Findings reveal a viral trade-off: the mutation attenuates epithelial replication and immunopathology but enhances DC uptake and disables mucosal immune functions, a strategy that likely contributed to the global predominance of PA-X N193S mutation in epidemic H5 subtype viruses.
Abstract
ABSTRACT In recent years, H5 subtype highly pathogenic avian influenza viruses (HPAIVs), especially clade 2.3.4.4b, have posed a global threat to poultry, cattle, and public health. Bioinformatics analysis of H5 subtype AIVs from 2000 to 2023 revealed a progressive increase in the PA-X N193S mutation, which became predominant in both avian and mammalian isolates of clade 2.3.4.4b. Using reverse genetics, we generated viruses with PA-X 193N (rWT) and 193S (rWT-N193S). The PA-X N193S mutation significantly inhibited viral polymerase activity while enhancing host shutoff. In vitro and vivo, rWT-N193S showed attenuated replication in avian and mammalian cells, reduced pathogenicity in mice, and suppressed cytokine storms. However, it enhanced uptake by dendritic cells (DCs), impairing DC maturation, activation, cytokine secretion, and CD4+ T cell proliferation. In murine nasal mucosal experiments, the PA-X N193S mutation reduced CCL5 expression, altered DC recruitment, and suppressed IL-17/MAPK signalling. These findings reveal a viral trade-off: the mutation attenuates epithelial replication and immunopathology but enhances DC uptake and disables mucosal immune functions, a strategy that likely contributed to the global predominance of PA-X N193S mutation in epidemic H5 subtype viruses.
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 (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.
K. Gaurav, Manoj Kumar, S. Nagarajan et al.· Journal of Pure and Applied...· 0 citations
ABSTRACT Some subtypes of avian influenza A viruses (IAVs) have been associated with human infections (e.g. H3N8, H5Ny, H7Ny, H9N2, and H10Ny), and the ability of these viruses to cause zoonotic infections further increases the public health risk of avian IAVs. Among them, H9N2 virus is one of particular importance, both in its own right and as a contributor of internal gene segments to other emerging zoonotic avian IAVs. In recent years, an increasing number of H9N2 strains have been observed to be highly pathogenic in mice without prior adaptation. In this study, we found that a naturally occurring H9N2 isolate, A/chicken/Jiangxi/198/2019 (JX198), is lethal in mice. To investigate the molecular basis for the high virulence of JX198 in mice, a series of reassortants and mutants were generated and tested. We found that the PA, especially D347G mutation in PA, is responsible for the increased pathogenicity of JX198 in mice. Notably, D347G in PA of JX198 significantly alters the polymerase activity, vRNA production, and plaque-formation. Thus, our data demonstrate that a novel molecular marker, D347G in PA, determines the virulence of H9N2 in mice, posing a potential risk of H9N2 with D347G in PA for public health and highlighting the significance of continuing surveillance of H9N2 field strains.
Zhehong Zhao, Ye Tian, Wenjie Jiang et al.· Virulence· 0 citations
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
ABSTRACT Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b, genotype D1.1, are responsible for widespread outbreaks in poultry and continue to cause sporadic, sometimes severe, human infections. Herein, we characterized a wild-type (WT) influenza A(H5N1) D1.1 isolate (BC-H5N1-WT) and its H275Y neuraminidase (NA) variant (BC-H5N1-H275Y), both of which emerged on farms in British Columbia, Canada, during the fall 2024 outbreak. In vitro analysis assessed replication kinetics in MDCK cells, with supernatants collected at different days post-infection (p.i.) and titrated by TCID50 and qRT-PCR. Neuraminidase inhibitor (NAI) susceptibility was determined by NA inhibition assays, whereas susceptibility to baloxavir acid (BXA) was evaluated by plaque reduction assay. In vivo virulence was evaluated in BALB/c mice infected with serial 10-fold dilutions of each virus to monitor weight loss and mortality. Viral titers in lungs, brain, nose, kidney, spleen, and heart were quantified at day 4 p.i. The BC-H5N1-WT virus was susceptible to the four antivirals tested, whereas BC-H5N1-H275Y displayed resistance to oseltamivir and peramivir but remained susceptible to zanamivir and BXA. The BC-H5N1-WT exhibited significantly higher viral replication titers than BC-H5N1-H275Y at all tested time points and showed larger plaque sizes. In mice, BC-H5N1-WT was more virulent with LD50 values of 1.78 × 103 PFUs compared to 8.71 × 104 PFUs for BC-H5N1-H275Y, and produced higher viral titers in lungs and other organs. Despite the reduced fitness of the resistant H5N1 D1.1 variant, its emergence in the absence of viral selection pressure underscores the need for continued surveillance.
Maxime Cochin, Yacine Abed, Robert Vendramelli et al.· Emerging Microbes and Infect...· 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
