Within- and between-host dynamics of highly pathogenic avian influenza in domestic birds from Pennsylvania farms and live bird markets

Since late 2021, highly pathogenic H5 avian influenza viruses have spread across the Americas, resulting in hundreds of outbreaks in agriculture and 71 human spillovers. In the United States, human infections overwhelmingly derive from exposure to infected agricultural animals. However, little is known about how these viruses evolve during outbreaks in agriculture, or how they spread locally, key information for understanding human infection risk. We produced high-quality deep sequence data from commercial and live bird market (LBM) outbreaks in Pennsylvania from 2023-2025. We uncover repeated spillovers from wild to domestic birds, and evidence of high circulating diversity in LBMs that likely moved across state lines. Samples collected from the same outbreak shared a significant fraction of viral variation, suggesting high circulating diversity and a strong signal of transmission linkage. Samples from poultry collectively harbored 9 known adaptive variants, including PB2 D701N, suggesting that rare, adaptive variants may circulate in settings where human exposure risk is high. Our data suggest that outbreaks in poultry pose opportunities for subsequent viral evolution, supporting continued efforts to stamp out transmission. We also find that LBMs experience outbreak risk from domestic and wild sources and promote viral mixing between species, suggesting that enhanced tracking and biosecurity may be useful in these settings. Finally, our data suggest the potential to use within-sample variation for enhanced outbreak linkage within these outbreak settings.