{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:08:51Z","timestamp":1785431331912,"version":"3.56.0"},"reference-count":53,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T00:00:00Z","timestamp":1780099200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["516634310"],"award-info":[{"award-number":["516634310"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"award":["516634310"],"award-info":[{"award-number":["516634310"]}],"id":[{"id":"https:\/\/ror.org\/018mejw64","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Volkswagen Stiftung","award":["9A874"],"award-info":[{"award-number":["9A874"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Air\u2013liquid interface (ALI) cultures recapitulate key features of the airway epithelium by driving basal cell differentiation into ciliated, club, and goblet cells and by generating a functional mucus barrier, thereby representing a highly relevant model of the human respiratory tract. Using a reduced factorial Design of Experiments (DoE) methodology, we simultaneously investigated the effects of seven variables on human coronavirus OC43 (HCoV-OC43) replication in air\u2013liquid interface (ALI)-cultured primary human bronchial epithelial cells (pHBECs) to identify robust conditions that support infection and viral replication. Epithelial differentiation was monitored by measuring transepithelial electrical resistance and determining expression levels of marker genes for basal, goblet, club, and ciliated cells using reverse transcription quantitative PCR (RT-qPCR). HCoV-OC43 replication was monitored by quantifying genomic and subgenomic RNA by RT-qPCR. Viral RNA peaked three days post-infection in cell lysates and four days post-infection in apical washes. Initiation of ALI conditions induced epithelial differentiation, which was complete after 21 days and emerged as the strongest determinant of viral replication. Differentiated pHBEC cultures showed significantly reduced viral RNA compared with undifferentiated cultures, particularly following apical infection. In contrast, basal infection resulted in lower viral RNA levels in undifferentiated cultures than apical infection but was less dependent on epithelial differentiation. However, productive infection following basal exposure was less consistent and more strongly dependent on viral inoculum size. We further demonstrate that repeated mucus washes prior to infection increased HCoV-OC43 replication in mature cultures. In summary, our findings show that epithelial differentiation negatively affects HCoV-OC43 replication and we identify conditions that maximize viral replication in fully differentiated pHBEC cultures.<\/jats:p>","DOI":"10.3390\/cells15111010","type":"journal-article","created":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T08:09:16Z","timestamp":1780301356000},"page":"1010","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Multifactorial Analysis Identifies Conditions for Robust HCoV-OC43 Replication in Primary Human Bronchial Epithelial Cells Cultured at Air\u2013Liquid Interface"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-0755-8599","authenticated-orcid":false,"given":"Natalie","family":"Fischhaber","sequence":"first","affiliation":[{"name":"Institute of Laboratory Medicine, Ludwig-Maximilians-University Hospital, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3213-6231","authenticated-orcid":false,"given":"Julian","family":"Vogler","sequence":"additional","affiliation":[{"name":"Institute of Laboratory Medicine, Ludwig-Maximilians-University Hospital, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivana","family":"Martan","sequence":"additional","affiliation":[{"name":"Institute of Laboratory Medicine, Ludwig-Maximilians-University Hospital, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4174-3316","authenticated-orcid":false,"given":"Thomas","family":"Michler","sequence":"additional","affiliation":[{"name":"Institute of Laboratory Medicine, Ludwig-Maximilians-University Hospital, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Harrison, C.M., Doster, J.M., Landwehr, E.H., Kumar, N.P., White, E.J., Beachboard, D.C., and Stobart, C.C. (2023). Evaluating the Virology and Evolution of Seasonal Human Coronaviruses Associated with the Common Cold in the COVID-19 Era. Microorganisms, 11.","DOI":"10.3390\/microorganisms11020445"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"6081","DOI":"10.1128\/JVI.03368-12","article-title":"Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism","volume":"87","author":"Dijkman","year":"2013","journal-title":"J. Virol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"7124","DOI":"10.1038\/s41598-018-25640-0","article-title":"Early events during human coronavirus OC43 entry to the cell","volume":"8","author":"Owczarek","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e2282","DOI":"10.1002\/rmv.2282","article-title":"An overview on the seven pathogenic human coronaviruses","volume":"32","author":"Kesheh","year":"2022","journal-title":"Rev. Med. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1595","DOI":"10.1128\/JVI.79.3.1595-1604.2005","article-title":"Complete genomic sequence of human coronavirus OC43: Molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event","volume":"79","author":"Vijgen","year":"2005","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e0169024","DOI":"10.1128\/spectrum.01690-24","article-title":"Infectivity duration of HCoV-OC43, a surrogate for SARS-CoV-2, in artificial bodily fluids on stainless steel simulating common nonporous surfaces","volume":"13","author":"Furin","year":"2025","journal-title":"Microbiol. Spectr."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"114317","DOI":"10.1016\/j.jviromet.2021.114317","article-title":"Improving human coronavirus OC43 (HCoV-OC43) research comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2","volume":"299","author":"Schirtzinger","year":"2022","journal-title":"J. Virol. Methods"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2000111","DOI":"10.1002\/anbr.202000111","article-title":"Air-liquid interface cultures of the healthy and diseased human respiratory tract: Promises, challenges and future directions","volume":"1","author":"Baldassi","year":"2021","journal-title":"Adv. Nanobiomed Res."