High pathogenicity avian influenza (HPAI) has impacted poultry and wild birds globally. The number of H5 HPAI virus (HPAIV) infection cases in wild birds in Hokkaido (Northern Japan) was high in the last two seasons, contributing to virus spillover to resident birds and poultry. Therefore, H5 HPAIVs in birds and mammals in Hokkaido in winter 2022–2023 and 2023–2024 were monitored and viruses were phylogenetically, antigenically, and pathogenetically characterized. Thirty HPAIV isolates were subtyped and pathotyped by sequencing the hemagglutinin (HA) gene of viruses. Phylogenetic analysis of the HA gene revealed that all isolated HPAIVs were categorized into clade 2.3.4.4b and divided into three groups (G2b, G2c, and G2d). Most isolates belonging to subgroup G2d clustered with isolates in winter 2021–2022 in Hokkaido. The other isolates were categorized into two subgroups, G2b and G2c, mainly composed of isolates in Honshu Island in winter 2021–2022 and 2022–2023, respectively. Two H5 HPAIVs isolated in Eastern Russia in spring and autumn 2022 were genetically close to most Hokkaido isolates (G2d), and a virus isolated in Hokkaido in November 2023 was also grouped in subgroup G2d. Further analysis of all eight gene segments identified six types of gene constellations. Cross‐hemagglutination inhibition test indicated that the antigenicity of H5 HPAIVs isolated in the last several seasons was similar within them but slightly different from that in the 2010s. Three chicken breeds were intranasally challenged with four representative isolates to assess their pathogenicity. All chickens except one broiler chicken were dead until 5‐day postchallenge with different pathogenicity of these viruses. The pathogenicity of one HPAIV strain was significantly lower in broiler chickens than in layer chickens. The mixture of multiple characteristics of HPAIVs in Hokkaido was confirmed by bird migration routes. Thus, many HPAIVs can be brought and scattered anywhere on Earth.

Influenza A viruses with fewer amino acids in the neuraminidase (NA) stalk domain are primarily isolated from chickens rather than wild ducks, indicating that a shortened NA stalk is considered an adaptation marker of avian influenza viruses (AIVs) to chickens. Experimental passages of an H7N7 nonpathogenic AIV (rgVac2-P0) in chickens resulted in a highly pathogenic variant (Vac2-P3L4) with a 34-amino-acid deletion in the NA stalk, encompassing five potential N -glycosylation sites. To investigate how amino acid truncation and deglycosylation in the NA stalk contribute to increased pathogenicity, a virus with glycosylation-deficient mutations at these sites (rgVac2-P3L4/P0NAΔGlyco) was constructed. Contrary to expectations, chickens inoculated with rgVac2-P3L4/P0NAΔGlyco exhibited variable clinical outcomes, attributed to the genetic instability of the virus. A single mutation stabilized the virus, and the mutant (rgVac2-P3L4/P0NAΔGlyco-Y65H) resulted in higher pathogenicity compared with a virus with restored glycosylation (rgVac2-P3L4/P0NA-Y65H). Glycan occupancy analysis revealed 3–4 glycans at the five potential sites. In functional analysis, glycosylation-deficient mutants, similar to the short-stalk NA virus, showed significantly reduced erythrocyte elution activity. Additionally, mutational analysis indicated variable contributions of N -glycans to elution activity across the sites. Moreover, the functionally most contributing sites of the five potential N -glycosylation motifs were consistently included in the amino acid deletions of the stalk-truncated NA in N7-subtyped field isolates, despite the varying truncation position or length. These findings suggest that the loss of glycosylation is functionally equivalent to a reduction in amino acids, and it plays a crucial role in enhancing pathogenicity in chickens and affecting NA function. IMPORTANCE: Avian influenza poses significant economic challenges to the poultry industry and presents potential risks to human health. Understanding the molecular mechanisms that facilitate the emergence of chicken-adapted avian influenza viruses (AIVs) from non-pathogenic duck-origin influenza viruses is crucial for improving AIV monitoring systems in poultry and controlling this disease. Amino acid deletions in the neuraminidase (NA) stalk domain serve as one of the molecular markers for AIV adaptation to Galliformes. This study highlights the critical role of N -glycosylation in the NA stalk domain in the pathogenesis of high pathogenicity avian influenza viruses in chickens. The findings propose a novel theory that the loss of glycosylation at the NA stalk domain, rather than a reduction in stalk length, is responsible for both NA function and increased virus pathogenicity in chickens.

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.

