猫细小病毒LZ-01株的分离鉴定及其VP2基因克隆和序列分析

Isolation and Identification of Feline Parvovirus LZ-Ol Strain and Cloning,Sequence Analysis of VP2 Gene

为了解猫细小病毒(FPV)在甘肃兰州地区的流行情况,将胶体金试纸条检测FPV阳性的粪便样品接种F81细胞,进行病毒分离鉴定,以FPV阳性病料基因组DNA为模板,对分离的FPV进行VP2基因的克隆和测序,并与国内外参考株进行同源性比较及遗传进化分析。结果显示,分离得到1株FPV,命名为FPV/LZ-01,其接种F81细胞可引起特异性的细胞病变,从出现细胞病变的F81细胞中提取病毒基因组DNA,用鉴定通用引物进行PCR鉴定,扩增出约为392 bp特异性基因条带;间接免疫荧光试验表明,FPV/LZ-01分离株接种到F81细胞48 h后可观察到特异性绿色荧光;序列比对结果显示,FPV/LZ-01分离株的VP2基因序列与22株国内外参考毒株的核苷酸同源性为99.2%~99.9%;进化树分析显示,FPV/LZ-01分离株与Beijing-01株(MK266797.1)亲缘关系最近,处于同一分支,与其他参考株遗传距离相对较远。研究结果丰富了我国FPV的流行病学资料,为进一步研发FPV疫苗提供了候选毒株。

In order to understand the prevalence of feline parvovirus(FPV)in Lanzhou,Gansu province,in this study,the faecal samples with FPV positive detected by colloidal gold test strip were inoculated into F81 cells for virus isolation and identification.The VP2 gene of isolated FPV was cloned and sequenced with the genomic DNA of FPV positive material as the template,and the homology and genetic evolution were compared with the reference strains at home and abroad,The results showed that a strain of FPV named FPV/LZ-01 was isolated and inoculated with F81 cells,which could cause specific cytopathic effect.The viral genomic DNA was extracted from F81 cells with cytopathosis and identified by PCR with universal primers.The specific gene band of 392 bp was amplified;Indirect immunofluorescence assay showed that the specific green fluorescence could be observed 48 hours after the strain of FPV/LZ-01 was inoculated into F8l cells;The results of sequence alignment showed that the nucleotide homology between the VP2 gene sequence of FPV/LZ-01 isolate and 22 domestic and foreign reference strains was 99.2%~99.9%;Phylogenetic tree analysis showed that the FPV/LZ-01 strain was closely related to Beijing-01 strain(MK266797.1),which was in the same branch and had a relatively long genetic distance from other reference strains.The results of this study enrich the epidemiological data of FPV in China and provide candidate strains for the further development of FPV vaccine.