Exploring respiratory virus-host interactions at the mucosal interface

Respiratory viruses, including influenza A viruses (IAVs) and coronaviruses (CoVs), pose a major global health burden, causing diseases ranging from mild upper respiratory tract infections to severe pneumonia, bronchiolitis, and acute respiratory distress syndrome. These infections lead to considerable morbidity and mortality worldwide, particularly among children, the elderly, and immunocompromised individuals. They also impose significant economic costs through medical care, lost productivity, and public health interventions. The emergence of SARS-CoV-2 underscored the pandemic potential of respiratory viruses beyond IAVs, highlighting the importance of understanding their biology using physiologically relevant in vitro models.

This thesis investigates the interplay between viruses and the respiratory mucosa, utilising human airway epithelial (HAE) air–liquid interface (ALI) cultures and the mucus they produce. These models replicate the architecture and function of the respiratory tract more accurately than conventional cell lines, supporting epithelial differentiation and mucus secretion, and thus provide a physiologically relevant system to study early virus–host interactions at the airway surface.

To establish infection, respiratory viruses must traverse the mucus layer to reach target epithelial cells, while avoiding entrapment and mucociliary clearance. Many viruses bind to sialoglycans on mucin proteins, and several—including IAVs and some CoVs—express envelope glycoproteins with glycan-cleaving activity to facilitate movement through mucus. Successful infection requires a functional balance between receptor-binding and -destruction activities, which must be recalibrated when adapting to new host species with distinct sialoglycomes (Chapter 2).

While the role of IAV neuraminidase (NA) in progeny release is well established, its role in entry remains debated. In Chapter 3, we systematically analysed NA’s role in entry, in relation to haemagglutinin (HA) receptor-binding preference, host receptor repertoire, and the presence of mucus decoy receptors. Using recombinant viruses differing only in HA–NA composition, we found that NA dependence during entry was primarily determined by HA properties. This dependence increased when preferred receptors were scarce, coinciding with greater inhibition by mucus decoy receptors.

Although mucus is recognised as a barrier to infection, the influence of its composition and donor variability on antiviral activity remains poorly defined. In Chapter 4, we characterised HAE ALI culture-derived mucus from different anatomical sites and donors using glycomic and proteomic profiling, alongside IAV binding and infection assays. Samples showed broad similarities in protein, glycan, and IAV inhibition profiles, though inhibition varied by IAV subtype in a manner dependent on HA receptor-binding preference—reinforcing observations in Chapter 3. These findings highlight the complex relationship between mucus composition and antiviral efficacy.

In Chapter 5, we compared HAE ALI cultures derived from primary nasal epithelial cells (HNECs) with those from immortalised airway basal cell lines—BCi-NS1.1 and hSABCi-NS1.1—for their ability to support IAV and CoV replication. IAVs that replicated similarly in conventional cells displayed distinct replication patterns in ALI cultures, with α2–6 sialoglycan-preferring viruses replicating more efficiently than α2–3 binders, a trend mirrored in swine ALI cultures. Clinical isolates of HCoV-OC43 replicated more robustly than a laboratory-adapted strain, with highest titres in the HNEC model. SARS-CoV-2 replication varied between models: Omicron replicated best in BCi-NS1.1 cultures, while the Wuhan strain favoured hSABCi-NS1.1. These results underscore the value of immortalised progenitor-derived ALI culture models and the importance of selecting appropriate systems for studying clinically relevant isolates.

Finally, Chapter 6 integrates these findings in the context of viral motility, mucus-mediated restriction, and the utility of HAE ALI cultures in respiratory virus research. Future studies should explore the HA–NA balance in greater depth and expand mucus characterisation, virus binding, and infection-inhibition assays across multiple species. Collectively, this work advances understanding of viral adaptation and the role of mucus in host specificity, and supports the development of improved strategies to predict, prevent, and mitigate respiratory viral infections.