北京地区犬冠状病毒的分离鉴定及遗传进化分析  

Isolation and identification of canine coronavirus and phylogenetic analysis of the isolate

作　　者：王静[1,2] 秦彤 由欣月[1,2] 刘畅 史利军 梁琳[1,2] 李金祥 崔尚金[1,2] WANG Jing;QIN Tong;YOU Xin-yue;LIU Chang;SHI Li-jun;LIANG Lin;LI Jin-xiang;CUI Shang-jin(Institute of Animal Science and Veterinary Medicine ,Chinese Academy of Agricultural Sciences ,Beijing 100193,China;Scientific Observing and Experiment Station of Veterinary Drugs and Diagnostic Technology,the Ministry of Agriculture, Beijing 100193,China;Chinese Academy of Agricultural Sciences ,Beijing 100081,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]农业部兽用药物与诊断技术北京科学观测实验站,北京100193 [3]中国农业科学院,北京100081

出　　处：《中国兽医科学》2019年第1期92-98,共7页Chinese Veterinary Science

基　　金：国家“十三五”重点研发计划项目(2016YFD0501003,2017YFD0501603);中国农业科学院创新工程项目(ASTIPIAS15);中国农业科学院基本科研业务费(2018-YWF-YTS-16)

摘　　要：采集北京地区宠物医院2~4周龄发生腹泻的幼犬粪便样品,经胶体金和PCR,将其初筛为犬冠状病毒(CCoV)阳性。然后,将其处理液接种于MDCK细胞和CRFK细胞,进行连续传代培养,出现特征性细胞病变。通过形态学观察、RT-PCR鉴定、间接免疫荧光试验,证明该分离株为犬冠状病毒,遂将其命名为CCoV BJ02株。针对M基因通过RT-PCR法鉴定分离株的基因型,证明其为CCoV-Ⅱ型。Reed-Muench法测得CCoV BJ02株的TCID50为10^-5.2/100μL。通过绘制一步生长曲线发现,该病毒在48小时左右收毒效果最好。遗传进化分析表明,CCoV BJ02株与日本分离株CCoV fc1株位于同一分支中,同源性最高,有较近亲缘关系。上述研究结果为深入了解北京地区CCoV的流行情况,给犬冠状病毒病的诊断、防治及后续研究奠定了基础。A faecal sample was collected from 2-4 weeks puppies with diarrhea from a pet hospital in Beijing.The sample showing canine coronavirus(CCoV) positive after initial screening by colloidal gold and PCR,was propagated in MDCK cells and CRFK cells.The cells in culture were passaged continuously until a specific cytopathic effect was observed.The sample was confirmed to be CCoV by morphological observation,RT-PCR and an indirect immunofluorescence assay,and it was named as CCoV BJ02 strain.The genotype of this strain was identified by RT-PCR for the M gene,and it was confirmed to be CCoV type Ⅱ(CCoV-Ⅱ).Virus titers were determined according to the method of Reed and Muench,and TCID50 of CCoV BJ02 isolate was 10^-5.2/100μL in CRFK cells.Forty-eight hours was shown to be the best time for virus collection based on the result of one-step growth curve.Phylogenetic analysis showed that the CCoV BJ02 strain has a very high homology with CCoV fcl strain,a Japan isolate,and both strains belong to the same branch.The results of the current study provide insights into the prevalence of CCoV in Beijing and lay a theoretical foundation for the diagnosis,prevention and treatment of canine coronavirus disease.

关 键 词：犬冠状病毒 分离鉴定 进化分析 

分 类 号：S852.655[农业科学—基础兽医学]