Mana Esaki

Clade 2.3.4.4b H5N1 high-pathogenicity avian influenza viruses (HPAIVs) continue to circulate extensively in wild-bird populations, yet temporal changes in genetically distinct viral populations within a single overwintering season remain poorly understood. We investigated H5N1 HPAIV dynamics in the Izumi Plain, Japan, during the 2024/25 winter season by combining virological surveillance of dead or debilitated wild birds with longitudinal virus isolation from crane roost water. Tracheal and cloacal swabs from 111 wild birds and 256 water samples collected weekly from two crane roosting sites were examined, and isolated viruses were characterized by phylogenetic and whole-genome genotype analyses. Among 103 cranes tested, 64 were positive for H5N1 HPAIV, and viruses were isolated from 58 birds. H5N1 HPAIVs were also isolated from two Eurasian Wigeons and one Northern Goshawk. All H5N1 HPAIV isolates belonged to the G2c or G2d subgroup within clade 2.3.4.4b. Both subgroups circulated in wild birds and the roost environment during 2024, whereas only G2c viruses were detected after January 2025. Whole-genome analysis classified the H5N1 HPAIV isolates into six genotypes and revealed complete replacement of the circulating genotypes around the turn of the year; all isolates obtained in 2025 belonged to genotype H5N1-VI, which had not been detected in 2024. H5N1-VI was characterized by a distinct PB1 gene segment that was not detected among low-pathogenicity AIVs isolated from the same roost environment. These findings demonstrate substantial within-season changes in H5N1 HPAIV populations at a major wild-bird overwintering site and highlight the value of integrating environmental and wild-bird surveillance with whole-genome analysis to characterize temporal changes and genotype turnover throughout the wintering season.

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.

Swine influenza A virus (swIAV) is an important pathogen with regard to both the swine industry and public health. The pandemic A(H1N1) 2009 outbreak was caused by the swine-origin pandemic A(H1N1) 2009 [A(H1N1)pdm09] virus. Several reports have shown that several amino acid substitutions in the hemagglutinin (HA) antigenic sites can alter HA antigenicity. However, the impact of the amino acid deletion at position 155 on HA antigenicity remains unknown. In this study, we have isolated 11 samples of swIAVs from seven pig farms in Japan and found an amino acid deletion at position 155 of the HA region in one of the isolates of the H1N2 subtype. To examine the impact of this amino acid deletion on viral replication and HA antigenicity, we generated recombinant influenza A viruses possessing the H1 HA gene encoding either an artificial insertion or deletion of glycine at position 155. The growth kinetics of these recombinant viruses in two different cell lines demonstrated that the effect of amino acid deletion at position 155 of H1 HA on viral replication is limited. In contrast, microneutralization assay-based neutralization titers revealed that amino acid deletion significantly altered HA antigenicity. These results demonstrate that a naturally occurring amino acid deletion at position 155 in an H1 HA antigenic site can markedly alter HA antigenicity with only a limited impact on replication in vitro , highlighting the need to monitor such variants in swine populations and to assess their zoonotic potential. IMPORTANCE: Influenza A viruses circulating in pigs are of major concern because they can reduce herd productivity and sometimes infect humans. Understanding how these viruses change their surface proteins is essential for predicting their evolution and preventing outbreaks. This study focuses on swine influenza A virus isolated from pig farms in Japan and investigates the effect of a specific amino acid deletion in the hemagglutinin protein, which plays a key role in viral recognition by the host immune system. The results show that this deletion does not greatly affect virus growth but markedly alters how the immune system recognizes the virus. Such antigenic changes may allow the virus to evade existing immunity in pig populations and potentially increase the risk of transmission to humans. These findings underscore the need for continued surveillance of such variants in pigs and for future studies to evaluate their possible zoonotic implications.

