Early detection and genetic characterization of clade 2.3.4.4b H5N1 and H5N9 highly pathogenic avian influenza viruses at the onset of fall migration in wild birds during october 2025 in South Korea.

Highly pathogenic avian influenza (HPAI) viruses of clade 2.3.4.4b continue to diversify through reassortment with co-circulating low-pathogenic avian influenza (LPAI) viruses and are repeatedly introduced into South Korea via migratory flyways. During national wild bird surveillance in October 2025, two HPAI viruses of different subtypes, H5N1 and H5N9, were detected in Common teals in the southwestern Korea. Whole-genome sequencing confirmed both isolates as clade 2.3.4.4b viruses belonging to the G2c sub-lineage. Phylogenetic analysis showed that the H5N1 virus possessed a genomic backbone related to the 22G4 genotype circulating in Korea during the 2022-2023 season, incorporating a PB1 segment derived from LPAI viruses. The H5N9 virus represented a distinct reassortant carrying an NA gene closely related to H11N9 LPAI viruses and internal segments associated with KorD and KorC genotypes prevalent in the same season. Bayesian time-scaled analysis indicated that both isolates originated from an East Asian H5Nx lineage with a common ancestor around 2019, and that the H5N1 virus diverged from a closely related Chinese strain in late 2023. Both viruses harbored multiple mammalian-adaptation markers, including substitutions commonly detected in recent East Asian HPAI strains. These findings demonstrate ongoing inter-lineage reassortment between regional HPAI and LPAI gene pools, emphasizing the continued role of migratory waterfowl in introducing emerging variants into Korea. The early-season detection of genetically distinct reassortants highlights the importance of sustained wild bird surveillance, rapid genomic characterization, and international data sharing to track the evolution and spread of newly emerging HPAI lineages.