中国畜牧兽医2024，Vol.51Issue(7)：3100-3108,9.DOI:10.16431/j.cnki.1671-7236.2024.07.034
1株牛源冠状病毒的分离鉴定及遗传进化分析
Isolation,Identification and Genetic Evolution Analysis of a Strain of Bovine Coronavirus
摘要
Abstract
[Objective]The experiment was aimed to obtain the Bovine coronavirus(BCoV)strain in Xinjiang,explore the best conditions for in vitro isolation of BCoV,understand the genetic evolution of BCoV Xinjiang strain,and provide candidate strain research basis for BCoV vaccine.[Method]The small intestine and the contents(n=185)of diarrhea calves from 7 cattle farms in Xinjiang were detected for BCoV by RT-PCR.The positive samples were inoculated into HCT-8 cells after treatment.The isolated virus was further identified by indirect immunofluorescence assay(IFA),Western blotting and transmission electron microscopy.Median tissue culture infective dose(TCID50)was determined by Reed-Muench method.The isolated virus was subjected to whole genome sequencing,and the sequencing results were subjected to genetic evolution analysis.[Result]The detection rate of BCoV in diarrhea samples was 64.9％.After inoculation with HCT-8 cells,a strain of BCoV was successfully isolated and named as XJ-SHZ-37(GenBank accession No.:C)R750853).The green specific fluorescence of the cells incubated with the virus could be observed by IFA,however,the control group did not.Western blotting test results showed that the isolated strain had a specific reaction with BCoV N protein polyclonal antibodies.The crown-like virus particles could be observed by transmission electron microscopy.The TCID50 of the isolates measured by Reed-Muench method was 10-7.9/0.1 mL.The results of genome sequencing and genetic evolution showed that XJ-SHZ-37 had the highest similarity with ZJNB2106,a BCoV isolate from the genus Coronaviruses of the Coronaviruses family,isolated from cattle.[Conclusion]There was a high detection rate of BCoV in 7 cattle farms in Xinjiang.A strain of virus that could be stably inherited and specifically bind to the polyclonal antibody of BCoV N protein was successfully isolated.The isolate had a high virus titer,and the genetic evolution relationship showed that it had the closest evolutionary relationship with the Chinese bovine strain ZJNB2106.关键词
牛冠状病毒(BCoV)/分离鉴定/全基因测序/遗传进化Key words
Bovine coronavirus(BCoV)/isolation and identification/whole gene sequencing/genetic evolution分类
农业科技引用本文复制引用
刘文锴,蒋松,夏俊,汪萍,金映红,薛晶,李月,梁纤纤,李晓卓,韩翔舒,郑启铭..1株牛源冠状病毒的分离鉴定及遗传进化分析[J].中国畜牧兽医,2024,51(7):3100-3108,9.基金项目
天山英才-青年科技拔尖人才项目(2022TSYCCX0046) （2022TSYCCX0046）
自治区重大科技专项——新疆畜禽疫病防控体系质量提升工程(2023A02007) （2023A02007）
