Detection of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Genotype D1.2 Virus in Swine after Experimental Inoculation

H5N1 Clade 2.3.4.4b Infections in Domestic Cats During an Avian Influenza Outbreak in Italy: Implications for One Health Surveillance

ABSTRACT Background H5Nx goose/Guangdong (Gs/GD) lineage highly pathogenic avian influenza (HPAI) viruses pose a significant public health threat due to their global spread, mutation accumulation, and expanding host range. The descendant clade 2.3.4.4b has been causing widespread infections in birds and increasing spillover events in mammals. Methods This report documents the first fatal case of highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b infection in a domestic cat in Italy, detected in early January 2025. The cat (CAT 1) resided on a backyard poultry farm experiencing a high pathogenicity avian influenza outbreak and succumbed rapidly following the onset of respiratory signs. A second exposed cat (CAT 2) developed clinical disease without fatal outcome. Comprehensive outbreak investigations were conducted, including pathological, serological, molecular analyses, and genomic characterization. Results Pathological examination of CAT 1 revealed acute necrotizing bronchointerstitial pneumonia, non‐suppurative meningoencephalitis, and disseminated foci of hepatic necrosis. Interestingly, the PB2‐E627K mutation associated with mammalian virus adaptation was observed in the feline viral isolate compared to avian isolates. Such polymerase complex mutations are key determinants of host range and increase pathogenicity in mammals. CAT 2 from the same farm tested negative for AIV genome detection but subsequently seroconverted for antibodies against NPA, H5, and N1. Conclusion Sharing these findings is crucial for surveillance aimed at enabling early identification of increased risks to human and animal health, preventing cross‐species viral transmission and mitigating the risk of potential spillover events.

Risk assessment of avian influenza A(H5N5) virus from the first human case using the ferret model

ABSTRACT The incursion of Eurasian-origin genotype A6 A(H5N5) virus into North America expanded the genetic diversity among North American highly pathogenic avian influenza viruses and heightened concern about zoonotic risk. Following a fatal human infection with the A(H5N5) virus A/Washington/2148/2025, viral replication was assessed in polarized human bronchial epithelial cells, and pathogenicity, transmissibility in direct contact and respiratory droplet models, and airborne virus shedding were evaluated in ferrets to inform pandemic risk assessment. A(H5N5) displayed robust replication in Calu-3 cells at 33°C and 37°C, showing kinetics and peak titers comparable to those of contemporary genotype B3.13 and D1.1 A(H5N1) viruses. In ferrets, A(H5N5) replicated efficiently in the respiratory tract, disseminated to extrapulmonary tissues, and caused fatal disease in all inoculated animals. Airborne transmission was not observed, and infrequent, low-level detection of virus in air samples paralleled that of A(H5) viruses that are not transmissible via air in ferrets. In a direct contact model, limited transmission was detected within 4 days of exposure, with evidence of lower respiratory tract replication in contact animals. These findings indicate that the A(H5N5) virus has the capacity for robust replication in an airway epithelial cell line and can cause severe systemic infection and mortality in ferrets but has not acquired adaptations for airborne spread in mammals. Collectively, these results underscore heterogeneity among clade 2.3.4.4b A(H5Nx) viruses in North America and the need for genotype-by-genotype evaluation of newly emerged viruses to understand public health risk. IMPORTANCE The emergence of Eurasian-origin genotype A6 highly pathogenic avian influenza A(H5N5) virus in North America has increased viral diversity and raised concerns about zoonotic and pandemic risk. In this study, we evaluated the replication kinetics, pathogenesis, and transmission of A/Washington/2148/2025 A(H5N5) virus, which was isolated from the first reported human infection with this influenza virus subtype, using polarized human bronchial epithelial cells and the ferret model. The A(H5N5) virus replicated efficiently in vitro at temperatures representative of the upper and lower respiratory tracts and caused fatal systemic disease in inoculated ferrets. Limited transmission was observed during 4 days of direct contact. Airborne virus detection was infrequent and did not result in airborne transmission. These findings show that A(H5N5) virus can replicate robustly in mammalian cells and cause severe disease but lacks adaptations supporting efficient airborne spread, informing assessment of the pandemic risk posed by genotype A6 influenza viruses. The emergence of Eurasian-origin genotype A6 highly pathogenic avian influenza A(H5N5) virus in North America has increased viral diversity and raised concerns about zoonotic and pandemic risk. In this study, we evaluated the replication kinetics, pathogenesis, and transmission of A/Washington/2148/2025 A(H5N5) virus, which was isolated from the first reported human infection with this influenza virus subtype, using polarized human bronchial epithelial cells and the ferret model. The A(H5N5) virus replicated efficiently in vitro at temperatures representative of the upper and lower respiratory tracts and caused fatal systemic disease in inoculated ferrets. Limited transmission was observed during 4 days of direct contact. Airborne virus detection was infrequent and did not result in airborne transmission. These findings show that A(H5N5) virus can replicate robustly in mammalian cells and cause severe disease but lacks adaptations supporting efficient airborne spread, informing assessment of the pandemic risk posed by genotype A6 influenza viruses.