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Rijsbergen, L.C., van Dijk, L.L.A., Engel, M.F.M., de Vries, R.D., and de Swart, R.L. (2021). In Vitro Modelling of Respiratory Virus Infections in Human Airway Epithelial Cells\u2014A Systematic Review. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.683002"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"500","DOI":"10.1038\/s41598-018-36735-z","article-title":"Optimization of Normal Human Bronchial Epithelial (NHBE) Cell 3D Cultures for in vitro Lung Model Studies","volume":"9","author":"Rayner","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Gonzalez-Rubio, J., Le-Trilling, V.T.K., Baumann, L., Cheremkhina, M., Kubiza, H., Luengen, A.E., Reuter, S., Taube, C., Ruetten, S., and Duarte Campos, D. (2023). SARS-CoV-2 particles promote airway epithelial differentiation and ciliation. Front. Bioeng. Biotechnol., 11.","DOI":"10.3389\/fbioe.2023.1268782"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.ejpb.2023.01.013","article-title":"Air-liquid interface (ALI) impact on different respiratory cell cultures","volume":"184","author":"Silva","year":"2023","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"943982","DOI":"10.1155\/2012\/943982","article-title":"Evaluation of differentiated human bronchial epithelial cell culture systems for asthma research","volume":"2012","author":"Stewart","year":"2012","journal-title":"J. Allergy"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1177\/2211068214561025","article-title":"TEER measurement techniques for in vitro barrier model systems","volume":"20","author":"Srinivasan","year":"2015","journal-title":"J. Lab. Autom."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"285","DOI":"10.7883\/yoken.JJID.2020.776","article-title":"Isolation of Human Coronaviruses OC43, HKU1, NL63, and 229E in Yamagata, Japan, Using Primary Human Airway Epithelium Cells Cultured by Employing an Air-Liquid Interface Culture","volume":"74","author":"Komabayashi","year":"2021","journal-title":"Jpn. J. Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Chan, R.W., Yuen, K.M., Yu, W.C., Ho, C.C., Nicholls, J.M., Peiris, J.S., and Chan, M.C. (2010). Influenza H5N1 and H1N1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0008713"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1159\/000530374","article-title":"Impact of Changes in Human Airway Epithelial Cellular Composition and Differentiation on SARS-CoV-2 Infection Biology","volume":"15","author":"Thaler","year":"2023","journal-title":"J. Innate Immun."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Mallek, N.M., Martin, E.M., Dailey, L.A., and McCullough, S.D. (2023). Liquid application dosing alters the physiology of air-liquid interface (ALI) primary human bronchial epithelial cell\/lung fibroblast co-cultures and in vitro testing relevant endpoints. Front. Toxicol., 5.","DOI":"10.3389\/ftox.2023.1264331"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1016\/j.bbrc.2021.09.015","article-title":"Air-liquid interphase culture confers SARS-CoV-2 susceptibility to A549 alveolar epithelial cells","volume":"577","author":"Sasaki","year":"2021","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1016\/j.virol.2012.11.002","article-title":"Biological characteristics and propagation of human rhinovirus-C in differentiated sinus epithelial cells","volume":"436","author":"Ashraf","year":"2013","journal-title":"Virology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"11255","DOI":"10.1128\/JVI.00947-10","article-title":"Culturing the unculturable: Human coronavirus HKU1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures","volume":"84","author":"Pyrc","year":"2010","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"14961","DOI":"10.1038\/s41598-021-94095-7","article-title":"High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro","volume":"11","author":"Gard","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"L926","DOI":"10.1152\/ajplung.00374.2020","article-title":"Human coronaviruses 229E and OC43 replicate and induce distinct antiviral responses in differentiated primary human bronchial epithelial cells","volume":"319","author":"Loo","year":"2020","journal-title":"Am. J. Physiol. Lung Cell Mol. Physiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.virol.2017.11.012","article-title":"Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry","volume":"517","author":"Shirato","year":"2018","journal-title":"Virology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e0042225","DOI":"10.1128\/msphere.00422-25","article-title":"Potential for prolonged replication of common acute respiratory viruses in air-liquid interface cultures of primary human airway cells","volume":"10","author":"Kawase","year":"2025","journal-title":"mSphere"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e2119680119","DOI":"10.1073\/pnas.2119680119","article-title":"SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13","volume":"119","author":"Morrison","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e2400329","DOI":"10.1002\/adbi.202400329","article-title":"Mucus Physically Restricts Influenza A Viral Particle Access to the Epithelium","volume":"9","author":"Kaler","year":"2025","journal-title":"Adv. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.chom.2016.01.001","article-title":"The Interaction between Respiratory Pathogens and Mucus","volume":"19","author":"Zanin","year":"2016","journal-title":"Cell Host Microbe"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1007\/s00163-002-0026-9","article-title":"A role for \u201cone-factor-at-a-time\u201d experimentation in parameter design","volume":"14","author":"Frey","year":"2003","journal-title":"Res. Eng. Des."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"kuae010","DOI":"10.1093\/jimb\/kuae010","article-title":"Using design of experiments to guide genetic optimization of engineered metabolic pathways","volume":"51","author":"Moon","year":"2024","journal-title":"J. Ind. Microbiol. Biotechnol."