Penetration of avian and human influenza A viruses through mucus gel networks in vitro

Influenza A viruses (IAVs) remain a public-health concern because of their continuous evolution and zoonotic transmission. Respiratory mucus is the first extracellular barrier encountered by inhaled viruses, yet standardized approaches for studying influenza virus passage through mucus and the infectivity of transferred virus populations remain limited. Here, we established an in vitro virus–mucus penetration assay to analyze IAV interactions with standardized mucin-containing matrices. We used mucus from human Calu-3 airway epithelial cells cultured under air–liquid interface conditions and bovine submaxillary mucin as surrogate matrices. Preparations were normalized by total protein and characterized using biochemical and biophysical methods. We compared a human seasonal H1N1pdm virus with a highly-pathogenic avian H5N1 virus. Viral genome transfer was quantified by RT-qPCR, while retained infectivity was assessed in susceptible cells. Both mucin-containing matrices restricted viral passage compared with control solutions. Viral RNA transfer and retained infectivity varied by matrix and virus, although no statistically significant difference between H1N1pdm and H5N1 was detected within either matrix. Together, these results establish a framework integrating mucus characterization with complementary measurements of viral transfer and infectivity. This approach provides a basis for investigating influenza virus–mucus interactions under controlled conditions and can be extended to other IAV strains and physiologically relevant mucus sources.