Transmission of influenza A viruses (IAVs) between pigs and humans can trigger pandemics but more often cease as isolated infections without further spread in the new host species population. In Denmark, a major pig-producing country, the first two detections of human infections with swine-like IAVs were reported during 2021. These zoonotic IAVs were reassortants of the H1N1 pandemic 2009 lineage (“H1N1pdm09”, H1 lineage 1A, clade 1A.3.3.2) introduced to swine farms in Denmark through humans approximately 11 years prior. However, predicting the likelihood and outcome of such IAV spillovers is challenging without a better understanding of the viral determinants.

This study traced the evolution of H1N1pdm09 from 207 sequenced genomes as the virus propagated across Danish swine farms over a decade. H1N1pdm09 diverged into several genetically distinct viral populations, largely prompted by reassortments with NA segments from other enzootic IAV lineages. The genomic segments encoding the viral envelope glycoproteins, HA and NA, evolved at the fastest rates, while the M and NS genomic segments were among the lowest evolutionary rates. The two zoonotic IAVs emerged from separate viral populations and shared the highest number of amino acid mutations in the PB2 and HA proteins. Acquisition of additional predicted glycosylation sites on the HA proteins of the zoonotic IAVs may have facilitated infection of the human patients. Ultimately, the analysis provides a foundation from which to further explore viral genetic indicators of host adaptation and zoonotic risk.