High pathogenicity avian influenza A (H5N1) viruses isolated from poultry and wild birds in Japan during the 2024–2025 season
ABSTRACT H5N1 high pathogenicity avian influenza (HPAI) viruses of clade 2.3.4.4b continue to spread globally among wild birds and poultry, and diversify through gene reassortment. Since 2024, spillovers of H5N1 viruses have been reported in dairy cattle in the United States. Given the numerous HPAI outbreaks in poultry and wild birds in Japan during the 2024–2025 season, we aimed to examine the genetic diversity, pathogenicity, and host range of these viruses. Phylogenetic analyses revealed that all viruses in the 2024–2025 season belonged to G2d and G2c subgroups in clade 2.3.4.4b and were divided into 11 genotypes (G2d: six genotypes, G2c: five genotypes). Of these, G2d-0 was the dominant genotype in both poultry and wild birds. Novel genotypes emerged through reassortment between G2d and G2c viruses. Experimental infection of chickens revealed comparable pathogenicity among genotypes, although the amounts of viral shedding were high in chickens inoculated with G2d-0 and G2c-13 viruses, which were genotypes confirmed in multiple outbreaks in the 2024–2025 season, suggesting high transmission potential. In vitro assays revealed markedly lower infectivity of Japanese isolates in bovine mammary epithelial cells compared with that of the B3.13 genotype responsible for dairy cattle outbreaks in the United States, indicating limited risk of cattle infection. Together, these findings underscore ongoing genetic diversification of HPAI viruses in G2d and G2c subgroups and highlight the importance of continued in vivo and in vitro characterization of these viruses to monitor changes in genotypes, pathogenicity, and potential risk of spillover to cattle. IMPORTANCE H5N1 high pathogenicity avian influenza (HPAI) viruses have spread in poultry and wild birds worldwide. H5N1 viruses have also spread in dairy cattle in the United States since 2024. Japan reported numerous HPAI cases during the 2024–2025 season. Herein, phylogenetic analyses using strains isolated from poultry and wild birds enabled a detailed investigation of their genetic backgrounds, which is essential for elucidating introduction routes and evolution. Investigation of pathogenicity may help predict the outbreak frequency. This study revealed that HPAI viruses of multiple genotypes had been introduced into Japan, and chickens infected with genotypes detected from continuous outbreaks exhibited high virus shedding. In addition, comparing infectivity in bovine cells revealed the risk of cattle infection. H5N1 infection had not been reported in cattle in Japan, and our results suggest that the risk remains low. These integrated analyses provide key insights for developing effective strategies against HPAI outbreaks. H5N1 high pathogenicity avian influenza (HPAI) viruses have spread in poultry and wild birds worldwide. H5N1 viruses have also spread in dairy cattle in the United States since 2024. Japan reported numerous HPAI cases during the 2024–2025 season. Herein, phylogenetic analyses using strains isolated from poultry and wild birds enabled a detailed investigation of their genetic backgrounds, which is essential for elucidating introduction routes and evolution. Investigation of pathogenicity may help predict the outbreak frequency. This study revealed that HPAI viruses of multiple genotypes had been introduced into Japan, and chickens infected with genotypes detected from continuous outbreaks exhibited high virus shedding. In addition, comparing infectivity in bovine cells revealed the risk of cattle infection. H5N1 infection had not been reported in cattle in Japan, and our results suggest that the risk remains low. These integrated analyses provide key insights for developing effective strategies against HPAI outbreaks.