The neuraminidase protein (NA) of influenza A viruses (IAVs) plays a role in the release of viruses from infected cells. NA on viral particles hydrolyzes sialylated glycans on the cell surface for viral budding. However, the maturation and intracellular functions of NA are poorly understood. To investigate how NA functions intracellularly, glycans displayed on IAV-infected cells were profiled by lectins, and global glycome alterations, accompanied by exposed terminal galactose and glycan recapping with α1-2 fucose, were found in the virus-infected cells. Since α1-2 fucosyltransferases are localized in the Golgi, these unique structures suggest a potential NA function in the IAV-infected cells. Functional analyses using antivirals and NA-expressing cells indicate that intracellular NA function is necessary for the glycome alterations. Time-course analyses in IAV-infected cells revealed that global desialylation and α1-2 fucosylation could be observed 5 h post-inoculation, corresponding to the timeframe of viral protein expression. These observations provide a novel theory of NA functions that NA obtains its enzymatic activity intracellularly before virus assembly and serves desialylated glycans for competitive glycosyltransferases, including α1-2 fucosyltransferases, as their acceptors, resulting in glycan recapping with α1-2 fucose. Hence, intracellular NA blocks the re-sialylation of glycans, promoting efficient virus release from infected cells by inhibiting the interaction between progeny virions and sialosides. This study further demonstrated the potential NA functions of limiting secondary IAV infection. These findings provide insights into the evolutionary strategies of IAVs for shaping the strict window of superinfection by NA functions under a balance between successful replication and reassortment. IMPORTANCE: Influenza A viruses (IAVs) exploit glycans for their replication cycle. The hemagglutinin protein uses sialic acid for viral attachment, and the neuraminidase protein (NA) hydrolyzes sialosides for virus release. However, the intracellular functions of NA are not well understood. This study demonstrated that intracellular NA induces global desialylation and glycan recapping with unique structures in IAV-infected cells. This suggests a novel mode of NA function during the IAV lifecycle, where virus particles are ready to be released at the assembly, and NAs no longer need to hydrolyze the sialic acids upon egress from the cells. Therefore, the present study provides novel and significant insights into the fundamental understanding of the lifecycle of IAV. Furthermore, as NA is a primary target for anti-influenza drugs, understanding the mechanism of intracellular NA function may also support the development of antivirals.

High pathogenicity avian influenza virus (HPAIV) infections have been frequently reported in wild birds since 2020. Because HPAIV infection has occasionally caused outbreaks in captive rare birds, treatment using antiviral drugs has been considered from the perspective of conservation medicine. In this study, the therapeutic efficacy of baloxavir marboxil (BXM) was evaluated using a duck model to support the establishment of post-infection strategies for captive avian species. Sixteen four-week-old ducks were divided into four groups and intranasally inoculated with the HPAIV strain, A/crow/Hokkaido/0103B065/2022 (H5N1). BXM was administered orally once daily at doses of 12.5, 2.5, 0.5, or 0 mg/kg to each of the four groups from 2 to 6 days post-infection. Blood samples were collected at 2, 8, and 24 hours after the initial BXM administration to measure the plasma concentrations of its active form, baloxavir acid (BXA). All ducks were monitored until 14 days post-infection, and oral and cloacal swabs were collected for virus recovery. All eight ducks administered either 12.5 or 2.5 mg/kg of BXM survived, demonstrating a significant reduction in virus recovery compared with the 0 mg/kg group. To further characterize the relationship between BXA exposure and virus shedding, an additional experiment was conducted in which ducks received a single oral administration of BXM at doses of 7.3, 4.3, or 0.5 mg/kg at 2 days post-infection. Pharmacokinetic/pharmacodynamic analysis of BXA across all dose groups showed that reductions in virus shedding from oral swabs were positively correlated with BXA exposure, and AUC _0–24hr exhibited the strongest association. These findings demonstrate that BXM administration within 48 hours post-HPAIV infection in ducks effectively reduces mortality and virus shedding. Comparison of pharmacokinetic parameters may facilitate the estimation of optimal BXM dosing options for captive avian species.

Background/Objectives: Biosecurity and stamping out are key control measures against H5 high pathogenicity avian influenza (HPAI) outbreaks. Vaccination in poultry is an additional tool to reduce disease risk and facilitate timely containment. This study aimed to establish a candidate vaccine strain against H5 HPAI in Asia and validate its protective efficacy. Methods: Based on genetic and antigenic analyses, a representative HPAI virus, A/duck/Vietnam/HU16-DD3/2023 (H5N1), collected in northern Vietnam, was selected to generate a candidate vaccine strain, rgPR8/VN23HA∆KRRK-NA (rgPR8/VN23; H5N1), using reverse genetics, followed by formulation of an inactivated oil-adjuvanted vaccine. Vaccine efficacy was evaluated by measuring humoral antibody responses after intramuscular vaccination and by assessing mortality and virus recovery following intranasal challenge with a clade 2.3.4.4b virus, A/Ezo red fox/Hokkaido/1/2022 (H5N1). Results were compared with those obtained using an antigenically homologous vaccine to the challenge strain and a Japanese stockpiled vaccine. Results: All vaccinated juvenile chickens developed sufficient immunity to survive the challenge at 21 days post-vaccination. The rgPR8/VN23 (H5N1) and homologous vaccines markedly reduced virus recovery, suggesting near-sterile protection, whereas low-titer viruses were transiently detected in chickens vaccinated with the stockpiled vaccine. The rgPR8/VN23 (H5N1) vaccine conferred clinical protection in juvenile chickens as early as 8 days post-vaccination. A single dose of the rgPR8/VN23 (H5N1) vaccine provided incomplete protection in laying hens, whereas a double-volume regimen improved protective efficacy. Conclusions: The rgPR8/VN23 (H5N1) vaccine conferred strong immunity to juvenile chickens; however, a refined vaccination strategy may be required to achieve complete protection in laying hens.