Exploring respiratory virus-host interactions at the mucosal interface

Summary

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Respiratory viruses, including influenza A viruses (IAVs) and coronaviruses (CoVs), pose a major global health burden, causing diseases that range from mild upper respiratory tract infections to severe conditions such as pneumonia, bronchiolitis, and acute respiratory distress syndrome. These infections result in substantial morbidity and mortality worldwide, particularly among young children, the elderly, and immunocompromised individuals. In addition to their health impact, they impose significant economic costs through medical care, lost productivity, and public health interventions. The emergence of SARS-CoV-2 has underscored the pandemic potential of respiratory viruses beyond IAVs, and highlighted the importance of understanding their fundamental biology, using optimal 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 more accurately replicate the structure and function of the human respiratory tract than conventional cell lines, supporting epithelial differentiation and mucus secretion, thus providing a physiologically relevant system to study (early) virus-host interactions at the airway surface.

To establish infection, respiratory viruses must first traverse the mucus layer to reach target epithelial cells, 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 a new host species with a different sialoglycome (reviewed in Chapter 2).

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

Although mucus is recognised as a barrier to infection, the influence of its composition and inter-donor variation on antiviral function remains poorly defined. In Chapter 4, we characterised HAE ALI culture-derived mucus from different anatomical sites and donors by glycomic and proteomic profiling, alongside IAV binding and infection assays. The 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 — consistent with observations in Chapter 3. These findings highlight the complex relationship between mucus composition and antiviral efficacy, warranting further investigation.

In Chapter 5, we compared HAE ALI models derived from primary nasal epithelial cells with those generated from two 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 MDCK-II cells showed distinct replication patterns in HAE ALI cultures. IAVs with α2–6-linked sialoglycan preference replicated more efficiently than those preferring α2–3, a trend mirrored in swine airway epithelial ALI cultures. Clinical isolates of HCoV-OC43 replicated more robustly than a laboratory-adapted Paris strain, with highest titres in the HNEC model. SARS-CoV-2 replication patterns varied between models: Omicron replicated best in BCi-NS1.1 cultures, while the Wuhan strain favoured hSABCi-NS1.1. These results underscore both the utility of immortalised progenitor-derived ALI 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 use of HAE ALI cultures in respiratory virus research. Future work should explore the HA–NA functional balance in greater depth and expand mucus characterisation, virus binding, and infection-inhibition assays to airway epithelial ALI cultures from multiple species. Combining these approaches, future studies can advance our understanding of virus adaptation and the role of mucus in host specificity. This work not only advances our fundamental knowledge of viral-host interactions but also supports the development of improved strategies to predict, prevent, and mitigate respiratory viral infections.

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