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

Abstract

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

CLC Number:

S852.659.6

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.

Figures/Tables 9

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Table 2

Table 3

References 35

1	CLARK M A . Bovine coronavirus[J]. Br Vet J, 1993, 149 (1): 51- 70. doi: 10.1016/S0007-1935(05)80210-6
2	HASOKSUZ M , LATHROP S L , AL-DUBAIB M A , et al. Antigenic variation among bovine enteric coronaviruses (BECV) and bovine respiratory coronaviruses (BRCV) detected using monoclonal antibodies[J]. Arch Virol, 1999, 144 (12): 2441- 2447. doi: 10.1007/s007050050656
3	HODNIK J J , JEŽEK J , STARIČ J . Coronaviruses in cattle[J]. Trop Anim Health Prod, 2020, 52 (6): 2809- 2816. doi: 10.1007/s11250-020-02354-y
4	BRUNAUER M , ROCH F F , CONRADY B . Prevalence of worldwide neonatal calf diarrhoea caused by bovine rotavirus in combination with bovine coronavirus, Escherichia coli K99 and Cryptosporidium spp.: A meta-analysis[J]. Animals (Basel), 2021, 11 (4): 1014.
5	SUZUKI T , OTAKE Y , UCHIMOTO S , et al. Genomic characterization and phylogenetic classification of bovine coronaviruses through whole genome sequence analysis[J]. Viruses, 2020, 12 (2): 183. doi: 10.3390/v12020183
6	POPOVA R , ZHANG X M . The spike but not the Hemagglutinin/Esterase protein of bovine coronavirus is necessary and sufficient for viral infection[J]. Virology, 2002, 294 (1): 222- 236. doi: 10.1006/viro.2001.1307
7	LU S , WANG Y Q , CHEN Y Z , et al. Discovery of a novel canine respiratory coronavirus support genetic recombination among betacoronavirus1[J]. Virus Res, 2017, 237, 7- 13. doi: 10.1016/j.virusres.2017.05.006
8	KEHA A , XUE L , YAN S , et al. Prevalence of a novel bovine coronavirus strain with a recombinant hemagglutinin/esterase gene in dairy calves in China[J]. Transbound Emerg Dis, 2019, 66 (5): 1971- 1981. doi: 10.1111/tbed.13228
9	HE Q F , GUO Z J , ZHANG B , et al. First detection of bovine coronavirus in Yak (Bos grunniens) and a bovine coronavirus genome with a recombinant HE gene[J]. J Gen Virol, 2019, 100 (5): 793- 803. doi: 10.1099/jgv.0.001254
10	YOO D , DEREGT D . A single amino acid change within antigenic domain Ⅱ of the spike protein of bovine coronavirus confers resistance to virus neutralization[J]. Clin Diagn Lab Immunol, 2001, 8 (2): 297- 302. doi: 10.1128/CDLI.8.2.297-302.2001
11	WOO P C Y , LAU S K P , HUANG Y , et al. Coronavirus diversity, phylogeny and interspecies jumping[J]. Exp Biol Med (Maywood), 2009, 234 (10): 1117- 1127. doi: 10.3181/0903-MR-94
12	WANG Y Q , LIU D , SHI W F , et al. Origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in China: phylogenetics and coalescence analysis[J]. mBio, 2015, 6 (5): e01280- 15.
13	MENACHERY V D , GRAHAM R L , BARIC R S . Jumping species-a mechanism for coronavirus persistence and survival[J]. Curr Opin Virol, 2017, 23, 1- 7. doi: 10.1016/j.coviro.2017.01.002
14	BAKKERS M J G , LANG Y F , FEITSMA L J , et al. Betacoronavirus adaptation to humans involved progressive loss of hemagglutinin-esterase Lectin activity[J]. Cell Host Microbe, 2017, 21 (3): 356- 366. doi: 10.1016/j.chom.2017.02.008
15	SAIF L J . Bovine respiratory coronavirus[J]. Vet Clin North Am Food Anim Pract, 2010, 26 (2): 349- 364. doi: 10.1016/j.cvfa.2010.04.005
16	VLASOVA A N , SAIF L J . Bovine coronavirus and the associated diseases[J]. Front Vet Sci, 2021, 8, 643220. doi: 10.3389/fvets.2021.643220
17	SAIF L J , REDMAN D R , MOORHEAD P D , et al. Experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts[J]. Am J Vet Res, 1986, 47 (7): 1426- 1432.
18	SOULES K R , RAHE M C , PURTLE L , et al. Bovine coronavirus infects the respiratory tract of cattle challenged intranasally[J]. Front Vet Sci, 2022, 9, 878240. doi: 10.3389/fvets.2022.878240
