Introduction: The unpredictable evolution of avian influenza viruses (AIVs) presents an ongoing threat to agricultural production and public and wildlife health. Severe outbreaks of highly pathogenic H5N1 viruses in US poultry and wild birds since 2022 highlight the urgent need to understand the changing ecology of AIV. Surveillance of gulls in marine coastal environments has intensified in recent years to learn how their long-range pelagic movements potentially facilitate inter-hemispheric AIV movements. In contrast, little is known about inland gulls and their role in AIV spillover, maintenance, and long-range dissemination. Methods: To address this gap, we conducted active AIV surveillance in ring-billed gulls (Larus delawarensis) and Franklin's gulls (Leucophaeus pipixcan) in Minnesota's natural freshwater lakes during the summer breeding season and in landfills during fall migration (1,686 samples). Results: Whole-genome AIV sequences obtained from 40 individuals revealed three-lineage reassortants with a mix of genome segments from the avian Americas lineage, avian Eurasian lineage, and a global "Gull" lineage that diverged more than 50 years ago from the rest of the AIV global gene pool. No poultry viruses contained gull-adapted H13, NP, or NS genes, pointing to limited spillover. Geolocators traced gull migration routes across multiple North American flyways, explaining how inland gulls imported diverse AIV lineages from distant locations. Migration patterns were highly varied and deviated far from assumed "textbook" routes. Discussion: Viruses circulating in Minnesota gulls during the summer breeding season in freshwater environments reappeared in autumn landfills, evidence of AIV persistence in gulls between seasons and transmission between habitats. Going forward, wider adoption of technological advances in animal tracking devices and genetic sequencing is needed to expand AIV surveillance in understudied hosts and habitats.
Keywords: franklin's gull; freshwater; influenza; migration; minnesota; ring-billed gull.
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