一株I型犬腺病毒的分离与鉴定  

Isolation and Identification of a Strain of Type I Canine Adenovirus

作　　者：易程 邓莉 唐青海 阳坤 滕威 谢璇 杨海 王芳宇 何丽芳 Yi Cheng;Deng Li;Tang Qinghai;Yang Kun;Teng Wei;Xie Xuan;Yang Hai;Wang Fangyu;He Lifang(College of Life Sciences and Environment,Institute of Animal Production Biotechnology & Institute of Biological Medicine,Hengyang Normal University,Hengyang,Hunan 421008,China)

机构地区：[1]衡阳师范学院生命科学与环境学院衡阳师范学院畜禽养殖生物工程技术研究所衡阳师范学院生物药物研究所,湖南衡阳421008

出　　处：《中国动物检疫》2019年第2期82-87,共6页China Animal Health Inspection

基　　金：国家级大学生创新创业训练计划项目(201810546002;201710546001);湖南省教育厅优秀青年项目(17B041);校企合作项目(ZXXQ201701;18H03);衡阳师范学院引进人才专项项目(16D20)

摘　　要：为给I型犬腺病毒(CAV-I)后续研究提供基础材料,采集疑似犬传染性肝炎患犬粪便,用PCR方法进行CAV-I核酸检测,并运用犬肾传代细胞(MDCK)进行病毒分离培养,采用PCR和免疫过氧化物酶单层细胞染色法(IPMA)检测病毒在细胞内的繁殖状态。结果显示:患犬粪便样品PCR检测为CAV-I核酸阳性,病料接种MDCK细胞培养24 h后出现明显的"葡萄串样"细胞病变效应(CPE);培养物血清学鉴定为CAV抗原阳性,不同代次病毒培养物均为CAV-I核酸强阳性;第9代病毒核酸序列与Gen Bank中CAV-I毒株核苷酸相似性在99.0%以上,与CAV-Ⅱ相似性仅为65.3%;该病毒与CAV-I毒株处于同一遗传分支;病毒接种MDCK后8 h内均无抗原检出,12 h后细胞核中分布大量的CAV抗原,24 h后细胞出现典型的"葡萄串样"病变,抗原在细胞中呈弥散分布。上述结果表明,分离到的毒株为CAV-I,命名为CAV-ZY180711。In order to isolate and identify type I canine adenovirus(CAV-I)so as to provide basic materials for subsequent researches,feces of dogs suspected of infecting CAV-I were sampled,and nucleic acid test for CAV-I was conducted with PCR,then madin-darby canine kidney cells(MDCK)were adopted for virus isolation and cultivation.What's more,PCR and immunoperoxidase mono-layer assay(IPMA)were applied to detect the proliferation of viruses in the cells.The results of PCR detection were positive for CAV-I nucleic acid.With 24 hours of cultivation for samples with MDCK,obvious botryoidal-shaped cytopathic effect(CPE)was observed.Culture serological identification found CAV antigen positive and virus cultures of various generations were all CAV-I nucleic acid positive.The identity of the nucleotide sequence homology between the 9th generation virus and the CAV-I strain published in GenBank exceeded 99.0%,while it only reached 65.3% comparing with CAV-II.The isolated virus strain and CAV-I strain belong to the same genetic branch.The dynamic detection of virus proliferation in the cells showed that no antigen was detected within 8 hours after virus inoculation,and a large amount of CAV-I antigens distributed in the nucleus 12 hours later.After 24 hours,the infected cells showed a typical botryoid shape and antigens were dispersed in the cells.In conclusion,the isolated virus was CAV-I,and finally it was named CAV-ZY180711 strain.

关 键 词：犬腺病毒 分离鉴定 PCR 免疫过氧化物酶单层细胞染色法 

分 类 号：S852.65[农业科学—基础兽医学]