Five decades of human H1N1 evolution reveal conserved glycan receptor specificity
H1N1 influenza viruses have circulated continuously among human populations for five decades. During evolution, the hemagglutinin has accumulated amino acid substitutions in and around the receptor-binding site (RBS), mainly due to antigenic pressure, potentially altering receptor recognition and binding profiles. Here, we investigated the glycan-binding properties of H1N1 strains collected from 1977 through 2024 using ELISA, flow cytometry, and tissue histochemistry to characterize changes in receptor-binding specificity over time. Across the strains examined, we observe a recurring preference for binding 2,6-sialylated di-LacNAc N-glycans. Binding to 2,3-sialylated glycans was generally undetectable, although A/Hongkong/117/1977 and A/New Caledonia/20/1999 bind chicken tracheal tissues. Interestingly, post-pandemic H1N1 viruses fluctuate in their relative binding avidities. The responsible amino acids, in turn, have different effects in pre-pandemic HA proteins. We also report a classical egg-adapted H1N1 mutant that shows null binding across platforms. These findings highlight the contribution of RBS mutations during initial adaptation and evolution of human H1N1 influenza viruses to glycan-binding properties, which, in contrast to H3N2 viruses, never fail to bind glycans that correlate with avian erythrocyte hemagglutination.