM-ORFV-3株。分离株经B2L基因PCR鉴定获得1137 bp的扩增产物,与预期一致;系统发育树及同源性分析显示,NMORFV-2株与NM-ORFV-3株遗传关系密切,处于同一分支,且与ORFV KP336704(中国)分离株的亲缘关系最近,同源性达到99.4%;NM-ORFV-1株与NM-ORFV-2株及NM-ORFV-3株处于不同分支,NM-ORFV-1株与NM-ORFV-2株的同源性为99.1%,与NM-ORFV-3株的同源性为99.0%,且与ORFV JQ904789(中国)疫苗株亲缘关系最近,同源性为99.6%。[结论]内蒙古地区规模化养殖场疑似羊口疮病例的病原是ORFV。

[Objective]To determine the etiologic agent causing the suspected cases of contagious ecthyma(orf)in large-scale sheep farms in Inner Mongolia.[Method]A total of 7 clinical samples were aseptically collected from the diseased sheep with suspected orf symptoms,and they were subsequently inoculated with goat skin fibroblasts(GSFs)for virus isolation after cutting,grinding,and centrifugation.The obtained viral strains were morphologically observed by electron microscope.B2L gene primers were used for specific PCR identification,and the product was then sequenced.The phylogenetic tree based on B2L gene was constructed,and homology analysis was performed as well.[Result]There were 3 clinical samples with capacity to cause evident cytopathic alterations in GSFs after inoculation for 48 hours.Morphological observation with electron microscope demonstrated that the isolated virus was oval in shape,and the viral particles were 220-250 nm long and 125-200 nm wide,which were consistent with the morphological characteristics of orf virus(ORFV).The 3 obtained ORFV strains were designated as NMORFV-1,NM-ORFV-2,and NM-ORFV-3,respectively.For each isolated viral strain,a product of 1137 bp was amplified by PCR assay targeting B2L gene,which was consistent with the expecting size.Phylogenetic tree and homology analysis revealed that NM-ORFV-2 strain was closely genetically related to NM-ORFV-3 strain,and they clustered in the same branch;NM-ORFV-2 strain shared the highest homology(99.4%)with ORFV KP336704(China)wild strain;NM-ORFV-1 strain clustered in different branch from NM-ORFV-2 strain and NM-ORFV-3 strain,and the homologies of NM-ORFV-1 strain shared with NM-ORFV-2 strain and NM-ORFV-3 strain were 99.1%and 99.0%,respectively;NM-ORFV-1 strain shared the highest homology(99.6%)with ORFV JQ904789(China)vaccine strain.[Conclusion]ORFV is identified as the pathogenic agent that causes the suspected cases of orf in large-scale sheep farms in Inner Mongolia.