H5N8 and H5N1 high pathogenicity avian influenza viruses (HPAIVs) caused outbreaks in poultry farms in Japan from November 2021 to May 2022. Hemagglutinin genes of these viruses belong to clade 2.3.4.4B and can be divided phylogenetically into the following groups: 20A, 20E, and 21E. In this study, we compared the infectivity and transmissibility of HPAIVs from three groups of chickens. Representative strains from 20A, 20E, and 21E groups are A/chicken/Akita/7C/2021(H5N8)(Akita7C), A/chicken/Kagoshima/21A6T/2021(H5N1)(Kagoshima6T), and A/chicken/Iwate/21A7T/2022(H5N1)(Iwate7T), respectively. Fifty percent lethal dose of Akita7C in chickens (10^3.83 fifty percent egg infectious dose (EID50)) was up to seven times lower than those of Kagoshima6T and Iwate7T (10^4.50 and 10^4.68 EID_50, respectively). Mean death times for Akita7C- and Kagoshima6T-infected chickens (3.45 and 3.30 days, respectively) were at least a day longer than that of Iwate7T (2.20 days). Viral titers of the trachea and cloaca of Iwate7T-infected chicken were the highest detected. The transmission rate of the Akita7C strain (100%) was markedly higher than those of the two strains (<50%). These data suggest that the infectivity and transmissibility of the Akita7C strain (H5N8) in chickens are higher than those of H5N1 viruses, providing fundamental information needed for formulating effective prevention and control strategies for HPAI outbreaks.

In winter 2021–2022, H5N1 and H5N8 high-pathogenicity avian influenza (HPAI) viruses (HPAIVs) caused serious outbreaks in Japan: 25 outbreaks of HPAI at poultry farms and 107 cases in wild birds or in the environment. Phylogenetic analyses divided H5 HPAIVs isolated in Japan in the winter of 2021–2022 into three groups—G2a, G2b, and G2d—which were disseminated at different locations and times. Full-genome sequencing analyses of these HPAIVs revealed a strong relationship of multiple genes between Japan and Siberia, suggesting that they arose from reassortment events with avian influenza viruses (AIVs) in Siberia. The results emphasize the complex of dissemination and reassortment events with the movement of migratory birds, and the importance of continual monitoring of AIVs in Japan and Siberia for early alerts to the intrusion of HPAIVs.

During the 2023–2024 winter, 11 high pathogenicity avian influenza (HPAI) outbreaks caused by clade 2.3.4.4b H5N1 and H5N6 HPAI viruses were confirmed in Japanese domestic poultry among 10 prefectures (n = 10 and 1, respectively). In this study, we aimed to genetically and pathologically characterize these viruses. Phylogenetic analysis revealed that H5N1 viruses were classified into the G2d-0 genotype, whereas the H5N6 virus was a novel genotype in Japan, designated as G2c-12. The G2c-12 virus shared PB2, PB1, PA, HA, and M genes with previous G2c viruses, but had NP and NS genes originating from avian influenza viruses in wild birds abroad. The N6 NA gene was derived from an H5N6 HPAI virus that was different from the viruses responsible for the outbreaks in Japan in 2016–2017 and 2017–2018. Experimental infections in chickens infected with H5N1(G2d-0) and H5N6(G2c-12) HPAI viruses showed no significant differences in the 50% chicken lethal dose, mean death time, or virus shedding from the trachea and cloaca, or in the histopathological findings. Different genotypes of the viruses worldwide, their introduction into the country, and their stable lethality in chickens may have triggered the four consecutive seasons of HPAI outbreaks in Japan.

During the winter of 2022/2023, Japan experienced its largest outbreak of high pathogenicity avian influenza (HPAI), affecting 84 poultry premises. In this study, we investigated the pathogenicity and antigenicity of A/chicken/Kagoshima/22A1T/2022 (Kagoshima/22A1T), a clade 2.3.4.4b H5N1 virus belonging to the G2b group. It was isolated from a poultry farm in Izumi City, where the largest number of consecutive cases was recorded. The 50% lethal dose, mean death time (MDT), amount of virus shed, and transmissibility in chickens of Kagoshima/22A1T were similar to those of A/chicken/Kagoshima/21A6T/2022 (Kagoshima/21A6T), the previous season’s isolate of the same group, indicating that their pathogenicities were comparable. However, the antigenicity of these isolates differed according to the hemagglutination inhibition (HI) test results. We found that the amino acid substitutions in residues 189 and 193, corresponding to antigenic site B in the H3 virus of the HA1 subunit, could have an impact on the HI cross‐reactivity of Kagoshima/21A6T. This study provides important insights into the factors contributing to the consecutive HPAI outbreaks on poultry farms in Izumi City during the 2022/2023 season and the prediction of antigenic changes in G2b group HPAI viruses.

Migratory water birds are considered to be carriers of high pathogenicity avian influenza viruses (HPAIVs). In Japan, mallards are often observed during winter, and HPAIV-infected mallards often shed viruses asymptomatically. In this study, we focused on mallards as potential carriers of HPAIVs and investigated whether individual wild mallards are repeatedly infected with HPAIVs and act as HPAIV carriers multiple times within a season. Mallards were experimentally infected with H5N1 and H5N8 HPAIVs that were isolated recently in Japan and phylogenetically belong to different hemagglutinin groups (G2a, G2b, and G2d). All of these strains are more infectious to mallards than to chickens, and the infected mallards shed enough virus to infect others, regardless of whether they exhibited clinical signs. Serum antibodies to the homologous antigen, induced by a single infection with a low virus dose (10 times the 50% mallard infectious dose), were maintained at detectable levels for 84 days. Immunity at 84 days post-inoculation fully protected the mallards from a challenge with the homologous strain, as demonstrated by a lack of viral shedding, and antibody levels did not increase significantly in most of these birds. Protection against heterologous challenge was also observed despite undetectable levels of antibodies to the challenge strain. Our findings suggest that repeated infections with homologous and heterologous HPAIV strains do not occur frequently in individual wild mallards within a season, particularly at low viral doses, and the frequency with which they act as carriers may be limited.