HE基因受体结合域变异对牛冠状病毒感染的影响

doi:10.11843/j.issn.0366-6964.2025.03.032

摘要/Abstract

摘要：

为研究HE基因受体结合域变异对牛冠状病毒(BCoV)感染的影响，构建了pEGFP-HE420、pEGFP-HE424和pEGFP-HE428重组质粒。三种重组质粒分别转染至HCT-8细胞，然后感染NX-2021-B2-HE424分离株通过RT-qPCR检测病毒mRNA在吸附、内化和复制阶段的表达水平以及通过TCID₅₀检测胞内和胞外的病毒滴度。结果显示，pEGFP-HE420、pEGFP-HE424和pEGFP-HE428三种重组质粒均能在HCT-8细胞中表达。与pEGFP-HE424相比，HE基因受体结合域缺失或插入不影响病毒的吸附、内化、复制及胞内病毒滴度；HE基因受体结合域缺失降低了BCoV胞外病毒滴度；HE基因受体结合域插入不影响BCoV胞外病毒滴度。本研究通过构建仅受体结合域变异的三种HE基因的重组质粒在HCT-8细胞上探究了其对牛冠状病毒感染的影响，为研究牛冠状病毒的感染机制提供了数据支撑。

关键词: 牛冠状病毒, HE基因, 受体结合域变异, 病毒感染

Abstract:

To study the effect of HE gene receptor binding domain variation on bovine coronavirus (BCoV) infection, recombinant plasmids pEGFP-HE420, pEGFP-HE424 and pEGFP-HE428 were constructed. Three recombinant plasmids were transfected into HCT-8 cells respectively and then infected with NX-2021-B2-HE424 to detect viral mRNA expression levels at adsorption, internalization and replication stages by RT-qPCR, and to detect viral intracellular and extracellular titers by TCID₅₀. The results showed that pEGFP-HE420, pEGFP-HE424 and pEGFP-HE428 could be expressed in HCT-8 cells. Compared with pEGFP-HE424, deletion or insertion of HE gene receptor binding domain did not affect the adsorption, internalization, replication and intracellular virus titer. Deletion of HE gene receptor binding domain decreased extracellular titers of BCoV. HE gene receptor binding domain insertion did not affect the BCoV extracellular virus titer. In this study, three recombinant plasmids of HE genes with only receptor binding domain variation was constructed to explore its effect on bovine coronavirus infection in HCT-8 cells, providing data support for the study of the infection mechanism of BCoV.

Key words: bovine coronavirus, HE gene, receptor binding domain vibration, viral infection

中图分类号:

S852.659.6

姜慧华, 赵龙, 郭抗抗. HE基因受体结合域变异对牛冠状病毒感染的影响[J]. 畜牧兽医学报, 2025, 56(3): 1336-1343.

JIANG Huihua, ZHAO Long, GUO Kangkang. Effect of HE Gene Receptor Binding Domain Variation on Bovine Coronavirus Infection[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1336-1343.

