Abbey J. McMurray, Queen's University

Abbey J. McMurray1, Che C. Colpitts1 1Department of Biomedical and Molecular Sciences, Queen's University, Kingston

Breaking & Entering

Highly pathogenic avian influenza (HPAI) subtype H5N1 causes widespread avian mortality, and recent spillover events with sustained mammal-to-mammal transmission have heightened pandemic concern. With ~50% reported mortality among human cases, defining mechanisms underlying mammalian host adaptation is urgent. Influenza A virus (IAV) entry is mediated by hemagglutinin (HA) binding to sialic acid (SA). Notably, avian IAVs preferentially bind α2,3-linked SA, whereas human IAVs favour α2,6-linkages. Shifts in binding preference toward α2,6-linked SA is critical to the emergence of human H5N1 infections. Here, we investigated the role of SA, and the functional requirement for α2,3- and α2,6-linked SA, during H5N1 entry into A549 human lung epithelial cells. Using a lentiviral pseudovirus model, we evaluated entry of the progenitor strain (A/goose/Guangdong/1/1996) and subsequent spillover strains, including a 2024 human isolate from British Columbia (A/BC/PHL-2032/2024). We also evaluated A/American wigeon/South Carolina/22-000345-001/2021, a contemporary North American avian strain. Pseudovirus entry was quantified by luciferase reporter activity. Recent avian and human H5N1 strains exhibited enhanced entry into A549 cells relative to earlier isolates. To assess SA dependence, we enzymatically removed surface SA via sialidase treatment, and generated A549-CMAS knockout (KO) cells to globally ablate SA expression. In both contexts, H5N1 entry was reduced, but not abrogated. Ongoing studies using ST3GAL4 and ST6GAL1 KO cells to disrupt α2,3- and α2,6-linked SA synthesis, respectively, will define linkage-specific contributions to entry. Collectively, these findings suggest that sialic acid usage may not be the only factor contributing to adaptation of H5N1 for entry in mammalian cells.