Standing HA phenotypic breadth shapes H5N1 cross-host potential

Linking genetic variation to functional phenotype remains a major barrier to assessing the cross-host potential of emerging viruses. Here, we reconstruct the evolution of predicted hemagglutinin (HA) phenotypic traits across ~13,000 highly pathogenic avian influenza A H5N1 clade 2.3.4.4b viruses circulating in North America. The wide geographic spread of avian influenza within North America triggered a wave of broad HA phenotypic diversity that was later refined by the selective sweeps in avian hosts. Following establishment in dairy cattle, however, viral populations exhibited renewed phenotypic diversification in HA, including increased permissiveness for $\alpha 2,6$ -linked sialoside engagement despite high conservation of the receptor-binding domain. These patterns indicate that cattle-associated HA phenotypes can draw from standing predicted HA phenotypic breadth within circulating viral populations while continuing to diversify during cattle-associated circulation, rather than following only a simple stepwise adaptive path through canonical receptor-binding substitutions. By linking viral sequence variation to predicted protein properties across naturally evolving populations, this framework provides a scalable strategy for prioritizing H5N1 variants with cross-host-relevant features for targeted surveillance and experimental follow-up.