Genotype- and cell type–specific differences in replication and host responses are indicated, with B3.3.4.13 showing enhanced tropism in bovine mammary cells, consistent with adaptation that may elevate zoonotic risk, and the value of comparative studies across host cell types and diverse viral isolates to inform risk assessments for emerging influenza viruses.
Abstract
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.
ABSTRACT Background The high‐pathogenicity avian influenza (HPAI) viruses represent a main threat to animal and human health, poultry productivity, and ecosystems. The newly evolved A(H5N1) clade 2.3.4.4b has become a worldwide concern for its expanding multihost ecology, including poultry and a wide range of mammalian species. Methods Avian influenza virus (AIV) was isolated from an outbreak in commercial quail farms associated with severe respiratory signs and high mortality. Molecular characterization of the detected virus was performed through gene sequencing via whole‐genome sequencing technology and phylogenetic analysis. Results Genetic and phylogenetic analyses revealed that the hemagglutinin (HA) gene clustered closely with a recent HPAI A(H5N1) clade 2.3.4.4b strain from live bird markets (LBMs) in Bangladesh, with a most recent common ancestor dated to July 2023. The NA gene and four internal genes (PB1, NP, MP, NS) were derived from contemporary A(H5N1) viruses of ducks in LBMs, while the PB2 and PA segments exhibited similarity to low pathogenicity avian influenza (LPAI) viruses, suggesting reassortment events within avian hosts. The presence of a multibasic cleavage site (PLREKRRKR↓GLF) with avian‐specific receptor‐binding residues (Q224 and G226) and five conserved glycosylation motifs, consistent with high‐pathogenicity phenotypes and strong avian receptor affinity. The PB2 gene harbored a rare I147T substitution; however, key mammalian adaptation markers such as E627K and D701N were absent. Conclusion The reassortant A(H5N1) clade 2.3.4.4b virus with high pathogenicity and genetic links to LBM‐origin strains is now circulating in the commercial poultry population in Bangladesh, increasing the risk of viral adaptation, the potential for cross‐species transmission, and threatening the poultry sector and public health.
M. A. Samad, Md Rezaul Karim, Amelia Coggon et al.· Influenza and Other Respirat...· 0 citations
Highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b virus continues to spread globally and sporadically transmits from avian reservoirs to mammalian hosts. In May 2024, H5N1 infections in young goats and alpacas in the United States were reported. Nevertheless, the overall susceptibility of camelids to clade 2.3.4.4b virus remains unclear. We conducted a controlled experimental infection study in 6 alpacas, assessing clinical signs, viral shedding, tissue distribution, and serologic responses after intranasal inoculation with HPAI H5N1 genotype B3.13 virus. Observed illness was generally mild; body temperature increased slightly and food intake reduced for up to 3 days postinfection. We detected viral RNA in nasal swab samples and confirmed infectious HPAI H5N1 virus. Immunohistochemistry and RNA in situ hybridization detected virus only in the nasopharyngeal tonsil and nasal conchae at 4 days postinfection. Our findings suggest alpacas are susceptible to productive H5N1 infection, highlighting implications for livestock surveillance and biosecurity in regions with ongoing circulation.
Jacob Schön, A. Breithaupt, N. Halwe et al.· Emerging Infectious Diseases· 0 citations
Abstract The emergence of the panzootic clade 2.3.4.4b highly pathogenic H5N1 avian influenza in 2020 marked a major expansion in the host range of influenza A viruses (IAVs), raising concerns about further cross-species transmission events and zoonotic spillover. The introduction of 2.3.4.4b viruses into US dairy herds has resulted in widespread circulation, accompanied by reduced milk yield, mastitis and high viral loads in milk. Notably, virus circulation in dairy cattle represents a novel route for mammalian adaptation and transmission that has already led to more than 40 human cases in the USA since 2024. As haemagglutinin (HA) is the primary determinant of viral host range and tissue tropism, we investigated whether viruses containing the glycoproteins of avian H5N1 subclade 2.3.4.4b viruses could infect mammary tissue from Aberdeen Angus, Holstein Friesian and Limousin cattle. Using mammary gland explants, we inoculated tissues with attenuated reassortant viruses expressing the HA and neuraminidase (NA) of three 2.3.4.4b viruses that predated the emergence of H5N1 in the US cattle: A/chicken/England/053052/2021 (AIV07), A/chicken/Scotland/054477/2021 (AIV09) and A/chicken/England/085598/2022 (AIV48). Detection of viral NP antigen by immunohistochemistry in explants from both the teat and gland cistern for all three breeds following inoculation with AIV09 and AIV48 indicates that mammary tissue from each of the three tested cattle breeds is susceptible to infection by viruses bearing H5N1 glycoproteins. Lectin staining showed expression of both α2,3- and α2,6-linked sialic acids in the mammary tissue of all donors, indicating that all three breeds have the potential to support infection with both avian- and mammalian-adapted IAVs. Together, these findings demonstrate that the mammary glands from both beef and dairy cattle breeds are susceptible to infection with viruses bearing avian-origin H5N1 glycoproteins and suggest that bovine mammary tissue may provide an environment in which diverse IAVs could co-infect the same tissue. This possibility underscores the need for further investigation and the inclusion of both beef and dairy cattle in ongoing H5N1 surveillance and risk-assessment frameworks.
