犬细小病毒DD株的分离鉴定和VP2基因序列分析

摘要

本实验室对从疑似犬细小病毒感染的发病犬分离的病毒采用同步培养法接种猫肾细胞（CRFK）增殖,通过PCR试验、IFA试验和VP2基因测序分析等方法进行鉴定并分型,获得一株犬细小病毒强毒株,命名为CPV-DD株。对分离病毒进行PCR扩增,可扩增出特异性DNA片段（1 163 bp）;盲传至第6代时,病毒液的HA效价为1∶1024,其血凝性能被特异性抗体抑制;IFA可见特异性亮绿色荧光,TCID50为105.5/0.1 mL;电镜观察可见20 nm左右的病毒颗粒,VP2蛋白氨基酸序列分析显示该毒株为CPV-2a型,氨基酸序列分析在324位出现Try→Ile替换,该位点处于病毒潜在的抗原表位。%A strain of canine parvovirus（CPV）was isolated from bloody feces of an ill puppy suspected of parvovirus infections in Beijing.The strain,designated as CPV-DD,was isolated by inoculating CRFK cells in synchronism and was identified as serotype CPV-2a by PCR,the HA,IFA,electron microscopy observation and sequencing of VP2 gene.A unique Ile-324 in VP2 of DD strain was found,contrasting to a Try-324 in the VP2 of other strains.After 6 passages in CRFK cells,the infected cell cultures could agglutinate red blood cells of pigs at the titer of 1∶ 1024.The haemagglutination could be inhibited by monoclonal antibody specific to CPV.Specific fluorescent in the nucleus of the infected cells was observed by IFA assay and the TCID50 of virus fluid was titred as 105.5/0.1mL.Concentrated virus particles with a diameter of 20nm were observed by transmission electron microscope.