Bats are natural reservoirs of numerous viruses, including members of the Rhabdoviridae family. Oita virus (OITV), classified within the genus Ledantevirus , was first isolated from the blood of a Japanese horseshoe bat ( Rhinolophus cornutus ) in the Oita Prefecture, Japan, in 1972. However, since then, no other OITV isolates have been reported. In this study, we isolated two OITV strains—OITV 321/2022 and OITV 326/2022—from oral swab samples of R. cornutus collected in the Katano Cave, Kagoshima Prefecture, Japan. Genetic analysis revealed >98% nucleotide identity with the previous isolate, OITV 296/1972. Phylogenetic analysis confirmed their classification within the Ledantevirus subgroup C. We assessed viral growth kinetics using 17 cell lines from diverse animal species, including bats, rodents, humans, and arthropods. OITV 321/2022 showed efficient replication in primate-derived, bat-derived, and human-derived cells, but not in tick or mosquito cell lines. Experimental infection of BALB/c mice demonstrated that OITV 321/2022 induced no overt clinical signs via either the intracerebral or intranasal routes. However, systemic infection and viral replication in the brain and lungs were observed by intracerebral inoculation. Intranasal inoculation with OITV 321/2022 and OITV 296/1972 resulted in transient pulmonary infection without systemic dissemination. This study provides evidence of OITV circulation in wild bat populations over 50 years, highlighting the need for continued surveillance of bat-associated rhabdoviruses in Japan. IMPORTANCE: Bats are known reservoirs of zoonotic viruses, and their proximity to humans raises concerns regarding zoonotic risks. We report the first isolation of the Oita virus (OITV), a bat-associated ledantevirus, that has circulated for over 50 years. Unlike a previous blood-derived isolate, our isolates were obtained from oral swabs, suggesting their potential for respiratory transmission. OITV could infect a wide range of mammalian cells, including human-derived cells, and induce systemic infection in mice without clinical symptoms. These findings indicate that OITV possesses a broad host tropism and may circulate among microbats through the respiratory tract. Although the pathogenicity of the newly isolated strain appears to be attenuated compared with that of a historical brain-passaged strain, its ability to replicate in human cells underscores its potential zoonotic relevance, necessitating active surveillance and functional characterization of bat-derived rhabdoviruses to better assess emerging infectious disease threats.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes significant economic losses in swine production. In May 2021 and March 2023, we detected PRRSV genes in serum samples from two weaned pigs with respiratory disorders on a farm in Japan. Partial gene sequences of these strains closely resembled those of a PRRS vaccine strain. We subsequently isolated two PRRSV field strains, KU-IG21-1 and KU-IG23-1, from the 2021 and 2023 samples, respectively. The KU-IG21-1 strain exhibited more pronounced cytopathic effects and significantly higher replication efficiency in cultured cells compared to both the vaccine and KU-IG23-1 strains. Despite these phenotypic differences, complete genome sequencing revealed high genetic similarity between the field isolates and the vaccine strain, with only 16 and 24 amino acid differences in the KU-IG21-1 and KU-IG23-1 strains, respectively. These findings suggest that the field strains likely emerged through the accumulation of point mutations in the vaccine strain rather than through homologous recombination. Furthermore, we identified three amino acid substitutions that may contribute to the enhanced replication of the KU-IG21-1 strain. This study underscores the potential impact of point mutations on PRRSV phenotypes and provides new insights into the complex evolutionary dynamics of PRRSV.

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.

