Kei Nabeshima

We established primary fibroblast cultures from a Japanese raccoon dog (Nyctereutes viverrinus) and a Japanese red fox (Vulpes vulpes japonica) and generated highly contiguous reference genome assemblies using Oxford Nanopore Technologies PromethION long-read sequencing. The Japanese raccoon dog assembly spanned 2.69 Gb in 813 scaffolds, with a scaffold N50 of 52 Mb and a Benchmarking Universal Single-Copy Orthologs completeness score of 98.2%. The Japanese red fox assembly spanned 2.47 Gb in 903 scaffolds, with a scaffold N50 of 139 Mb and a Benchmarking Universal Single-Copy Orthologs completeness score of 97.5%. Phylogenomic analysis placed the Japanese raccoon dog in a lineage distinct from the continental raccoon dog, supporting its evolutionary differentiation within Nyctereutes. The Japanese red fox formed a distinct lineage within the red fox clade, consistent with its recognized regional differentiation. These genome assemblies and associated fibroblast cultures provide resources for studies of canid systematics, population history, local adaptation, comparative genome evolution, and conservation genetics.

Environmental surveillance of antimicrobial resistance (AMR) in wildlife remains limited, despite increasing recognition that resistance determinants can circulate across human, livestock, and natural ecosystems. Migratory waterbirds move long distances and aggregate at shared stopover and wintering sites, potentially facilitating the acquisition and redistribution of antimicrobial resistance genes (ARGs) across regions. However, nationwide evidence describing the breadth of ARGs carried by winter migratory birds in Japan is scarce. We assessed the diversity and distribution of ARGs in pooled fecal samples from winter migratory birds across Japan. We analyzed pooled fecal DNA collected at migratory bird habitats across 12 local governments during the 2021-2022 and 2022-2023 winter seasons (24 pools). Avian host origin was inferred by DNA metabarcoding, and ARGs were profiled by probe-based target enrichment with read-based detection (ARG detected at ≥ 10 reads). Ducks (Anas spp. and Mareca spp.) were the predominant inferred hosts. ARGs were detected in all areas and included genes associated with resistance to multiple antibiotic classes used in livestock production. Across the two seasons, genes associated with resistance to gentamicin, cephalosporins, macrolides, tetracyclines, fosfomycin, clindamycin, penicillins, streptogramins, sulfonamides/trimethoprim, colistin, erythromycin, chloramphenicol, rifampicin, and isoniazid were detected in all 12 areas in at least one season. Genes associated with resistance to agents restricted for use in Japanese livestock production, including colistin, erythromycin, chloramphenicol, and rifampicin, were also detected in all 24 pools. Isoniazid-, gentamicin-, meropenem-, and tigecycline-associated genes were detected in 23/24, 20/24, 11/24, and 9/24 pools, respectively. These data indicate widespread environmental occurrence of diverse ARGs and support the possibility that migratory birds could contribute to long-distance dispersal of ARGs. Culture-based isolation, phenotypic testing, and quantitative analyses will be needed to identify host bacteria and assess clinical relevance.

The Bonin flying fox (Pteropus pselaphon) is an endemic species of the Ogasawara Islands, Japan, and is classified as Critically Endangered. Here, we report a genome assembly of P. pselaphon, constructed using Nanopore long reads and Omni-C scaffolding. The final assembly spans 2.16 Gb across 929 scaffolds and achieves a Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness score of C:98.2% [S:94.3%, D:4.0%], F:0.7%, M:1.0%. Gene prediction with Helixer software identified 20,647 protein-coding sequences, with a BUSCO score of C:92.2% [S:88.9%, D:3.3%], F:3.3%, M:4.5%. This genome provides a valuable resource for the conservation management of this endangered species, as well as for evolutionary genomics and studies of host–pathogen interactions in flying fox.

Genetic and antigenic analyses were performed on highly pathogenic avian influenza viruses (HPAIVs) isolated in Hokkaido, northern Japan, during the winter of 2024–2025. Ninety-eight HPAIVs were isolated from feces of waterfowl, tracheal swabs from dead wild birds, or lung homogenates from dead chickens. Phylogenetic analysis of the hemagglutinin (HA) gene from 47 representative isolates revealed that all sequences belonged to the G2d subgroup of clade 2.3.4.4b H5HA, which has been the dominant lineage in Hokkaido since the winter of 2021–2022. These isolates were further divided into three major groups within the subgroup. The HPAIVs isolated in the Republic of Korea, China, and North America were genetically closely related to the Hokkaido isolates, whereas no HPAIVs genetically related to European strains or those detected in North American cattle were identified. Furthermore, HPAIVs isolated from seabirds were genetically closely related to those found in dead marine mammals along the eastern coast of Hokkaido in the spring of 2025. No apparent antigenic differences were observed between the HPAIVs isolated in this study and those from previous seasons. These findings highlight the wide distribution of HPAIVs in Hokkaido, particularly from Asian and North American lineages, and underscore the importance of continuous surveillance.

In the fall of 2022, high pathogenicity avian influenza viruses (HPAIVs) were detected from raptors and geese in Japan, a month earlier than in past years, indicating a shift in detection patterns. In this study, we conducted a phylogenetic analysis on H5N1 HPAIVs detected from six wild birds during the 2022/2023 season to determine their genetic origins. Our findings revealed that these HPAIVs belong to the G2 group within clade 2.3.4.4b, with all isolates classified into three subgroups: G2b, G2d, and G2c. The genetic background of the G2b virus (a peregrine falcon-derived strain) and G2d viruses (two raptors and two geese-derived strains) were the same as those detected in Japan in the 2021/2022 season. Since no HPAI cases were reported in Japan during the summer of 2022, it is probable that migratory birds reintroduced the G2b and G2d viruses. Conversely, the G2c virus (a raptor-derived strain) was first recognized in Japan in the fall of 2022. This strain might share a common ancestor with HPAIVs from Asia and West Siberia observed in the 2021/2022 season. The early migration of waterfowl to Japan in the fall of 2022 could have facilitated the early invasion of HPAIVs.

Many high pathogenicity avian influenza (HPAI) cases in wild birds due to H5N1 HPAI virus (HPAIV) infection were reported in northern Japan in the winter of 2021–2022. To investigate the epidemiology of HPAIVs brought to Japan from surrounding areas, a genetic analysis of H5 HPAIVs isolated in northern Japan was performed, and the pathogenicity of the HPAIV in chickens was assessed by experimental infection. Based on the genetic analysis of the hemagglutinin gene, pathogenic viruses detected in northern Japan as well as one in Sakhalin, the eastern part of Russia, were classified into the same subgroup as viruses prevalent in Europe in the same season but distinct from those circulating in Asia in winter 2020–2021. High identities of all eight segment sequences of A/crow/Hokkaido/0103B065/2022 (H5N1) (Crow/Hok), the representative isolates in northern Japan in 2022, to European isolates in the same season could also certify the unlikeliness of causing gene reassortment between H5 HPAIVs and viruses locally circulating in Asia. According to intranasal challenge results in six-week-old chickens, 50% of the chicken-lethal dose of Crow/Hok was calculated as 10^4.5 times of the 50% egg-infectious dose. These results demonstrated that the currently prevalent H5 HPAIVs could spread widely from certain origins throughout the Eurasian continent, including Europe and the Far East, and implied a possibility that contagious viruses are gathered in lakes in the northern territory via bird migration. Active monitoring of wild birds at the global level is essential to estimate the geographical source and spread dynamics of HPAIVs.