一株羊肠道病毒的分离鉴定及其基因组特征分析

Identification and genomic characteristics of a novel caprine enterovirus stain

本研究利用宏基因组测序技术,从死亡湖羊淋巴组织中检测出小反刍兽疫病毒与羊肠道病毒混合感染,为了分离羊肠道病毒,采取病毒总RNA转染BHK21细胞方法,连续传代后,通过致细胞病变、测序、TCID_(50)测定和病毒一步生长曲线、电镜观察等鉴定分离出病毒,并与参考毒株比对分析分离病毒的基因特征。结果显示,转染病毒总RNA的BHK21细胞传至第2代就出现了明显的致细胞病变效应,测序证明培养物中仅含羊肠道病毒,其基因组全长为7446 nt,其中5′UTR长821 nt,3′UTR长109 nt,ORF长6516 nt,与参考毒株比较可划分为G种肠道病毒,病毒粒子大小为20~30 nm,第6代细胞毒感染8 h时TCID_(50)值最高,为10^(-4.2)/0.1 mL,命名为JSNT/1/2022株。本研究首次利用组织病料RNA转染细胞的方法成功分离出1株羊肠道病毒,说明羊肠道病毒核酸具有感染性,为羊肠道病毒的分离鉴定探索了一种更为可靠的方法,也为羊肠道病毒相关基础与应用研究奠定了病原学基础。

In this study,we identified the mixed infection of peste des petits ruminants virus(PPRV)and CEV from the lymphoid tissue of a dead sheep by metagenome sequencing technology.To isolate CEV,the total viral RNA from the mesenteric lymph node tissue was used to transfect into BHK21 cells.After continuous passage,the virus was isolated and identified by cytopathogenic observation,sequencing,TCID_(50),one-step growth curve of virus,and electron microscope observation.The genetic characteristics of the isolated virus were compared with the reference strains.The results indicated that BHk2l cells transfected with total viral RNA showed obvious CPE in the second passage and sequencing showed that the culture only contained CEV,and its total genome length was 7446 nt,which possess 5'UTR length of 821 nt,3'UTR of 109 nt and the ORF of 6516 nt.The isolated virus was named JSNT/1/2022 strain and divided into enterovirus G(Ev-G),compared with the reference strains.The virus particle was measured 2030 nm in diameter,and the TCID_(50) value of the 6 th generation virus was 10^(-4.2)/0.1 mL at 8 h.In this study,a strain of CEV was firstly isolated by transfection of tissue material RNA into cells,indicating that the nucleic acid of CEv has infectious,which provides a more reliable method for the isolation and identification of CEv.The isolated virus will lay the etiological foundation for the basic and applied research on CEV.