1株牛源冠状病毒的分离鉴定及遗传进化分析

doi:10.16431/j.cnki.1671-7236.2024.07.034

Abstract

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.:OR750853). 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.

Key words: Bovine coronavirus (BCoV); isolation and identification; whole gene sequencing; genetic evolution

CLC Number:

S852.65⁺3

LIU Wenkai, WANG Ping, JIN Yinghong, XUE Jing, LI Yue, LIANG Qianqian, LI Xiaozhuo, HAN Xiangshu, ZHENG Qiming, JIANG Song, XIA Jun. Isolation,Identification and Genetic Evolution Analysis of a Strain of Bovine Coronavirus[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 3100-3108.

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Isolation,Identification and Genetic Evolution Analysis of a Strain of Bovine Coronavirus

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