一株牛呼吸道冠状病毒的分离鉴定及部分生物学特征OA北大核心CSTPCD

Bovine coronavirus(BCoV)is one of the important pathogens causing respiratory and digestive tract diseases in cattle.The objective of this study was to isolate and investigate its partial biological characteristics of the bovine respiratory coronavirus(BRCV).Nasal swabs were collected from beef cattle with outbreak of respiratory diseases,and the samples tested positive for BRCV by real-time qPCR were inoculated into human cecal adenocarcinoma cells(HCT-8)for virus isolation and purification.Then sequencing and phylogenetic analysis were performed on the S and HE gene of the isolates which could cause cytopathic effect(CPE).Furthermore,the pathogenicity of the BRCV isolate was evaluated in BALB/c mice.The results showed that one strain exhibiting noticeable CPE was successfully isolated,and was confirmed as BRCV through real-time qPCR,indirect immunofluorescence(IF)assay and transmission electron microscopy(EM).After plaque purification,the TCID50 of the BRCV isolate was 10-8.46·0.1mL-1.The complete S and HE gene sequences of the isolate were successfully obtained,and recombination events were observed in both genes.Comparative analysis with all available S and HE genes in GenBank revealed two unique amino acid(aa)mutations(A33G and A975V)in the S protein,which were located in the S1 subunit receptor binding domain and S2 subunit,and HE gene had two unique amino acid mutations(L235R and L365F),which were located in the lectin domain and near membrane terminal domain,respectively.The BRCV isolate demonstrated strong pathogenicity in 4-week-old BALB/c mice,causing clinical symptoms such as accelerated respiration and diarrhea.Postmortem examination and histopathological analysis revealed hemorrhage in lung and intestinal tissues.Tissue distribution analysis revealed that the virus predominantly localized in the lung and intestinal tissues,with the highest viral load observed in the lung tissue.This study reports the first isolation of a BRCV from beef cattle with respiratory disease outbreak in China.The S and HE proteins of the isolate exhibit unique amino acid mutations.Moreover,a mouse infection model was successfully established,providing a valuable reference for further investigation into the biological characteristics and genetic evolution of BCoV.