Characterization and Genetic Evolution of H6N2 Subtype AIV Isolates from Aquatic Birds

Birds serve as the primary natural reservoirs for avian influenza viruses (AIVs), harboring nearly all known AIV subtypes. The seasonal migratory movements of wild birds play a significant role in the transmission and dissemination of AIVs. Jianhu Lake in Dali, Yunnan Province, serves as a vital congregation point along avian migratory routes, providing an ideal habitat for birds. In this study, a total of 619 avian samples were collected from the Jianhu area, from which four H6N2 subtype AIV strains were successfully isolated. Among these, A/grey heron/Jianhu/JH-89/2024 (hereafter referred to as JH-89) and A/grey heron/Jianhu/JH-91/2024 (JH-91) were isolated from grey herons (Ardea cinerea); A/mareca penelope/Jianhu/JH-2-11/2025 (JH-2-11) from a Eurasian wigeon (Mareca penelope); and A/duck/Jianhu/JH-1-1/2025 (JH-1-1) from a domestic duck (Anas platyrhynchos domesticus). Genomic analyses revealed that these four H6N2 isolates belong to the Eurasian lineage, with all eight gene segments originating from complex reassortment events among diverse Asian isolates. In vitro assays demonstrated that the representative strain JH-2-11 replicated efficiently in various human- and animal-derived cell lines. In vivo infection models revealed that, without prior adaptation, the JH-2-11 strain successfully infected BALB/c mice, resulting in suppressed body weight gain and severe pathological lesions in the respiratory tract (nasal turbinates, trachea, and lungs), without causing mortality or extrapulmonary dissemination. Collectively, although these H6N2 viruses evolve primarily within avian hosts, they exhibit potential for mammalian adaptation and require continuous epidemiological monitoring.