While geographic distance often restricts the spread of pathogens via hosts, this barrier may be compromised when host species are mobile. Migratory waterfowl in the Order Anseriformes are important reservoir hosts for diverse populations of avian-origin influenza A viruses (AIVs) and are assumed to spread AIVs during their annual continental-scale migrations. However, support for this hypothesis is limited and rarely tested using data from comprehensive surveillance efforts incorporating both the temporal and spatial aspects of host migratory patterns. Over three autumn migratory seasons we conducted intensive AIV surveillance in waterfowl using the North American Mississippi Migratory Flyway (MMF). Viral isolates (n=297) from multiple host species were sequenced and analyzed for patterns of gene dispersal between northern staging and southern wintering locations. Using a phylogenetic and nucleotide identity framework, we observed a greater amount of gene dispersal within rather than between the other three longitudinally identified North American flyways. Across seasons, we observed patterns of regional persistence of diversity for each genomic segment along with the limited survival of dispersed AIV gene lineages. Reassortment increased with both time and distance resulting in transient AIV constellations. This study shows that within the MMF,AIV gene flow favors spread along the migratory corridor within a season and that intensive surveillance during bird migration is important to identify virus dispersal on time scales relevant to pandemic responsiveness. In addition, this study indicates that comprehensive monitoring programs to capture AIV diversity are critical to provide insight into AIV evolution and ecology in a major natural reservoir.

IMPORTANCE: Migratory birds are a reservoir for antigenic and genetic diversity of influenza A viruses and are implicated in the spread of virus diversity that has contributed to previous pandemic events. Evidence for dispersal of avian-origin influenza A viruses (AIV) by migratory birds is rarely examined on temporal scales relevant to pandemic or panzootic threats. Therefore, characterizing AIV movement by hosts within a migratory season is important for implementing effective surveillance strategies. We conducted surveillance following birds along a major North American migratory route and observed that within a migratory season avian-origin AIVs rapidly reassorted and gene lineages were dispersed primarily within the migratory corridor. Patterns of regional persistence were observed across seasons for each gene segment. We show that dispersal of AIV gene lineages by migratory birds occur quickly along migratory routes and that surveillance for AIV threatening human and animal health should focus attention on these routes.