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参考文献 23

1	沈思思, 陈亮, 冯万宇, 等. 牛冠状病毒研究进展[J]. 动物医学进展, 2022, 43 (1): 112- 116. doi: 10.3969/j.issn.1007-5038.2022.01.019
	SHEN S S , CHEN L , FENG W Y , et al. Progress on bovine coronaviruse[J]. Progress in Veterinary Medicine, 2022, 43 (1): 112- 116. doi: 10.3969/j.issn.1007-5038.2022.01.019
2	THOMAS C J , HOET A E , SREEVATSAN S , et al. Transmission of bovine coronavirus and serologic responses in feedlot calves under field conditions[J]. Am J Vet Res, 2006, 67 (8): 1412- 1420. doi: 10.2460/ajvr.67.8.1412
3	罗鹏飞, 夏俊, 张凌, 等. 犊牛腹泻病因分析及诊断防控综述[J]. 草食家畜, 2023 (5): 30- 38.
	LUO P F , XIA J , ZHANG L , et al. Analysis of the etiology of infectious calf diarrhea and its diagnosis, prevention and control[J]. Grass-feeding Livestock, 2023 (5): 30- 38.
4	CHOULJENKO V N , LIN X Q , STORZ J , et al. Comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia[J]. J Gen Virol, 2001, 82 (Pt 12): 2927- 2933.
5	ROSENTHAL P B , ZHANG X D , FORMANOWSKI F , et al. Structure of the haemagglutinin-esterase-fusion glycoprotein of influenza C virus[J]. Nature, 1998, 396 (6706): 92- 96. doi: 10.1038/23974
6	ZENG Q H , LANGEREIS M A , VAN VLIET A L W , et al. Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution[J]. Proc Natl Acad Sci U S A, 2008, 105 (26): 9065- 9069. doi: 10.1073/pnas.0800502105
7	SCHULTZE B , WAHN K , KLENK H D , et al. Isolated HE-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor- destroying and receptor-binding activity[J]. Virology, 1991, 180 (1): 221- 228. doi: 10.1016/0042-6822(91)90026-8
8	DE GROOT R J . Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses[J]. Glycoconj J, 2006, 23 (1-2): 59- 72. doi: 10.1007/s10719-006-5438-8
9	GÉLINAS A M , BOUTIN M , SASSEVILLE A M J , et al. Bovine coronaviruses associated with enteric and respiratory diseases in Canadian dairy cattle display different reactivities to anti-HE monoclonal antibodies and distinct amino acid changes in their HE, S and ns4. 9 protein[J]. Virus Res, 2001, 76 (1): 43- 57. doi: 10.1016/S0168-1702(01)00243-X
10	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
11	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
12	KO C K , KANG M I , LIM G K , et al. Molecular characterization of HE, M, and E genes of winter dysentery bovine coronavirus circulated in Korea during 2002-2003[J]. Virus Genes, 2006, 32 (2): 129- 136. doi: 10.1007/s11262-005-6867-3
13	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
14	WORKMAN A M , MCDANELD T G , HARHAY G P , et al. Recent emergence of bovine coronavirus variants with mutations in the hemagglutinin-esterase receptor binding domain in U. S. cattle[J]. Viruses, 2022, 14 (10): 2125. doi: 10.3390/v14102125
15	HASOKSUZ M , ALEKSEEV K , VLASOVA A , et al. Biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe[J]. J Virol, 2007, 81 (10): 4981- 4990. doi: 10.1128/JVI.02361-06
16	ALEKSEEV K P , VLASOVA A N , JUNG K , et al. Bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences[J]. J Virol, 2008, 82 (24): 12422- 12431. doi: 10.1128/JVI.01586-08
17	CHUNG J Y , KIM H R , BAE Y C , et al. Detection and characterization of bovine-like coronaviruses from four species of zoo ruminants[J]. Vet Microbiol, 2011, 148 (2-4): 396- 401. doi: 10.1016/j.vetmic.2010.08.035
18	ZHANG X M , HERBST W , KOUSOULAS K G , et al. Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child[J]. J Med Virol, 1994, 44 (2): 152- 161. doi: 10.1002/jmv.1890440207
19	SIMON-LORIERE E , HOLMES E C . Why do RNA viruses recombine?[J]. Nat Rev Microbiol, 2011, 9 (8): 617- 626. doi: 10.1038/nrmicro2614
20	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
21	BAHOUSSI A N , SHAH P T , GUO Y Y , et al. Evolutionary adaptation of bovine coronavirus (BCoV): screening of natural recombinations across the complete genomes[J]. J Basic Microbiol, 2023, 63 (5): 519- 529. doi: 10.1002/jobm.202200548
22	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. doi: 10.11843/j.issn.0366-6964.2023.02.024
	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. doi: 10.11843/j.issn.0366-6964.2023.02.024
23	DESFORGES M , DESJARDINS J , ZHANG C S , et al. The acetyl-esterase activity of the hemagglutinin-esterase protein of human coronavirus OC43 strongly enhances the production of infectious virus[J]. J Virol, 2013, 87 (6): 3097- 3107. doi: 10.1128/JVI.02699-12

引物名称 Primer name	引物序列(5′→3′) Primer sequence(5′→3′)
HE-F(Xho Ⅰ)	CCG$\underline{{\rm{CTCGAG}}}$ATGTTTTTGCTTCCTAGATTTG
HE-R(EcoR Ⅰ)	CG$\underline{{\rm{GAATTC}}}$GAGCATCATGCAGCCTAGTAC
Lig-HE-F	AAGGCTACTGTTTTGTCAAATACAAAGTATTATGATGATAGTC
Lig-HE-R	AACAGTAGCCTTAAACTTGCCATTAAAAATACAAAGTGGTAC
GAPDH-F(human)	ATTCCATGGCACCGTCAAGG
GAPDH-R(human)	TGGACTCCACGACGTACTCA
N-F	CGAGTTGAACACCCAGAT
N-R	GAGACGGGCATCTACACT

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