Rebecca A. Ross, Sarah K. Walsh, Hannah Montgomery et al.· Journal of General Virology· 0 citations
Abstract Avian influenza A(H5N1) is a highly pathogenic virus causing significant out-breaks in birds and sporadic human infections since 1997. The virus is classified into various clades based on genetic characteristics, and recently, concerns have been raised over the risk ofclade 2.3.4.4b becoming widespread and infecting humans. Since 1997, more than 1,000 infections with highly pathogenic avian influenza A(H5N1) virus among humans have been reported globally. Given ongoing A(H5N1) outbreaks in animals, understanding the frequency of A(H5N 1) virus infections ‘among asymptomatic persons can inform public health risk assessments and infection prevention guidance. We performed a literature search focusing on reported infections amona humans with Highly Pathogenic Avian Influenza (HPAI). This study was conceived as a narrative review focusing on clade 2.3.4.4b H5N1, but was expanded to all reported H5Nx and other zoonotic subtypes (H5N6, H6N1, H7N9, H10N3) when clade-specific data were sparse. Although not a formal systematic review, the search, screening, and appraisal followed the PRISMA 2020 Checklist for search reporting where applicable.
P. Velikov, T. Valkov· Acta Medica Bulgarica· 0 citations
H5N1 clade 2.3.4.4b avian influenza A viruses pose a significant threat to wild animal populations, domesticated animals, and potentially, the human population. For H5N1s to infect and transmit among mammalian species, mutations for improved utilization of mammalian receptors and enhanced replication at the lower temperatures of the upper respiratory tract need to be acquired. A human H1N1pdm09-like virus was compared to H5N1 genotypes B3.13 and D1.1 for replication at 33°C, 37°C, and 39°C – temperatures consistent with the upper and lower respiratory tract in humans, and dairy cow udder tissue. All H5N1 viruses had increased plaque sizes on MDCK cells at 37°C and 39°C compared to H1N1pdm09. In primary, differentiated human nasal and bronchial epithelial cultures, all H5N1 viruses show restricted infectious virus production compared to H1N1 at 33°C. While H5N1 D1.1 also showed restricted replication at 37°C and 39°C, the H5N1 B3.13 replicated to nearly equivalent titers as H1N1pdm09. All H5N1 viruses demonstrated similar cell tropism in cells from the upper and lower respiratory tract, infecting more ciliated than non-ciliated cells relative to H1N1pdm09. H1N1, H5N1 B3.13 D1.1 infection induced similar innate immune factors, with nasal epithelial cells producing higher levels compared to bronchial epithelial cells. These data suggest that genotype B3.13 and D1.1 H5N1 viruses show different temperature dependent replication patterns compared to H1N1pdm09. IMPORTANCE The ongoing H5N1 outbreaks have increased the number of mammalian infections with H5N1, thus increasing the chances of the virus adapting to mammals. Two H5N1 genotypes that have recently infected dairy cows, commercial poultry flocks, and a limited number of humans in the United States showed decreased replication at 33°C but not 37°C or 39°C compared to human H1N1 virus. H5N1 virus replication was detected at all temperatures, suggesting temperature and not host species may be the primary barrier preventing H5N1 infection in humans.
Anne P. Werner, J. Sachithanandham, E. Akin et al.· bioRxiv· 0 citations
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.
Ahmed M. Elsayed, Ramya S. Barre, Mahmoud Bayoumi et al.· bioRxiv· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
