Abstract
The arrival of H5N1 clade 2.3.4.4b genotype B3.2 in South America marks an unprecedented geographic expansion of highly pathogenic avian influenza viruses. The continent remains the only region with sustained H5N1 transmission in wild marine mammals. To investigate the evolution of H5N1 in this novel context, we conducted genomic surveillance in marine wildlife along the northern Patagonian coast in Argentina (August 2023 - February 2024). Phylogenetic analyses revealed two B3.2 subclades: an avian subclade linked to poultry outbreaks in central Argentina, and a marine mammal subclade that persisted locally and repeatedly spilled back into seabirds, causing mortality in terns. Notably, most seabird viruses retained mammalian-adaptive mutations; however, one tern cluster exhibited reversion only at PB2-N701D. These findings suggest the ability of South American seabirds to sustain mammal-adapted H5N1 viruses, potentially enabling long-distance spread and establishment in novel wildlife reservoirs, thereby threatening biodiversity and increasing risks to animal and public health.