犬细小病毒DD株的分离鉴定和VP2基因序列分析

Isolation and Identification of Canine Parvovirus DD Strain and Sequence Analysis of the VP2 GeneLi Run, Ling Ning, Yin Chunsheng, Xue Qinghong, Zhi Haibing

关键词:  犬细小病毒  VP2  分离  鉴定

Key words:  canine parvovirus  VP2  isolation  identification

摘要:

采集北京地区疑似细小病毒(CPV)感染病犬血样粪便,采用同步培养法接种猫肾细胞(CRFK)分离病毒,通过PCR试验、HA试验、HI试验、IFA试验和电镜观察鉴定为犬细小病毒,命名为CPV-DD株。分离毒株可扩增出特异DNA片段(1163bp)；盲传至第2代时,病毒液可凝集猪红细胞,血凝价为1:8,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10^3.0／0.1mL；电镜观察可见20nm左右的病毒颗粒,VP2基因序列分析显示该毒株为CPV-2a型,氨基酸序列在324位出现Try→Ile。

Abstract:

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 2 passages in CRFK cells, the infected cell cultures could agglutinate red blood cells of pigs at the titer of 1:8. 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 10^3.0/0.1ml. Concentrated virus particles with a diameter of 20nm were observed by transmission electron microscope.