Abstract
A human infection with clade 2.3.4.4b H5N1 influenza A virus in Canada revealed minority variants E190D and Q226H in the hemagglutinin (HA) receptor-binding site (RBS). Because mutations at positions 190 and 226 have been associated with altered receptor specificity in other influenza subtypes, we investigated their impact on receptor binding in H5 HA. Using a recombinant protein approach and an ELISA-based glycan-binding assay, we assessed binding to representative avian- and human-type sialylated glycans. Both single mutations and their combination resulted in a complete loss of detectable binding to the tested glycans. To evaluate whether this phenotype was background-dependent, Q226H was additionally introduced into two other H5 HA proteins, each representing a distinct clade. In both cases, the mutation similarly abolished receptor binding. These findings independently validate recent glycan microarray observations and demonstrate that the patient-derived E190D and Q226H substitutions severely impair receptor-binding capacity across multiple H5 backgrounds. Single mutations at key RBS residues in H5 often disrupt receptor binding rather than confer human-type receptor specificity, confirming complex mutational pathways required for adaptation to human-type receptors.