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Environmental water‐targeted surveillance of migratory aquatic birds at overwintering sites is potentially one of the most effective approaches for understanding the ecology of avian influenza viruses (AIVs). In this study, we improved the method for AIV isolation from environmental water samples by making a minor modification to our previously reported process. We experimentally demonstrated that the AIV recovery efficiency of the modified method was 10–100‐fold higher than that of the original method. This improved isolation method allowed us to isolate a considerably larger number of AIV isolates from environmental water samples collected at an overwintering site for tens of thousands of migratory aquatic birds in Japan during the 2018/2019 winter season, compared with those during previous winter seasons. Genetic and phylogenetic analyses revealed that AIVs of the same subtypes with multiple genetic constellations were circulating in a single overwintering site during a single winter season. These findings indicate that our improved isolation method contributes to enhance environmental water‐targeted surveillance and to a better understanding of AIV ecology in migratory aquatic bird populations by monitoring ongoing AIV circulation. [ABSTRACT FROM AUTHOR]

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site for migratory ducks and endangered cranes. We have surveyed avian influenza viruses (AIVs) in this area since 2012 and isolated low-pathogenic AIVs (LPAIVs) of various subtypes every winter season. H3N8 LPAIVs were isolated during the 2012/13 and 2016/17 seasons, and H4N6 LPAIVs were isolated during the 2012/13 and 2013/14 seasons. In the 2017/18 season, one H3N8 and two H4N6 LPAIV strains were isolated from environmental water samples. Genetic and phylogenetic analysis for each gene segment from these H3N8 and H4N6 LPAIVs suggested that our isolates were genetic reassortants generated by intermixing between AIVs circulating not only in Eurasia but also in Africa and/or North America. Comparison of the genetic constellations of our three isolates with their counterparts isolated during previous seasons from the Izumi plain revealed a drastic transition in the genetic constellations of both subtypes. These findings emphasize the importance of continuous surveillance of AIVs on the Izumi plain. [ABSTRACT FROM AUTHOR]