Using the ferret model of influenza infection and transmission, it is demonstrated that the currently circulating B3.3.13 is already well adapted for mammalian infection and transmission whereas D1.1 rapidly acquires mammalian-adaptive mutations after a single infection and/or transmission event, highlighting its evolutionary potential.
Abstract
Since its emergence in 2020, multiple genotypes of the H5N1 clade 2.3.4.4b have been identified, with B3.13 and D1.1 emerging in the USA as two major and concerning genotypes. However, their relative pathogenicity and transmissibility in mammals have not been fully elucidated. We compared the pathogenicity and transmissibility of the first two human H5N1 clade 2.3.4.4b cases caused by B3.13 in Texas (A/Texas/37/2024; HPhTX B3.13) and D1.1 in Louisiana (A/Louisiana/12/2024; HPhLA D1.1) in a ferret model of infection and transmission. HPhTX B3.13 infection resulted in more severe clinical disease and enhanced viral shedding, with evidence of increased transmission relative to HPhLA D1.1. Histopathological analysis revealed more extensive lung pathology in animals infected with HPhTX B3.13, consistent with increased viral loads and inflammatory responses. Importantly, both genotypes showed no significant differences in reactivity to ferret sera raised against candidate vaccine virus (CVV) strains, receptor binding properties, or neuraminidase (NA) activity and thermostability. Whole-genome sequencing revealed no adaptive mutations in HPhTX B3.13 following infection or transmission. In contrast, HPhLA D1.1 showed rapid acquisition of the mammalian-adaptive mutation E627K in infected ferrets and both E627K and Q194K in the only fatal contact animal. Both mutations were associated with enhanced polymerase activity and computational analyses suggested that they enhance interactions with the mammalian host factors ANP32A and B. Our findings indicate that B3.13 is already well adapted for mammalian infection and transmission whereas D1.1 retains evolutionary potential through the rapid acquisition of adaptive mutations, highlighting important genotype-specific differences relevant to zoonotic risk assessment and pandemic preparedness. Significance Influenza H5N1 viruses continue to diversify genetically while expanding into mammalian hosts, increasing opportunities for viral adaptation and zoonotic transmission, including humans. However, whether the predominant clade 2.3.4.4b genotype differs in its capacity to infect, transmit, and evolve in mammals remains poorly understood. Using the ferret model of influenza infection and transmission, we demonstrated that the currently circulating B3.13 and D1.1 genotypes exhibit distinct pathogenic and transmission characteristics despite retaining similar receptor-binding characteristics, NA functions, and antigenic profiles. While B3.13 readily infects and transmits in ferrets and does not develop further adaptive mutations associated with increased replication and transmission, D1.1 rapidly acquires mammalian-adaptive mutations after a single infection and/or transmission event, highlighting its evolutionary potential. These findings show that genotype-specific biological properties can influence zoonotic risk independently of antigenic similarity and emphasize the importance of integrating phenotypic characterization with genomic surveillance to improve pandemic preparedness and guide public health risk assessment.
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.
