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.

The fowl glioma-inducing virus prototype (FGVp) and its variants, which belong to avian leukosis virus subgroup A (ALV-A), induce cardiomyocyte abnormalities and gliomas in chickens. However, the molecular mechanisms underlying these myocardial changes remain unclear, and ALV-induced tumorigenesis, which is caused by proviral insertional mutagenesis, does not explain the early development of cardiac changes in infected chickens. We established a quantitative PCR (qPCR) assay to measure ALV-A proviral loads in the brains and hearts of FGV-infected Japanese bantam chickens and compared these results with morphologic lesions. Four of 22 bantams had both gliomas and cardiac lesions. Hearts with cardiac lesions had a higher proviral load (10.3 ± 2.7 proviral copies/nucleus) than those without cardiac lesions (0.4 ± 0.4), suggesting that the proviral load in hearts is correlated with the frequency of myocardial changes. Our qPCR method may be useful in the study of ALV-induced cardiomyocyte abnormalities.

We previously described cardiomyocyte abnormality caused by Km_5666 strain, a variant of fowl glioma-inducing virus (FGV) prototype, which is an avian leukosis virus (ALV). However, the cardiac involvement appeared to be eradicated from the flock after a few years. An epidemiological survey from 2017 to 2020 was performed to elucidate the current prevalence of the cardiopathogenic strains in this flock. Four of the 71 bantams pathologically examined showed both glioma and cardiomyocyte abnormality, from which three ALV strains were detected. DNA sequencing revealed that several different ALV strains coexisted in each bantam and that the conserved Km_5666 virus fluid also contained at least two different ALV strains. We generated three infectious molecular clones from these samples, named KmN_77_clone_A, KmN_77_clone_B, and Km_5666_clone. The envSU of KmN_77_clone_A shared high sequence identity with that of Km_5666 (94.1%). In contrast, the envSU of KmN_77_clone_B showed >99.2% nucleotide similarity with that of an FGV variant without cardiopathogenicity. Furthermore, Km_5666_clone experimentally reproduced both gliomas and cardiomyocyte abnormality in chickens. From these results, it is suggested that the pathogenic determinant of cardiomyocyte abnormality is located in envSU similar to that of Km_5666. The cloning technique described here is beneficial for evaluating the viral pathogenicity in cases where affected birds are coinfected with several different ALV strains.

Calving difficulty may lead to traumatic peripheral nerve injury. A male, 8-mo-old, Japanese Black calf with a history of secondary dystocia as a result of fetal gigantism had lameness and gait disturbance. At autopsy, multifocal dural thickening with adhesions to the adjacent spinal cord was observed at T12–13 and L4–5 vertebral levels. Microscopically, numerous traumatic neuroma-like fascicles of nerve twigs were embedded in the dura mater with abundant collagenous connective tissue. By immunohistochemistry, axons and Schwann cells were confirmed in each nerve fascicle. Our observations suggest that avulsion injuries in the preganglionic fibers of the spinal nerve roots, and secondary spinal cord compression, resulted in the development of neurologic signs.