Isolation and genetic characterization of multiple genotypes of both H5 and H7 avian influenza viruses from environmental water in the Izumi plain, Kagoshima prefecture, Japan during the 2021/22 winter season.

In the 2021/22 winter, one H5N1 and nine H5N8 high pathogenicity avian influenza viruses (HPAIVs) of clade 2.3.3.4b were isolated from the water in crane roosts on the Izumi plain, Japan. Additionally, we isolated low pathogenicity avian influenza viruses (LPAIVs) of five subtypes: H1N1, H4N2, H4N6, H7N7, and H10N4. H5N8 HPAIVs belonging to the G2a group were isolated throughout winter, whereas H5N1 HPAIV belonging to the G2b group were isolated only in early winter. These findings suggest co-circulation of both G2a and G2b HPAIVs in early winter. Although two H7N7 LPAIVs were isolated from cranes' roost water collected on the same day, the gene constellations of the two isolates were clearly different, indicating the contemporary invasion of at least two different genotypes of H7N7 LPAIVs in the Izumi plain. This study underscores the importance of monitoring both HPAIVs and LPAIVs to understand avian influenza virus ecology in migratory waterfowl populations.

Newly emerged genotypes of highly pathogenic H5N8 avian influenza viruses in Kagoshima prefecture, Japan during winter 2020/21.

During the 2020/21 winter season, 29 and 10 H5N8 high pathogenicity avian influenza viruses (HPAIVs) were isolated from environmental water and wild birds, respectively, in Kagoshima prefecture, Japan. Furthermore, seven subtypes of low pathogenicity avian influenza viruses (LPAIVs) were also isolated; H1N1, H2N9, H3N2, H3N6, H3N8, H4N6, and H6N6 subtypes. While the H5 hemagglutinin (HA) genes of the G1 cluster were isolated throughout the winter season, those of the G2 cluster were also detected in late winter, suggesting that H5 HPAIVs possessing H5 HA genes from the two different clusters were individually introduced into Kagoshima prefecture. Intriguingly, genetic constellations revealed that the H5N8 HPAIVs could be classified into six genotypes, including four previously reported genotypes (E1, E2, E3, and E7), and two new genotypes (tentatively named E8 and E9). The PB1 and PA gene segments of genotypes E8 and E9 shared high similarity with those of LPAIVs, whereas the remaining gene segments were close to those of genotype E1. Furthermore, LPAIVs whose PA gene segment was close to that of genotype E9 were isolated from the environmental water. Overall, we revealed that various HPAIV genotypes circulated in Kagoshima prefecture during the 2020/21 winter season. This study highlights the importance of monitoring both HPAIV and LPAIV to better understand AIV ecology in migratory waterfowl populations.

Prevalence and organ tropism of crane-associated adenovirus 1 in cranes overwintering on the Izumi plain, Japan.

Crane-associated adenovirus 1 (CrAdV-1) is a proposed novel virus in the genus Aviadenovirus, first detected in faecal samples from hooded cranes (a vulnerable crane species) on the Izumi plain, a major overwintering site for migratory cranes in Japan. CrAdV-1 was genetically characterized in that study; however, its virological characteristics remain largely unclear. To investigate the prevalence and organ tropism of CrAdV-1, we collected swab and organ samples from dead or debilitated cranes on the Izumi plain. CrAdV-1 gene was detected in 47% (45/95) of tested cranes, comprising mainly hooded cranes but also white-naped and sandhill cranes. These results indicate that CrAdV-1 shedding is widespread among cranes overwintering on the Izumi plain. Phylogenetic analyses revealed that the 68 nucleotide sequences determined from the positive swabs formed a single cluster, suggesting phylogenetic differences between CrAdV-1 and other aviadenoviruses. CrAdV-1 prevalence showed a significant linear increase with time through the overwintering period (November to February), especially among juveniles. These findings indicate that CrAdV-1 spreads mainly by transmission between juveniles progressively through the overwintering period. The CrAdV-1 gene-positive rate was significantly higher in cloacal swabs than conjunctival or tracheal swabs. Copy numbers for the partial CrAdV-1 gene sequence were markedly high in the colon samples from three of the four cranes investigated for organ tropism. We also detected relatively high copy numbers in the cerebrum, trachea, lung and heart, suggesting that CrAdV-1 mainly targets these four organs and transmitted via the faecal-oral route and airborne transmission. These results contribute to further understanding of the virological characteristics of CrAdV-1.

Genetically Diverse Highly Pathogenic Avian Influenza A(H5N1/H5N8) Viruses among Wild Waterfowl and Domestic Poultry, Japan, 2021.

Genetic analyses of highly pathogenic avian influenza H5 subtype viruses isolated from the Izumi Plain, Japan, revealed cocirculation of 2 genetic groups of clade 2.3.4.4b viruses among migratory waterfowl. Our findings demonstrate that both continuous surveillance and timely information sharing of avian influenza viruses are valuable for rapid risk assessment.

Chondrosarcoma with undifferentiated neoplastic cell proliferation around the distal tibiotarsus bone in a wild Hooded Crane (Grus monacha).

An adult male Hooded Crane was found dead on the Izumi plane. At autopsy, subcutaneous nodules were found around the medial and lateral sides of the left distal tibiotarsus bone. The largest cross-section of the masses revealed a multilobular pattern, with small amounts of viscous mucus. Histopathologically, the nodules were composed of three types of neoplastic cells: chondrocytic cells with abundant lightly basophilic cartilaginous matrices, mesenchymal cells and a small portion of the neoplastic tissue consisted of undifferentiated neoplastic cells exhibiting a high mitotic count and frequent multinucleation. This is the first case of a chondrosarcoma including undifferentiated neoplastic cell proliferation in a wild Hooded Crane.

Phylogenetic variations of highly pathogenic H5N6 avian influenza viruses isolated from wild birds in the Izumi plain, Japan, during the 2016-17 winter season.

During the 2016-2017 winter season, we isolated 33 highly pathogenic avian influenza viruses (HPAIVs) of H5N6 subtype and three low pathogenic avian influenza viruses (LPAIVs) from debilitated or dead wild birds, duck faeces, and environmental water samples collected in the Izumi plain, an overwintering site for migratory birds in Japan. Genetic analyses of the H5N6 HPAIV isolates revealed previously unreported phylogenetic variations in the PB2, PB1, PA, and NS gene segments and allowed us to propose two novel genotypes for the contemporary H5N6 HPAIVs. In addition, analysis of the four gene segments identified close phylogenetic relationships between our three LPAIV isolates and the contemporary H5N6 HPAIV isolates. Our results implied the co-circulation and co-evolution of HPAIVs and LPAIVs within the same wild bird populations, thereby highlighting the importance of avian influenza surveillance targeting not only for HPAIVs but also for LPAIVs.