J. Pulit-Penaloza, J. Belser, N. Brock et al.· Journal of Virology· 0 citations
ABSTRACT Highly pathogenic avian influenza A(H5N1) (HPAI H5N1) viruses of clade 2.3.4.4b have recently been detected in U.S. dairy cattle following multiple spillover events from avian reservoirs. In December 2025, HPAI H5N1 virus was identified in a dairy herd in Wisconsin through the National Milk Testing Strategy. Here, we report the isolation of a clade 2.3.4.4b, genotype D1.1 H5N1 virus, A/dairy cow/Wisconsin/25G05743-001/2025 (WI5743-H5N1), from bulk milk associated with the affected herd, describe its phylogenetic relationships, and assess its pathogenicity in mice. Infectious virus was recovered following blind passage in embryonated chicken eggs. Phylogenetic analysis demonstrated that WI5743-H5N1 is distinct from previously reported D1.1 viruses detected in dairy cattle in Nevada and Arizona, supporting an independent introduction into cattle, and indicating a likely local avian source. Compared with closely related avian viruses, WI5743-H5N1 encoded the mammalian-adapting substitution PB2-E627K and additional amino acid differences in HA, PB1-F2, and NS1. In mice, WI5743-H5N1 replicated efficiently in respiratory tissues and was detectable in the brain but exhibited lower lethality relative to other recent clade 2.3.4.4b, genotype B3.13 viruses. Together, these findings highlight the genetic and phenotypic diversity of HPAI H5N1 viruses infecting dairy cattle and underscore the importance of continued surveillance and functional characterization of emerging strains. IMPORTANCE Highly pathogenic avian influenza A(H5N1) viruses have recently entered U.S. dairy cattle through multiple spillover events from avian reservoirs, creating new opportunities for viral adaptation in mammals. Here, we describe the isolation and characterization of a clade 2.3.4.4b, genotype D1.1 H5N1 virus from bulk milk collected during a spillover event in Wisconsin in December 2025. Phylogenetic analyses demonstrated that this virus represents an independent introduction into dairy cattle distinct from previously reported D1.1 viruses identified in Nevada and Arizona. Although the virus encoded the mammalian-adapting PB2-E627K substitution, it exhibited comparatively low lethality in mice, highlighting the complexity of mammalian adaptation and pathogenicity in H5N1 viruses. These findings expand current understanding of the genetic and phenotypic diversity of H5N1 viruses infecting dairy cattle and emphasize the importance of continued surveillance and functional characterization of emerging strains. Highly pathogenic avian influenza A(H5N1) viruses have recently entered U.S. dairy cattle through multiple spillover events from avian reservoirs, creating new opportunities for viral adaptation in mammals. Here, we describe the isolation and characterization of a clade 2.3.4.4b, genotype D1.1 H5N1 virus from bulk milk collected during a spillover event in Wisconsin in December 2025. Phylogenetic analyses demonstrated that this virus represents an independent introduction into dairy cattle distinct from previously reported D1.1 viruses identified in Nevada and Arizona. Although the virus encoded the mammalian-adapting PB2-E627K substitution, it exhibited comparatively low lethality in mice, highlighting the complexity of mammalian adaptation and pathogenicity in H5N1 viruses. These findings expand current understanding of the genetic and phenotypic diversity of H5N1 viruses infecting dairy cattle and emphasize the importance of continued surveillance and functional characterization of emerging strains.
Daria Mezhenskaia, Lavanya Babujee, Ailam Lim et al.· Journal of Virology· 1 citation
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.
G. D’Annunzio, Ana Moreno, Giovanni Tosi et al.· Influenza and Other Respirat...· 0 citations
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.
N. Brock, H. Zeng, C. Pappas et al.· Microbiology spectrum· 1 citation
Highly pathogenic avian influenza clade 2.3.4.4b virus continues to circulate in North America and has caused severe human disease. That clade includes genotype D1.1, which became dominant in birds in late 2024. Recent phylodynamic reconstructions place D1.1 emergence in mid-2024 but differ on its inferred origin and early dissemination pathways. We combined targeted surveillance of wild birds in Arizona with publicly available US clade 2.3.4.4b hemagglutinin sequences to estimate when D1.1 genotype emerged and to infer its diffusion among the 4 major US flyways. Phylodynamic analyses showed transitions concentrated among adjacent flyways regions, consistent with stepwise dissemination during fall 2024 and limited support for long-distance Pacific–Atlantic exchange. The Pacific Flyway showed patterns consistent with an early source and the Central Flyway with a secondary hub linked to onward spread. Our findings support coordinated genomic surveillance across adjacent flyways to reduce detection delays and improve situational awareness during rapid viral expansion.
M. Scotch, T. Faleye, Angelica Urquidez-Negrete et al.· Emerging Infectious Diseases· 0 citations
It is suggested that identifying infection in commercial swine without overt respiratory signs could be difficult, and consistent detection in nasal swab specimens, combined with subclinical respiratory infection, suggest that identifying infection in commercial swine without overt respiratory signs could be difficult.
Hannah Seger, Amy L. Baker, Alexandra C. Buckley et al.· Emerging Infectious Diseases· 0 citations
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