水貂肠炎病毒山东株分离鉴定及生物学特性分析  

Isolation and biological characterization of mink enteritis virus in Shandong province

作　　者：于永乐 姚延珠 张传美 秦志华 杨瑞梅 张洪亮 段笑笑 单虎 YU Yongle;YAO Yanzhu;ZHANG Chuanmei;QIN Zhihua;YANG Ruimei;ZHANG Hongliang;DUAN Xiaoxiao;SHAN Hu(Shandong Provincial Key Laboratory of Preventive Veterinary Medicine,College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,Shandong,China;College of Plant Health and Medicine,Qingdao Agricultural University,Qingdao 266109,Shandong,China;Qingdao Animal Disease Prevention and Control Center,Qingdao 266000,Shandong,China)

机构地区：[1]青岛农业大学动物医学院,山东省预防兽医学重点实验室,山东青岛266109 [2]青岛农业大学植物医学学院,山东青岛266109 [3]青岛市动物疫病预防控制中心,山东青岛266000

出　　处：《微生物学报》2022年第5期1832-1842,共11页Acta Microbiologica Sinica

基　　金：在青岛高校服务青岛重点学科(兽医学)(025/1119002);青岛农业大学高层次人才科研基金(663/1120015)。

摘　　要：【目的】本研究旨在研究水貂肠炎病毒(mink enteritis virus,MEV)的基因组遗传进化特征。【方法】对采自山东境内水貂养殖场的109份水貂腹泻样品进行MEV的分离和鉴定,利用血凝和血凝抑制试验、多步生长曲线绘制以及蛋白的三级结构模拟等,对分离毒株生物学特性进行分析,通过重叠PCR对分离株进行全基因扩增,使用MegAlign进行序列同源性比对分析,利用DNAMANV6对基因组5’末端和3’末端回文结构进行预测,应用MEGAV6进行遗传进化分析。【结果】共分离得到5株病毒,经电镜观察和间接免疫荧光试验鉴定为MEV毒株,分别命名为MUTQS-1-5,GenBank登录号分别为OK275645、OK275646、OK275647、OK275648和OK275649;各分离株5’-和3’-UTR分别由长回文序列组成,具有典型的细小病毒基因组末端的茎环样结构,NS1和VP2基因的推导氨基酸序列存在多个非同义突变位点,其中NS1蛋白的E/Q545V位氨基酸突变,以及VP2蛋白的F267Y、Y324I位氨基酸突变为首次在MEV上发现;生物学特性分析表明,上述突变并未明显改变病毒的血凝及血凝抑制效价、生长趋势和病毒粒子的空间构象。全基因序列系统发育进化树也显示,本次分离株处于同一分支上,与山东分离株SDNH亲缘关系最近。【结论】本研究报道了包含新型变异位点的MEV毒株的基因组特征,试验初步证实了新型变异位点的出现并未改变病毒的血凝性、抗原性以及在易感细胞内的增殖能力。以上研究结果为进一步开展病毒的流行病学与变异规律研究奠定了基础。[Objective] This study aims to investigate the genomic characteristics of mink enteritis virus(MEV) in Shandong province, China. [Methods] A total of 109 fecal samples were collected from mink farms for the isolation and identification of MEV strains. Biological characterization of the isolates was carried out based on hemagglutination and hemagglutination inhibition tests, multistep growth curves, and protein structure prediction. The genomic DNA sequences of the isolates were amplified, cloned, and sequenced. MegAlign was employed for multiple sequence alignment and DNAMAN V6 for the prediction of the inverted terminal repeats of 5’-untranslated region(UTR) and 3’-UTR. The neighbor-joining phylogenetic tree was constructed in MEGA V6. [Results] Five strains were isolated and identified as MEV by electron microscopy and indirect immunofluorescence assay(IFA), which were named as MUTQS-1, MUTQS-2, MUTQS-3, MUTQS-4, and MUTQS-5,respectively. The genomic sequences were submitted to GenBank and got the accession numbers OK275645, OK275646, OK275647, OK275648, and OK275649, respectively. The 5’-and 3’-UTRs consisted of long palindromic sequences with a typical stem-loop-like structure at the end of parvovirus genome. The deduced amino acid sequences of NS1 and VP2 genes had several nonsynonymous mutations, among which E/Q545V in NS1 protein and F267Y and Y324I in VP2 protein had not been reported. Biological characterization showed that the above mutations did not significantly alter the hemagglutination and hemagglutination inhibition titers, growth trend, and spatial conformation of viral particles. The phylogenetic tree suggested that the five MEV isolates shared the same clade and were closely related to the Shandong isolate SDNH. [Conclusion] We reported the genomic characteristics of MEV strains containing novel mutation sites and confirmed that the presence of these sites did not alter the hemagglutination, antigenicity, or proliferation in susceptible cells.

关 键 词：水貂肠炎病毒 遗传变异 生物学特性 

分 类 号：S852.65[农业科学—基础兽医学]