包头医学院,公共卫生学院;传染病溯源预警与智能决策全国重点实验室国家卫生健康委医学病毒和病毒病重点实验室中国疾病预防控制中心病毒病预防控制所;
本研究旨在明确HCoV-229E在多种细胞中的感染特征与复制动力学。首先比较了33℃和37℃下HCoV-229E在MRC-5中的致细胞病变效应、核酸增殖与感染活性等特征，确定病毒培养温度；通过RT-PCR(Real-time reverse transcription,rRT-PCR）比较了HCo -229E在不同种属与组织来源的13种细胞系（Huh7.5、He La、MRC-5、A549、RD、Hep-2、Calu3、Ha Cat-WT、Vero、LLC-MK2、MDCK、BHK-21、Mv1Lu）中的病毒RNA复制水平，评估了上述细胞对病毒的易感性；进一步探究其在易感细胞系中的致细胞病变效应（Cytopathic effect,CPE）、半数组织感染剂量（50%of tissue culture infectious dose,TCID50）复制动力学和噬斑形成特性。研究发现，HCoV-229E以同等MOI感染MRC-5后，病毒RNA与TCID50的复制增殖均表现为33℃优于37℃。病毒RNA复制动力学结果显示，13种细胞中的6种人源细胞（Huh7.5、HeLa、MRC-5、RD、A549、Hep-2）对HCo V-229E易感，但在Hep-2中的复制较差。CPE结果表明，Huh7.5与He La均在感染后48h观察到CPE，并于72h出现细胞变圆脱落，与MRC-5趋势一致；而A549和RD分别于72h和96h观察到CPE，但细胞脱落死亡情况较轻。病毒活性复制动力学显示，0.01与0.001MOI感染剂量下，HCo V-229E在Huh7.5中分别于24h和48h达到复制高峰（106.96 TCID50/m L和106.65 TCID50/mL,P<0.05）；在HeLa中分别于48h与72h达到复制高峰（106.8 TCID50/mL和106.56 TCID50/mL,P<0.05）；在A549中均在96h到达复制高峰（约106.3 TCID50/mL,P>0.05）；在RD细胞中均在72h到达复制高峰（106.58 TCID50/m L和106.65 TCID50/mL,P>0.05）。HCoV-229E可在Huh7.5、HeLa、A549、RD细胞中产生不同形态的空斑，其中He La细胞时间短（96h）、噬斑清晰易于计数，是HCoV-229E空斑实验的良好选择。本研究通过RT-PCR、CPE、病毒活性和噬斑特性系统展示了HCoV-229E在13种细胞中表现出不同感染特征与复制动力学，为深入研究HCoV-229E病原生物学和抗病毒药物筛选等提供了参考。
| 66 | 0 | 67 |
| 下载次数 | 被引频次 | 阅读次数 |
[1] Zhou Z, Qiu Y, Ge X. The taxonomy, host range and pathogenicity of coronaviruses and other viruses in the Nidovirales order[J]. Anim Dis, 2021, 1(1):5. DOI:10. 1186/s44149-021-00005-9.
[2] Kesheh MM, Hosseini P, Soltani S, et al. An overview on the seven pathogenic human coronaviruses[J]. Rev Med Virol, 2022, 32(2):e2282. DOI:10. 1002/rmv. 2282.
[3] Alejandra Tortorici M, Walls AC, Joshi A, et al.Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein[J]. Cell, 2022, 185(13):2279-2291. e17. DOI:10. 1016/j. cell. 2022. 05. 019.
[4] Lednicky JA, Tagliamonte MS, White SK, et al.Independent infections of porcine deltacoronavirus among Haitian children[J]. Nature, 2021, 600(7887):133-137. DOI:10. 1038/s41586-021-04111-z.
[5] Kim MI, Lee C. Human coronavirus OC43 as a lowrisk model to study COVID-19[J]. Viruses, 2023, 15(2):578. DOI:10. 3390/v15020578.
[6] Hoffmann HH, Sánchez-Rivera FJ, Schneider WM, et al. Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors[J]. Cell Host Microbe, 2021,29(2):267-280. e5. DOI:10. 1016/j.chom. 2020. 12. 009.
[7] Hamre D, Procknow JJ. A new virus isolated from the human respiratory tract[J]. Proc Soc Exp Biol Med,1966, 121(1):190-193. DOI:10. 3181/00379727-121-30734.
[8] Yeager CL, Ashmun RA, Williams RK, et al. Human aminopeptidase N is a receptor for human coronavirus229E[J]. Nature, 1992, 357(6377):420-422. DOI:10. 1038/357420a0.