Background: The accurate determination of bird sex is crucial in various biological fields, including ecology, behavioral research, and conservation. However, this task remains challenging in species in which males and females exhibit similar external morphologies, such as owls. Although polymerase chain reaction (PCR)-based molecular sexing techniques that target the chromodomain helicase DNA-binding protein 1 gene found on sex chromosomes Z (CHD1-Z gene) and W (CHD1-W gene) are widely used, we encountered atypical banding patterns when applying the previously reported primers 2550F and 2718R to four wild owls of unknown sex. This study aims to reveal the owl-specific genetic structure of the CHD1 gene. Methods: We developed a new primer set and determined the nucleotide sequences—including the binding sites for the primers 2550F and 2718R—within both the CHD1-Z and CHD1-W genes. Results: Sequencing analysis, conducted using a newly developed primer set that successfully amplified both Z- and W-derived CHD1 products across various owl species, revealed a unique genetic insertion of approximately 600 bp in intron 17 of the CHD1-W gene. This insertion reversed the usual length relationship between PCR products from the chromosomes Z and W. Additionally, mutations identified in the 2550F primer binding site of the CHD1-Z gene in certain owl species may explain the failure to amplify CHD1-Z-derived PCR products. Conclusion: These findings provide valuable insights for improving molecular sexing in owls.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), has a significant impact on the global pork industry. It results in reproductive failure in sows and respiratory issues in pigs of all ages. Despite the availability of vaccines, controlling the PRRSV remains challenging, partly owing to the limitations of cell culture systems. Current methods largely rely on primary porcine alveolar macrophages (PAMs), which must be harvested from piglets and have limited proliferative capacity. Although some simian cell lines support PRRSV replication, their inability to express porcine CD163, which is a key receptor for PRRSV entry, compromises their effectiveness, because the virus replicates differently in these non-target cells. To address these issues, we established an immortalized PAM cell line, PAM-T43, using SV40 large T antigen for immortalization and porcine serum as a culture supplement. PAM-T43 cells maintain essential macrophage functions, including CD163 expression and phagocytic activity, and exhibit high sensitivity to the PRRSV, efficiently supporting viral replication. This novel cell line offers significant potential for advancing PRRSV research, particularly in vaccine development and field strain isolation, by overcoming the limitations of current systems.

Avian influenza (AI) is an extremely contagious viral disease of domestic and wild birds that can spread rapidly among bird populations, inducing serious economic losses in the poultry industry. During the winter season 2021–2022, we isolated seventeen highly pathogenic avian influenza (HPAI) H5N8 viruses from outbreaks involving ducks in Egypt, occurring in both backyard and farm settings. The aim of this study was to pinpoint genetic key substitutions (KSs) that could heighten the risk of a human pandemic by influencing the virus’s virulence, replication ability, host specificity, susceptibility to drugs, or transmissibility. To understand their evolution, origin, and potential risks for a human pandemic, whole-genome sequencing and phylogenetic analysis were conducted. Our analysis identified numerous distinctive mutations in the Egyptian H5N8 viruses, suggesting potential enhancements in virulence, resistance to antiviral drugs, and facilitation of transmission in mammals. In this study, at least five genotypes within one genome constellation of H5N8 viruses were identified, raising concerns about the potential emergence of novel viruses with altered characteristics through reassortment between different genotypes and distinct groups. These findings underscore the role of ducks in the virus’s evolutionary process and emphasize the urgent need for enhanced biosecurity measures in domestic duck farms to mitigate pandemic risk.

Highly pathogenic avian influenza viruses (HPAIVs) have caused outbreaks in both domestic and wild birds during the winter seasons in several countries in the Northern Hemisphere, most likely because virus-infected wild ducks overwinter and serve as the primary source of infection for other birds in these countries. Several chemical disinfectants are available to deactivate these viruses outside a living organism. However, their virucidal activity is known to be compromised by various factors, including temperature and contamination with organic matter. Hence, the effectiveness of virucidal activity under winter field conditions is crucial for managing HPAIV outbreaks. To investigate the impact of the winter field conditions on the virucidal activity of disinfectants against AIVs, we assessed the stability of the virucidal activity of seven representative disinfectants that are commercially available for poultry farms in Japan against both LPAIVs and HPAIVs under cold and/or organic contamination conditions. Of the seven disinfectants examined, the ortho-dichlorobenzene/cresol-based disinfectant exhibited the most consistent virucidal activity under winter field conditions, regardless of the virus pathogenicity or subtype tested.