北京市1株猫传染性腹膜炎病毒的分离鉴定及遗传进化分析

Isolation,Identifcation and Genetic Evolutionary Analysis of a Strain of Feline Infectious Peritonitis Virus in Beijing City

为掌握北京市猫传染性腹膜炎病毒(FIPV)与国内外毒株基因型之间的差异性,无菌采集北京市宠物医院4例疑似患有猫传染性腹膜炎病例的腹水和组织等病料样本,将经RT-PCR诊断为猫冠状病毒(FCoV)阳性的病料样本接种于猫肾细胞(CRFK)进行连续传代培养,结果分离到1株毒株。对分离毒株进行形态学观察,RT-PCR检测,TCID_(50)测定以及基因测序及遗传演化分析。结果显示:该毒株在CRFK上出现典型细胞病变效应(CPE),且经连续细胞接种,可以得到稳定增殖;RT-PCR扩增获得与预期目的基因片段大小一致的条带,证实为FCoV,将其命名为FCoV BEJ5-2021(B5)。对该毒株S、3c、7ab蛋白编码基因片段进行测序发现:在S蛋白基因分型上,该分离毒株属FCoV I型,与意大利参考分离株VGCaFn@14同源性为99.8%;基于3c蛋白基因分析,该毒株与德国参考分离毒株FIPV16F同源性为98.9%;基于7ab蛋白基因分析,该毒株与美国参考分离毒株FIPV-UCD 15b-1同源性为99.1%。不同组织病料分离毒株TCID_(50)测定显示,肠管、脾等组织病料中的病毒滴度相对较高,气管中的滴度最低。结果表明:FCoV分离毒株不同基因分型对应不同血清型和生物型,因而基因进化树同源性分析只能确定遗传进化关系;分离毒株对猫肠、脾组织等有较强嗜性。本研究丰富了我国FIPV分子流行病学资料,为该病防控提供了参考。

In order to identify the genotype diference between Beijing feline infectious peritonitis virus(FIPV)and other strains in and out China,ascites and tissue samples of four cases suspected of FIPV in Beijing Pet Hospital were aseptically collected.The samples diagnosed as feline coronavirus(FCoV)positive by RT-PCR were inoculated into feline kidney cells(CRFK)for continuous subculture,one virus strain was isolated for morphological observation,RT-PCR detection,TCID_(50) determination,gene sequencing and genetic evolution analysis.The results showed that typical cytopathic efect(CPE)of the strain could be observed on CRFK,and stable proliferation could be obtained after continuous cell inoculation;a band with the same length as the expected target gene fragment was obtained by RT-PCR amplifcation,which was confrmed to be FCoV and named as FCoV BEJ5-2021(B5).It was found that,through sequencing the gene fragments coding S,3c and 7ab protein,for S protein genotyping,the strain belonged to FCoV type I,and was 99.8%homologous to Italian reference isolate VGCaFn@14;the strain was with 98.9%homology with German reference isolate FIPV16F based on the analysis of gene coding 3C protein,99.1%with US reference isolate FIPV-UCD 15b-1 based on analysis of gene coding 7ab protein.It was shown that,by the results of genetic evolution analysis,the relationship of genetic evolution could be only determined by homology analysis of gene evolutionary tree;and by the results of determination of TCID_(50) of isolates from diferent samples,the isolates had stronger tropism to cat intestine and spleen tissues,but poorer to stomach and bladder tissues.