19	ABI K M , ZHANG Q , ZHANG B , et al. An emerging novel bovine coronavirus with a 4-amino-acid insertion in the receptor-binding domain of the hemagglutinin-esterase gene[J]. Arch Virol, 2020, 165 (12): 3011- 3015. doi: 10.1007/s00705-020-04840-y
20	何琪富, 汤承, 郭紫晶, 等. 牦牛源牛冠状病毒部分基因的扩增、序列分析及病毒分离鉴定[J]. 畜牧兽医学报, 2019, 50 (2): 343- 353.
	HE Q F , TANG C , GUO Z J , et al. Amplification, sequence analysis of bovine coronavirus genes and isolation of the viruses from yak[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50 (2): 343- 353.
21	关新, 朴范泽, 侯喜林. 牛冠状病毒的分离鉴定[J]. 黑龙江八一农垦大学学报, 2007, 19 (2): 55- 58.
	GUAN X , PIAO F Z , HOU X L . Isolation and identification of bovine coronavirus[J]. Journal of Heilongjiang August First Land Reclamation University, 2007, 19 (2): 55- 58.
22	张莹钰, 张迎春, 王青青, 等. 新疆南疆牛冠状病毒BCV-Aks-01株分离及基因型鉴定[J]. 中国兽医杂志, 2018, 54 (2): 12-14, 18.
	ZHANG Y Y , ZHANG Y C , WANG Q Q , et al. Isolation and genotype identification of bovine coronavirus BCV-Aks-01 strain in southern Xinjiang[J]. Chinese Journal of Veterinary Medicine, 2018, 54 (2): 12-14, 18.
23	谭烁, 汤承, 何琪富, 等. 牛冠状病毒TaqMan荧光定量RT-PCR检测方法的建立及初步应用[J]. 中国预防兽医学报, 2020, 42 (9): 918- 923.
	TAN S , TANG C , HE Q F , et al. Establishment and application of a TaqMan-based real-time RT-PCR assay for detecting Bovine coronavirus[J]. Chinese Journal of Preventive Veterinary Medicine, 2020, 42 (9): 918- 923.
24	STOKSTAD M , KLEM T B , MYRMEL M , et al. Using biosecurity measures to combat respiratory disease in cattle: the norwegian control program for bovine respiratory syncytial virus and bovine coronavirus[J]. Front Vet Sci, 2020, 7, 167.
25	FRUCCHI A P S , AGNOL A M D , BRONKHORST D E , et al. Bovine coronavirus Co-infection and molecular characterization in dairy calves with or without clinical respiratory disease[J]. Front Vet Sci, 2022, 9, 895492.
26	ZHOU Y , SHAO Z , DAI G , et al. Pathogenic infection characteristics and risk factors for bovine respiratory disease complex based on the detection of lung pathogens in dead cattle in Northeast China[J]. J Dairy Sci, 2023, 106 (1): 589- 606.
27	杨海峰, 粟灵琳, 王婧, 等. 我国14省市呼吸道综合征患牛冠状病毒感染的检测[J]. 畜牧与兽医, 2019, 51 (11): 92- 96.
	YANG H F , SU L L , WANG J , et al. Detection of bovine coronavirus infection in calves with respiratory disease syndrome in 14 provinces of China[J]. Animal Husbandry & Veterinary Medicine, 2019, 51 (11): 92- 96.
28	孙飞雁, 叶京飞, 魏宇, 等. 吉林省肉牛冠状病毒感染状况调查及分析[J]. 畜牧兽医学报, 2023, 54 (2): 673- 682.
	SUN F Y , YE J F , WEI Y , et al. Epidemiological investigation and analysis of bovine coronavirus in beef cattle in Jilin Province[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54 (2): 673- 682.
29	ZHU Q H , LI B , SUN D B . Advances in bovine coronavirus epidemiology[J]. Viruses, 2022, 14 (5): 1109.
30	JACKSON C B , FARZAN M , CHEN B , et al. Mechanisms of SARS-CoV-2 entry into cells[J]. Nat Rev Mol Cell Biol, 2022, 23 (1): 3- 20.
31	LANGEREIS M A , ZENG Q H , HEESTERS B A , et al. The murine coronavirus hemagglutinin-esterase receptor-binding site: a major shift in ligand specificity through modest changes in architecture[J]. PLoS Pathog, 2012, 8 (1): e1002492.
32	CHANNAPPANAVAR R , FEHR A R , VIJAY R , et al. Dysregulated Type Ⅰ interferon and inflammatory monocyte-macrophage responses cause lethal Pneumonia in SARS-CoV-infected mice[J]. Cell Host Microbe, 2016, 19 (2): 181- 193.
33	ZHUANG Z , LAI X M , SUN J , et al. Mapping and role of T cell response in SARS-CoV-2-infected mice[J]. J Exp Med, 2021, 218 (4): e20202187.
34	SHEAHAN T P , SIMS A C , ZHOU S T , et al. An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice[J]. Sci Transl Med, 2020, 12 (541): eabb5883.
35	刘振格, 侯喜林, 余丽芸, 等. 牛冠状病毒DQ株感染BALB/c小鼠的敏感性研究[J]. 中国预防兽医学报, 2014, 36 (1): 15- 18.
	LIU Z G , HOU X L , YU L Y , et al. Study on the sensitivity of BALB/c mice to bovine coronavirus[J]. Chinese Journal of Preventive Veterinary Medicine, 2014, 36 (1): 15- 18.

