一株牛呼吸道冠状病毒的分离鉴定及部分生物学特征

doi:10.11843/j.issn.0366-6964.2024.07.028

摘要/Abstract

摘要：

牛冠状病毒(bovine coronavirus, BCoV)是引起牛呼吸道疾病和消化道疾病的重要病原之一。本研究旨在分离牛呼吸道冠状病毒(BRCV)并研究其部分生物学特性。采集暴发呼吸道疾病的肉牛鼻拭子，经荧光定量PCR鉴定为BCoV阳性。将阳性样本接种人盲肠腺癌细胞(HCT-8)，分离纯化后，对引起细胞病变的病毒分离株的S和HE基因进行序列测定和系统发育分析，并将病毒分离株接种BALB/c小鼠进行致病性研究。结果显示，成功分离到1株致细胞病变的毒株，经荧光定量PCR、间接免疫荧光和电镜观察鉴定为BRCV，蚀斑纯化后计算病毒TCID₅₀为10^-8.46·0.1 mL^-1。S基因和HE基因序列分析表明两种基因均发生重组事件；S蛋白具有2个独特的氨基酸突变(A33G和A975V)，分别位于S1亚基的受体结合域和S2亚基；HE基因具有2个独特的氨基酸突变(L235R和L365F)，分别位于凝集素域和近膜端域。分离株对4周龄BALB/c小鼠具有较强的致病性，能够引起小鼠呼吸加快和腹泻等临床症状，剖检和组织病理学显示肺和肠道出血，病毒主要分布在肺和肠组织，以肺组织中病毒载量最高。本研究首次在国内肉牛中分离到1株BRCV，S和HE基因分子有独特氨基酸突变，成功建立了小鼠感染模型，为进一步研究BCoV的生物学特性和遗传进化提供了参考。

关键词: 牛呼吸道冠状病毒, S基因, HE基因, 小鼠, 致病性

Abstract:

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.

Key words: bovine respiratory coronavirus, S gene, HE gene, mouse, pathogenicity

中图分类号:

S852.659.6

班玛王清, 陈曦, 岳怡, 苏玉蓉, 岳华, 汤承. 一株牛呼吸道冠状病毒的分离鉴定及部分生物学特征[J]. 畜牧兽医学报, 2024, 55(7): 3094-3104.

Wangqing BAN MA, Xi CHEN, Yi YUE, Yurong SU, Hua YUE, Cheng TANG. Isolation, Identification and Partial Biological Characteristics of a Bovine Respiratory Coronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3094-3104.

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引物名称 Primer Name	引物序列(5′→3′) Primer Sequence(5′→3′)	扩增片段/bp Amplified fragment size	退火温度/℃ Annealing temperature
SA	F：GATGCCCTACTGCTGCTGAT R：AAATCGCTCTTACAATCAAC	1 050	49
SB	F：ACTATGGCATTGGGATACAG R：AAGAGTCAACAGACCAACCC	1 229	50
SC	F：TGCCATAATGCTGCCCAATG R：ACCCTTCCTGAATAGCACCA	1 525	55
SD	F：GTGCTATTCAGGAAGGGTTT R：AACAACCACCACATTTCTTA	960	50
HE1	F：ATTTCCGTAAGTCTTGTC R：TACGCTTGTTAAGACAGATTGC	926	46
HE2	F：TATGTTTACAATGGCTCTG R：CTGCCATAGGGGTAGAG	635	46

组别Group	组织Tissue	结果Result
灌胃组 Gavage group	肺	炎性细胞浸润(+)
	十二指肠	黏膜固有层坏死(+)，炎性细胞浸润(+)
	结肠	黏膜固有层坏死(+)，炎性细胞浸润(+)
滴鼻组 Nasal drip group	肺	肺泡隔增厚(+)，炎性细胞浸润(+)
	十二指肠	黏膜固有层坏死(++)，炎性细胞浸润(+)
	结肠	黏膜固有层坏死(+)，炎性细胞浸润(+)

组织 Tissue	接种途径 Vaccination route	感染后时间/d Post infection time
		3		6		9
		阳性率	病毒拷贝数/(Copies·μL^-1)	阳性率	病毒拷贝数/(Copies·μL^-1)	阳性率	病毒拷贝数/(Copies·μL^-1)
肺 Lung	滴鼻	3/3	228	3/3	292	3/3	351
肺 Lung	灌胃	3/3	2570	3/3	5 298	3/3	5 459
十二指肠 Duodenum	滴鼻	3/3	121	1/3	111	1/3	201
十二指肠 Duodenum	灌胃	1/3	141	1/3	156	1/3	180
结肠 Colon	滴鼻	3/3	111	1/3	177	2/3	154
结肠 Colon	灌胃	2/3	289	2/3	169	3/3	161

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