[9] Gerna G, Campanini G, Rovida F, et al. Genetic variability of human coronavirus OC43-，229E-，and NL63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients[J]. J Med Virol, 2006,78(7):938-949. DOI:10. 1002/jmv. 20645.
[10]Pene F, Merlat A, Vabret A, et al. Coronavirus 229Erelated pneumonia in immunocompromised patients[J].Clin Infect Dis, 2003, 37(7):929-932. DOI:10. 1086/377612.
[11]申梁，朱娜，牛军伟，等.人冠状病毒OC43易感细胞系的高效筛选[J].病毒学报，2017, 33(3):307-312.DOI:10. 13242/j. cnki. bingduxuebao. 003151.
[12]Joel Funk C, Wang J, Ito Y, et al. Infection of human alveolar macrophages by human coronavirus strain 229E[J]. J Gen Virol, 2012, 93(Pt 3):494-503. DOI:10. 1099/vir. 0. 038414-0.
[13]Gong Q, Jiang R, Ji L, et al. Establishment of a human organoid-based evaluation system for assessing interspecies infection risk of animal-borne coronaviruses[J]. Emerg Microbes Infect, 2024, 13(1):2327368.DOI:10. 1080/22221751. 2024. 2327368.
[14]Li P, Wang Y, Lamers MM, et al. Recapitulating infection, thermal sensitivity and antiviral treatment of seasonal coronaviruses in human airway organoids[J].EBioMedicine, 2022, 81:104132. DOI:10. 1016/j.ebiom. 2022. 104132.
[15]Wang G, Deering C, Macke M, et al. Human coronavirus 229E infects polarized airway epithelia from the apical surface[J]. J Virol, 2000, 74(19):9234-9239. DOI:10. 1128/jvi. 74. 19. 9234-9239. 2000.
[16]Pasquereau S, Nehme Z, Haidar Ahmad S, et al.Resveratrol inhibits HCoV-229E and SARS-CoV-2coronavirus replication in vitro[J]. Viruses, 2021, 13(2):354. DOI:10. 3390/v13020354.
[17]Andreu S, Ripa I, López-Guerrero JA, et al. Human coronavirus 229E uses clathrin-mediated endocytosis as a route of entry in huh-7 cells[J]. Biomolecules, 2024, 14(10):1232. DOI:10. 3390/biom14101232.
[18]Kwon EB, Kim B, Kim YS, et al. Anastrozole protects against human coronavirus infection by ameliorating the reactive oxygen species-mediated inflammatory response[J]. Antioxidants(Basel), 2024, 13(1):116. DOI:10. 3390/antiox13010116.
[19]Lambert F, Jacomy H, Marceau G, et al. Titration of human coronaviruses, HcoV-229E and HCoV-OC43,by an indirect immunoperoxidase assay[J]. Methods Mol Biol, 2008, 454:93-102. DOI:10. 1007/978-1-59745-181-9_8.
[20]Siragam V, Maltseva M, Castonguay N, et al.Seasonal human coronaviruses OC43, 229E, and NL63induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics[J].Microbiol Spectr, 2024, 12(7):e0422023. DOI:10. 1128/spectrum. 04220-23.
[21]Fausto A, Otter CJ, Bracci N, et al. Improved culture methods for human coronaviruses HCoV-OC43, HCoV-229E, and HCoV-NL63[J]. Curr Protoc, 2023, 3(10):e914. DOI:10. 1002/cpz1. 914.
[22]Tang BSF, Chan KH, Cheng VCC, et al.Comparative host gene transcription by microarray analysis early after infection of the Huh7 cell line by severe acute respiratory syndrome coronavirus and human coronavirus 229E[J]. J Virol, 2005, 79(10):6180-6193. DOI:10. 1128/JVI. 79. 10. 6180-6193. 2005.
[23]Weil T, Lawrenz J, Seidel A, et al. Immunodetection assays for the quantification of seasonal common cold coronaviruses OC43, NL63, or 229E infection confirm nirmatrelvir as broad coronavirus inhibitor[J]. Antiviral Res, 2022, 203:105343. DOI:10. 1016/j.antiviral. 2022. 105343.
[24]Hu Y, Ma C, Wang J. Cytopathic effect assay and plaque assay to evaluate in vitro activity of antiviral compounds against human coronaviruses 229E, OC43,and NL63[J]. Bio Protoc, 2022, 12(3):e4314. DOI:10. 21769/BioProtoc. 4314.
[25]Bracci N, Pan HC, Lehman C, et al. Improved plaque assay for human coronaviruses 229E and OC43[J].PeerJ, 2020, 8:e10639. DOI:10. 7717/peerj. 10639.