Genetic and Antigenic Characterization of a Clade 2.3.4.4b Highly Pathogenic H5N1 Avian Influenza Virus Isolated from a Free-Range Layer Duck in India

Highly pathogenic avian influenza (HPAI) H5N1 virus poses a significant threat to the global poultry sector. The report highlights the genetic and antigenic features of a clade 2.3.4.4b HPAI H5N1 virus isolated from a free-range layer duck in the Kuttanad delta of Kerala, India, in 2022. The virus is similar to an H5N1 virus detected in Russia in October 2021 and antigenically distinct from clade 2.3.2, showing a 16-fold reduction in hemagglutination inhibition titer and 23.95% amino acid differences at the HA antigenic sites. The duck virus shares genetic similarities with H5N1 viruses found in Eurasian wild birds, suggesting a role for these birds in its introduction. The study underscores the importance of strengthening avian influenza surveillance to monitor virus evolution and spread.

Cellular tropism of highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b viruses in human respiratory and bovine mammary epithelial cells

ABSTRACT The recent introduction and spread of HPAI A(H5N1) clade 2.3.4.4b viruses to U.S. dairy cattle demonstrate their ability to adapt to new mammalian hosts. Although multiple A(H5N1) genotypes have caused human infections, only two clade 2.3.4.4b genotypes (B3.13 and D1.1) have been detected in cattle, suggesting clade- or genotype-specific tropism. Understanding how these viruses replicate in human airway and bovine mammary epithelial cells is important for assessing viral evolution, mammalian adaptation, and associated public health risks. Here, we characterized replication kinetics and immune gene expression in human bronchial epithelial (Calu-3) cells and bovine mammary epithelial (MAC-T) cells infected with A(H5N1) clade 2.3.4.4b human isolates representing the B3.13, D1.1, and B3.2 genotypes, an A(H5N1) clade 1 virus, and an A(H1N1)pdm09 strain. All viruses replicated efficiently in Calu-3 cells, although replication was delayed at 33°C compared to 37°C. Clade 2.3.4.4b viruses induced moderate expression of type I interferon and proinflammatory response in Calu-3 cells, but at lower levels than the A(H1N1)pdm09 and A(H5N1) clade 1 strains. Replication and immune activation varied markedly among clade 2.3.4.4b genotypes in MAC-T cells. B3.13 viruses achieved higher titers and triggered stronger induction of innate immune response genes than other A(H5N1) genotypes or clades. These findings indicate genotype- and cell type–specific differences in replication and host responses, with B3.13 showing enhanced tropism in bovine mammary cells, consistent with adaptation that may elevate zoonotic risk. Our results highlight the value of comparative studies across host cell types and diverse viral isolates to inform risk assessments for emerging influenza viruses. IMPORTANCE Influenza A viruses cross species barriers through a combination of factors, including the ability to bind to and infect permissive cells, replicate efficiently, and modulate host immune responses. Since 2024, A(H5N1) clade 2.3.4.4b viruses have continued to evolve, infecting a broad range of avian and mammalian species, including cattle in the United States, and causing sporadic human infections. Here, we evaluated a panel of A(H5N1) clade 2.3.4.4b viruses isolated from humans to assess their replication and host responses in two relevant mammalian cell types: human bronchial epithelial cells and bovine mammary gland epithelial cells. While all genotypes replicated efficiently in human bronchial epithelial cells, only B3.13 viruses showed strong replication and broad host response induction in bovine mammary epithelial cells. These results underscore variation in evolution, tissue tropism, and host adaptation among A(H5N1) clade 2.3.4.4b viruses and highlight the need for continued surveillance and close monitoring of B3.13 genotype viruses. Influenza A viruses cross species barriers through a combination of factors, including the ability to bind to and infect permissive cells, replicate efficiently, and modulate host immune responses. Since 2024, A(H5N1) clade 2.3.4.4b viruses have continued to evolve, infecting a broad range of avian and mammalian species, including cattle in the United States, and causing sporadic human infections. Here, we evaluated a panel of A(H5N1) clade 2.3.4.4b viruses isolated from humans to assess their replication and host responses in two relevant mammalian cell types: human bronchial epithelial cells and bovine mammary gland epithelial cells. While all genotypes replicated efficiently in human bronchial epithelial cells, only B3.13 viruses showed strong replication and broad host response induction in bovine mammary epithelial cells. These results underscore variation in evolution, tissue tropism, and host adaptation among A(H5N1) clade 2.3.4.4b viruses and highlight the need for continued surveillance and close monitoring of B3.13 genotype viruses.

Highly pathogenic avian influenza A(H5N1) in domestic cats: epidemiology, clinical manifestations, and One Health implication.

High pathogenicity avian influenza A (H5N1) viruses isolated from poultry and wild birds in Japan during the 2024–2025 season

ABSTRACT 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. 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.