{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,13]],"date-time":"2026-09-13T20:37:41Z","timestamp":1789331861749,"version":"build-2803163510"},"posted":{"date-parts":[[2026,7,19]]},"group-title":"Microbiology","reference-count":74,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,19]],"date-time":"2026-07-19T00:00:00Z","timestamp":1784419200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["R01 HL182101"],"award-info":[{"award-number":["R01 HL182101"]}],"id":[{"id":"https:\/\/ror.org\/012pb6c26","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01 HL160540"],"award-info":[{"award-number":["R01 HL160540"]}],"id":[{"id":"https:\/\/ror.org\/012pb6c26","id-type":"ROR","asserted-by":"publisher"}]},{"award":["F31 HL176146"],"award-info":[{"award-number":["F31 HL176146"]}],"id":[{"id":"https:\/\/ror.org\/012pb6c26","id-type":"ROR","asserted-by":"publisher"}]},{"award":["F31 HL182183"],"award-info":[{"award-number":["F31 HL182183"]}],"id":[{"id":"https:\/\/ror.org\/012pb6c26","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["R01 AI170596"],"award-info":[{"award-number":["R01 AI170596"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["T32 AI089621"],"award-info":[{"award-number":["T32 AI089621"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["CBET 2129624"],"award-info":[{"award-number":["CBET 2129624"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,19]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Respiratory viruses are a significant cause of morbidity and mortality world-wide and an important trigger of acute exacerbation in chronic lung disease. Secreted airway mucus \u2013 a front-line defense system against respiratory virus infection \u2013 is largely composed of glycosylated mucins that promote virus trapping via steric and adhesive interactions. Still, the degree to which mucus can trap specific viruses is unclear. Further, mucin expression is altered in chronic lung disease with undefined impacts on host susceptibility to infection. Here, we devised an inverse Transwell assay (ITA) to specifically probe the barrier function of mucus towards infection without the confounding effects of ongoing mucus secretion and transport on viral dynamics. Using the ITA, we assessed the barrier function of human airway epithelial (HAE) culture-derived mucus towards influenza (IAV), rhinovirus (RV), adenovirus, and parainfluenza virus. Results showed differences in mucus penetration efficiency between viruses, with IAV being the most inhibited relative to no mucus controls. Notably, IAV and RV penetration efficiency was similar between normal mucus and mucus sourced from an in-vitro model of asthmatic epithelium. To further explore the role of specific mucins during infection, we employed CRISPR\/Cas9-modified HAE cultures lacking either MUC5B or MUC5AC expression. Direct infection in these cultures with IAV yielded higher viral titers compared to control HAE, suggesting both MUC5B and MUC5AC contribute to antiviral defense. Application of mucus harvested from specific mucin-knockout or control HAE in the ITA revealed that while RV penetration was similar across conditions, IAV was more efficient in breaching MUC5AC-depleted gels. Subsequent biophysical analysis of these mucus gels revealed a larger pore structure in the absence of MUC5AC. Together, these data indicate mucus-mediated restriction is virus dependent and highlight the contribution of MUC5AC to mucus structure and antiviral capabilities. Further, they establish the ITA as a tunable platform enabling investigation into mucus penetration by diverse viruses and the effects of altered mucus composition on barrier function.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Clinical Relevance<\/jats:title>\n                  <jats:p>Mucus composition is altered in chronic lung disease states and during inflammation with unknown consequences on its barrier function towards respiratory virus infection. Using an inverse Transwell assay, we describe virus-specific kinetics through mucus representative of health and disease and identify a critical role for MUC5AC in defense towards influenza A virus. This work can inform future strategies to improve infection prevention or mucus targeted therapies, and may help explain differences in susceptibility to viral infections across the population.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.07.19.739437","type":"posted-content","created":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T05:05:11Z","timestamp":1784523911000},"source":"Crossref","is-referenced-by-count":1,"title":["An Inverse Transwell Assay for Airway Mucus Barrier Function Reveals both Virus- and Mucin-Specific Impacts on Infection"],"prefix":"10.64898","author":[{"given":"Maria","family":"Corkran","sequence":"first","affiliation":[{"name":"Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, MD, USA"},{"name":"Molecular and Cellular Biology Program, University of Maryland, College Park, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth M.","family":"Engle","sequence":"additional","affiliation":[{"name":"Molecular and Cellular Biology Program, University of Maryland, College Park, MD, USA"},{"name":"Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heng","family":"Pan","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA"},{"name":"Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sahana","family":"Kumar","sequence":"additional","affiliation":[{"name":"Molecular and Cellular Biology Program, University of Maryland, College Park, MD, USA"},{"name":"Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allison","family":"Boboltz","sequence":"additional","affiliation":[{"name":"Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey R.","family":"Johnson","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA"},{"name":"Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gregg A.","family":"Duncan","sequence":"additional","affiliation":[{"name":"Molecular and Cellular Biology Program, University of Maryland, College Park, MD, USA"},{"name":"Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Margaret A.","family":"Scull","sequence":"additional","affiliation":[{"name":"Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, MD, USA"},{"name":"Molecular and Cellular Biology Program, University of Maryland, College Park, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026072315351086000_2026.07.19.739437v1.1","doi-asserted-by":"crossref","unstructured":"White DO , Brown LE. 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