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

[1]	Weizhe LIU, Chenggang LUO, Rong YUAN, Yijie LIAO, Yimin WEN, Ying SUN, Enbo YU, Sanjie CAO, Xiaobo HUANG. Isolation and Identification of a Highly Pathogenic Strain of Porcine Epidemic Diarrhea Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3049-3063.
[2]	LIU Bohua, FU Hanyu, WANG Yuheng, Suolangsizhu, NIU Jiaqiang, BAO Yuhua, LI Jiakui, XU Yefen. Isolation, Identification and Genome Analysis of Type B Pasteurella multocida Isolated from Yak in Tibetan Nakchu City [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3105-3118.
[3]	Kun YANG, Jingwen MA, Xinrui ZHOU, Liezhu LUO, Zhe LIU, Ziqiang HU, Xingchen WU, Libin LIANG, Shimin GAO. Pathogenicity of Three Recombinant Strains of Infectious Bursal Disease Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2550-2559.
[4]	Ning ZHOU, Cheng TANG, Jia XU, Hua YUE, Xi CHEN. Pathogenicity and Genomic Characteristics of Feline Panleukopenia Virus A91S Variant in Cats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2560-2568.
[5]	XIONG Ting, HE Xianming, ZHAO Xiya, ZHUANG Tingting, HUANG Meizhen, LIANG Shijin, YU Chuanzhao, LIANG Xuejing, CHEN Ruiai. Whole Genome Analysis of Three Predominant Epidemic Strains of Chicken Infectious Bronchitis Virus and Their Pathogenicity [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2109-2122.
[6]	ZHENG Rui, LIU Zishi, ZHANG Kangyou, YAN Yong, WEI Ling, ZEREN Wengmu, DINGZE Demi, HUANG Jianjun, WANG Li, WEI Yong. Isolation, Identification and Biological Characterization of Colletotrichum jasminigenum in Stems of Peanuts [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2206-2213.
[7]	QIN Yi, HU Wenjie, FANG Xiaowei, GUO Qian, TIAN Lanxin, LIU Fang, FANG Chun. Effect of Deletion of the Lipoteichoic Acid Synthase ltaS Gene on the Pathogenicity of Listeria monocytogenes [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 670-679.
[8]	WU Zihao, CAI Yilong, TUO Haixin, CHEN Wei. Pathogenicity Analysis of a PVL⁺ ST22 Staphylococcus aureus Isolated from Equine Raw Milk [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 718-726.
[9]	ZHUANG Cuicui, HAN Bo. Mechanism of Mitochondrial Damage in Bovine Mammary Epithelial Cells and Mouse Mammary Gland Infected with Escherichia coli Isolated from Bovine Mastitis [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 822-833.
[10]	CHEN Yuetong, LIU Xiaohan, WANG Zhiyang, ZHAO Yuxin, ZHOU Tiezhong, HU Zengjin, ZHU Yue, WANG Shaohui, TIAN Mingxing, DING Siyu, QI Jingjing, YU Shengqing. Isolation, Identification, Pathogenicity and Drug Susceptibility of Mycoplasma gallisepticum from Dead Chicken Embryos in Large-scale Chicken Farms in Guangdong Province [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 290-299.
[11]	LIANG Rui, FAN Xiaorui, ZHANG Jinqiang, PANG Quanhai. Effects of Mouse Melanocyte Silencing and Overexpression of Pigment Epithelium-Derived Factor on Melanin Synthesis [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3916-3930.
[12]	CHEN Xihong, LU Guicong, WANG Haolei, GOU Shaoxiao, YU Yongxiong, LIN Tao, JIANG Caode. Isochlorogenic Acid C Inhibits Mammary Inflammatory Response through NF-κB Signaling Pathway Using Bovine Mammary Gland Cells and Mouse Mammary Gland Tissue [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3931-3940.
[13]	ZHANG Yi, WANG Chenyan, YANG Xiao, SHAO Guoqing, HOU Bo. Phylogenetic Analysis and Pathogenicity of an Avian Infectious Laryngotracheitis Virus Strain WF03 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3435-3444.
[14]	HUANG Jiang, LI Chuang, CUI Yueqi, YUAN Xueying, ZHAO Zhicheng, LIU Yu, ZHOU Yulong, ZHU Zhanbo, ZHANG Zecai. Study on the Effect of Gut Microbiota Disturbance on Susceptibility to BVDV Based on a Mouse Model [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3466-3473.
[15]	WANG Jiali, ZHOU Ning, CHEN Xi, YUE Hua, TANG Cheng. Isolation, Identification and Pathogenicity of Canine Adenovirus Type 2 Strain with Natural Deletion in E3 Gene [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2982-2990.

Isolation, Identification and Partial Biological Characteristics of a Bovine Respiratory Coronavirus

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 9

References 35

Related Articles 15

Recommended Articles

Metrics

Comments