上海地区3株猫冠状病毒ORF3、ORF7和S2基因的序列测定及进化分析

Sequencing and phylogenetic analysis of ORF3,ORF7 and S2 gene of three feline coronavirus in Shanghai

根据病毒的致病性,猫冠状病毒(feline coronavirus,FCoV)可分为猫肠道冠状病毒(feline enteric coronavirus,FECV)和猫传染性腹膜炎病毒(feline infectious peritonitis virus,FIPV)两种生物型。为分析上海地区FIPV部分基因的序列特征,本研究对3株FIPV的非结构蛋白基因ORF3(3a、3b和3c)、ORF7(7a和7b)、S2基因序列进行测序及进化分析。收集上海某宠物医院于2018年7月~2018年9月接诊的3例经临床初步诊断患猫传染性腹膜炎(feline infectious peritonitis,FIP)的猫腹水样品,参照试剂盒说明书提取猫腹水中病毒RNA,采用反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)对3株FIPV的非结构蛋白基因ORF3、ORF7和S2基因序列进行扩增并测序,构建进化树进行分析。结果显示:3株FIPV的非结构蛋白基因ORF 3a、ORF 3b和ORF 3c的变异主要表现为点突变,ORF 7a基因在第47位发生氨基酸替换,2个具有完整的ORF 7b基因,而1个存在3个核苷酸(nucleotide,nt)的缺失;S基因的S1/S2裂解位点处有连续的R-R-S/A-R-R-S序列模体,符合FIPV裂解位点的氨基酸序列特征,且第1045位甲硫氨酸被亮氨酸替换。遗传进化树显示,3株FIPV的非结构蛋白基因ORF 3c、ORF 7b及S2基因序列与血清型Ⅰ型毒株的亲缘关系最近,表明这3株病毒属于血清型Ⅰ型毒株。推测S基因的突变可能参与了FCoV组织嗜性的改变,这为FIPV的分子流行病学调查提供了资料。

In order to understand the molecular characteristics of a feline infectious peritonitis virus(FIPV)isolated in Shanghai,genes encoding three non-structural proteins ORF3(3a,3b and 3c),ORF7(7a and 7b)and S2 of three FIPV strains were amplified by RT-PCR,sequenced and analyzed.On ORF3a,ORF3b and ORF3c substitutional mutations leading to amino acid changes were identified.On ORF7a gene,mutations were found at the 47th amino acid.A 3 nt deletion in ORF7b gene was identified in one strain.At the S1/S2 cleavage site of the S gene,there were continuous R-R-S/A-R-R-S sequence modules,which were consistent with the protein sequence characteristics of the FIPV cleavage site.The methionine was replaced by leucine at 1045th amino acid in all samples.The phylogenetic tree showed that the sequences of FIPV ORF3c,ORF7b and S2 genes were closely related to serotypeⅠstrains,indicating that the three strains belonged to serotypeⅠ.The mutations in S genes may be involved in the change of tissue tropism in FCoV.