},{"key":"ref_31","unstructured":"JMP Statistical Discovery, LLC (2024). JMP\u00ae 18 Fitting Linear Models, JMP Statistical Discovery, LLC."},{"key":"ref_32","unstructured":"Norman, R., and Draper, H.S. (1998). Applied Regression Analysis, Wiley. [3rd ed.]."},{"key":"ref_33","unstructured":"JMP Statistical Discovery, LLC (2024). JMP\u00ae 18 Design of Experiments Guide, JMP Statistical Discovery, LLC."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1177\/32.1.6418790","article-title":"An immunologic study of the secretory products of rat Clara cells","volume":"32","author":"Singh","year":"1984","journal-title":"J. Histochem. Cytochem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1177\/36.1.3275712","article-title":"Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein","volume":"36","author":"Singh","year":"1988","journal-title":"J. Histochem. Cytochem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1042\/bj3180319","article-title":"Different mucins are produced by the surface epithelium and the submucosa in human trachea: Identification of MUC5AC as a major mucin from the goblet cells","volume":"318","author":"Hovenberg","year":"1996","journal-title":"Biochem. J."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1146\/annurev.physiol.70.113006.100441","article-title":"Regulation of airway mucin gene expression","volume":"70","author":"Thai","year":"2008","journal-title":"Annu. Rev. Physiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1242\/dmm.006031","article-title":"Airway basal stem cells: A perspective on their roles in epithelial homeostasis and remodeling","volume":"3","author":"Rock","year":"2010","journal-title":"Dis. Model. Mech."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1186\/s12931-016-0404-x","article-title":"Detection and quantification of epithelial progenitor cell populations in human healthy and IPF lungs","volume":"17","author":"Smirnova","year":"2016","journal-title":"Respir. Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1038\/ng.727","article-title":"The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation","volume":"43","author":"Zohn","year":"2011","journal-title":"Nat. Genet."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1037\/a0015826","article-title":"Design of experiments with multiple independent variables: A resource management perspective on complete and reduced factorial designs","volume":"14","author":"Collins","year":"2009","journal-title":"Psychol. Methods"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1093\/nar\/gkab1248","article-title":"Targeting genomic SARS-CoV-2 RNA with siRNAs allows efficient inhibition of viral replication and spread","volume":"50","author":"Ambike","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2074","DOI":"10.1016\/j.jaci.2017.07.018","article-title":"Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures","volume":"141","author":"Brito","year":"2018","journal-title":"J. Allergy Clin. Immunol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Persson, B.D., Jaffe, A.B., Fearns, R., and Danahay, H. (2014). Respiratory syncytial virus can infect basal cells and alter human airway epithelial differentiation. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0102368"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"95","DOI":"10.3109\/08820139309063393","article-title":"HLA class I antigen serves as a receptor for human coronavirus OC43","volume":"22","author":"Collins","year":"1993","journal-title":"Immunol. Investig."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1007\/978-1-4615-1899-0_60","article-title":"Analysis of cellular receptors for human coronavirus OC43","volume":"380","author":"Krempl","year":"1995","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"L181","DOI":"10.1152\/ajplung.00321.2010","article-title":"Mucociliary interactions and mucus dynamics in ciliated human bronchial epithelial cell cultures","volume":"301","author":"Sears","year":"2011","journal-title":"Am. J. Physiol. Lung Cell Mol. Physiol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"5094","DOI":"10.1096\/fj.13-234476","article-title":"Vasoactive intestinal peptide regulates sinonasal mucociliary clearance and synergizes with histamine in stimulating sinonasal fluid secretion","volume":"27","author":"Lee","year":"2013","journal-title":"FASEB J."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1038\/s41557-021-00655-9","article-title":"Synthetic O-acetylated sialosides facilitate functional receptor identification for human respiratory viruses","volume":"13","author":"Li","year":"2021","journal-title":"Nat. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2681","DOI":"10.1073\/pnas.1809667116","article-title":"Human coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved receptor-binding site in spike protein domain A","volume":"116","author":"Hulswit","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1038\/s41594-019-0233-y","article-title":"Structural basis for human coronavirus attachment to sialic acid receptors","volume":"26","author":"Tortorici","year":"2019","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Math\u00e9, J., Brochu, S., Adam, D., Brochiero, E., and Perreault, C. (2024). Sex and Disease Regulate MHC I Expression in Human Lung Epithelial Cells. bioRxiv.","DOI":"10.1101\/2024.06.20.599895"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"e00379-16","DOI":"10.1128\/mSphere.00379-16","article-title":"Distribution of O-Acetylated Sialic Acids among Target Host Tissues for Influenza Virus","volume":"2","author":"Wasik","year":"2017","journal-title":"mSphere"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/11\/1010\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T04:15:05Z","timestamp":1780632905000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/11\/1010"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,30]]},"references-count":53,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["cells15111010"],"URL":"https:\/\/doi.org\/10.3390\/cells15111010","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,30]]}}}