[26]Lie LK, Synowiec A, Mazur J, et al. An engineered A549 cell line expressing CD13 and TMPRSS2 is permissive to clinical isolate of human coronavirus 229E[J]. Virology, 2023, 588:109889. DOI:10. 1016/j.virol. 2023. 109889.
[27]叶永丽，李梓暄，刘丽娟，等.病毒感染性检测方法研究进展[J].病毒学报，2025, 41(1):202–210. DOI:10. 13242/j. cnki. bingduxuebao. 004651.
[28]Niu P, Shen J, Zhu N, et al. Two-tube multiplex realtime reverse transcription PCR to detect six human coronaviruses[J]. Virol Sin, 2016, 31(1):85-88.DOI:10. 1007/s12250-015-3653-9.
[29]Vassilara F, Spyridaki A, Pothitos G, et al. A rare case of human coronavirus 229E associated with acute respiratory distress syndrome in a healthy adult[J]. Case Rep Infect Dis, 2018, 2018:6796839. DOI:10. 1155/2018/6796839.
[30]Otter CJ, Fausto A, Tan LH, et al. Infection of primary nasal epithelial cells differentiates among lethal and seasonal human coronaviruses[J]. Proc Natl Acad Sci USA, 2023, 120(15):e2218083120. DOI:10. 1073/pnas. 2218083120.
[31]Devhare PB, Desai S, Lole KS. Innate immune responses in human hepatocyte-derived cell lines alter genotype 1 hepatitis E virus replication efficiencies[J].Sci Rep, 2016, 6:26827. DOI:10. 1038/srep26827.
[32]Sekrecka A, Kluzek K, Sekrecki M, et al. Timedependent recruitment of GAF, ISGF3 and IRF1complexes shapes IFNα and IFNγ-activated transcriptional responses and explains mechanistic and functional overlap[J]. Cell Mol Life Sci, 2023, 80(7):187. DOI:10. 1007/s00018-023-04830-8.
[33]Schmidt OW, Cooney MK, Kenny GE. Plaque assay and improved yield of human coronaviruses in a human rhabdomyosarcoma cell line[J]. J Clin Microbiol,1979, 9(6):722-728. DOI:10. 1128/jcm. 9. 6. 722-728. 1979.
[34]Channappanavar R, Perlman S. Age-related susceptibility to coronavirus infections:role of impaired and dysregulated host immunity[J]. J Clin Invest,2020, 130(12):6204-6213. DOI:10. 1172/JCI144115.
[35]Lachance C, Arbour N, Cashman NR, et al.Involvement of aminopeptidase N(CD13)in infection of human neural cells by human coronavirus 229E[J]. J Virol, 1998, 72(8):6511-6519. DOI:10. 1128/JVI. 72. 8. 6511-6519. 1998.
[36]Wong AHM, Tomlinson ACA, Zhou D, et al.Receptor-binding loops in alphacoronavirus adaptation and evolution[J]. Nat Commun, 2017, 8(1):1735.DOI:10. 1038/s41467-017-01706-x.
[37]Anonymous. ANPEP protein expression summary-the human protein atlas[EB/OL]([2025–02–17])[2025–02–17]. https：//www. proteinatlas. org/ENSG00000166825-ANPEP.
[38]Hirose R, Watanabe N, Bandou R, et al. A cytopathic effect-based tissue culture method for HCoV-OC43titration using TMPRSS2-expressing VeroE6 cells[J].mSphere, 2021, 6(3):e00159-21. DOI:10. 1128/mSphere. 00159-21.
[39]Lau SKP, Lung DC, Wong EYM, et al. Molecular evolution of human coronavirus 229E in Hong Kong and a fatal COVID-19 case involving coinfection with a novel human coronavirus 229E genogroup[J]. mSphere,2021, 6(1):e00819-20. DOI:10. 1128/mSphere. 00819-20.
[40]李丰辰，蒋豪然，夏百成，等.急性呼吸道感染病例中人冠状病毒229E感染情况及病毒基因特征分析[J].病毒学报，2024, 40(1):13-26. DOI:10. 13242/j.cnki. bingduxuebao. 004458.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250030
中图分类号:R373
引用信息:
[1]刘冠雅,吴依依,刘士元等.人冠状病毒229E体外感染与复制特征研究[J].病毒学报,2025,41(02):383-394.DOI:10.13242/j.cnki.bingduxuebao.250030.
基金信息:
国家重点研发计划（项目号：2022YFC2304100）,题目：新冠与流感等大流行风险病原体多肽疫苗的研制;国家重点研发计划（项目号：2021YFA1201003）,题目：抗病毒纳米药物的体内外功能评价;国家重点研发计划（项目号：2022YFC2303401）,题目：潜在高危新病原风险识别的新技术体系研究~~