#Mutations in severe #human #H5N1 cases facilitate #evasion from human mucus and #antivirals

 

Abstract

In late 2024, two individuals in Canada and the United States were treated in intensive care for acute respiratory distress caused by infection with the avian Influenza A Virus H5N1 2.3.4.4b genotype D1.1. Viral sequence data obtained from sampling these patients indicated mixed alleles at haemagglutinin (HA) positions 190 and 226. Mutations at these positions are key determinants of HA usage of α2,6-linked sialic acids (SA), the most abundant influenza receptors in human upper respiratory tracts. Thus, these mutations raised concerns about human adaptation and pandemic potential of the H5N1 virus. In this study, we investigated the impact of the mutations at residues 190 and 226 in H5 HA. We studied the receptor binding properties, cell entry phenotypes and fitness impacts of the mutations using recombinant proteins, pseudotyped lentiviruses, and in the context of influenza viruses using reverse genetics. The mutations did not confer any detectable α2,6-linked sialic acid receptor usage either alone or in combination. Rather, viruses carrying these mutations exhibit weakened binding towards α2,3-linked sialic acid receptors. This correlated with an enhanced capacity to evade human airway mucus, and a reduced susceptibility to oseltamivir and zanamivir. This research underscores that in addition to the way HA interacts with SA as entry receptors, other factors that impact the HA/NA balance might influence the evolutionary trajectory of a zoonotic virus in the human respiratory tract. This study presents a new paradigm for the evolutionary drivers of HA, where reduced sialic acid binding can serve as an advantage for escape from host barriers and antivirals.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

Medical Research Council, https://ror.org/03x94j517, MR/Y03368X/1, MR/Y015061/1, CC2127

Biotechnology and Biological Sciences Research Council, BB/Y007298/1, APP104179, BBS/E/PI/23NB000, BBS/E/PI/23NB0003

Wellcome Trust, https://ror.org/029chgv08, CC2127, 218304/Z/19/Z

Department for Environment Food and Rural Affairs, BB/Y007298/1

The Pirbright Institute, BBS/E/PI/230002A, BBS/E/PI/230001C, BBS/E/PI/230002B

Cancer Research UK, CC2127

UK Research and Innovation, https://ror.org/001aqnf71, UKRI3602

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.07.28.740943v1

____

#H5N1 #influenza binding and cell entry via #human class II #MHC, and blocking by cross-reactive #antibodies

 

Abstract

Highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses are currently responsible for a multi-species outbreak affecting wild birds, poultry, numerous mammalian species, and humans. Influenza A viruses typically initiate infection through binding to sialic acid, although select bat and human influenza viruses can also exploit class II major histocompatibility complex (MHC–II) molecules for cell entry. Here we show that emerging H5N1 clade 2.3.4.4b viruses, but not historical H5 lineages, bind human MHC–II HLA-DR and mediate sialic acid-independent cell entry. Hemagglutinin binding to primary human immune cells varies with MHC–II expression and is further shaped by HLA–DR allelic variation, identifying host genetic determinants that may influence susceptibility to infection. Mammalian-adaptive substitutions within the hemagglutinin sialic acid receptor-binding domain reduce MHC–II binding, suggesting this interaction is remodeled during clade 2.3.4.4b H5 adaptation to a human host. Lastly, cross-reactive monoclonal antibodies isolated from clade 2.3.4.4b H5–naive humans can block the hemagglutinin–MHC–II interaction. These findings identify a previously unrecognized receptor pathway in contemporary H5N1 viruses and reveal that both human genetic variation and pre-existing humoral immunity can modulate this interaction, with implications for host range, cellular tropism, spillover risk, and therapeutic intervention.

Competing Interest Statement

S.D.B. has consulted for Regeneron, Sanofi, Novartis, Genentech, Pfizer, Visterra, and Otsuka on topics unrelated to the research presented here; owns stock in AbCellera Biologics; and is a scientific cofounder of Immunera, Inc.; S.E.H reports receiving consulting fees from Sanofi, Pfizer, Lumen, Novavax, and Merck.

Funder Information Declared

NIH/NIAID CEIRR contract, 75N93021C00015

NIH, 1U54CA260517

HIPC, U19AI057266

P01 grant, 5P01AI153559

David Crown Foundation endowment

Early Postdoc Mobility Fellowship Stipend from the Swiss

National Institutes of Health NRSA T32, T32OD011121

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.07.22.739677v1

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#MHC-II acts as a #fusion-triggering #receptor for #bat #influenza virus

 

Abstract

Influenza A virus hemagglutinin is a prototypical class I viral fusion protein that binds sialylated glycans and is activated by low pH in endosomes. In contrast, bat-derived IAV subtypes H17N10 and H18N11 use major histocompatibility complex class II (MHC-II) as an entry receptor, but how this receptor contributes to membrane fusion remains unknown. We find that MHC-II-dependent hemagglutinin subtypes H17, H18, and H19 possess an increased negative net charge relative to canonical HAs. Using cryo-electron tomography, we demonstrate that H18N11 morphology remains stable and H18 is in prefusion conformation at strongly acidic pH. Remarkably, H18 undergoes fusion-relevant conformational changes only when both MHC-II binding and low pH are present. By reconstitution of H18N11 fusion with liposomes and purified MHC-II, we show that receptor engagement is required to trigger the fusion activity of H18. These findings identify MHC-II as a receptor that directly triggers membrane fusion and reveal a previously unrecognized receptor-dependent mechanism of influenza virus entry.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 240245660 – P19, 537227910

European Research Council, 882631—Bat Flu

Excellence Initiative of the German Research Foundation, GSC-4, Spemann Graduate School

Hans A. Krebs Medical Scientist Programme

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.07.08.737388v1

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Lactylation of #Influenza Virus #Polymerase Acidic Protein Promotes Viral #Replication and #Pathogenicity

 

Abstract

Influenza virus poses a potential risk of triggering the next global pandemic. In-depth investigation into the mechanisms underlying influenza virus replication and pathogenicity will provide robust support for controlling influenza virus infection. Although post-translational modifications are known to regulate viral infection, the role of lactylation in influenza virus replication remains elusive. In this study, influenza virus ribonucleoprotein complex subunits are found to be lactylated. Specifically, ATAT1 promotes viral polymerase acidic protein (PA) lactylation and enhances viral replication. In contrast, SIRT1 mediates de-lactylation of PA and exerts an inhibitory effect on viral replication. Further investigations reveal lactylation of PA at residues K605 and K609 is essential for viral replication and pathogenicity. Mechanistically, PA K605/609 residues are localized at the interaction interface of the ANP32-mediated polymerase asymmetric dimer; mutation at these residues inhibits polymerase asymmetric dimerization, thereby impairing RNA production during viral genome replication. Collectively, this study uncovers a novel mechanism by which influenza virus hijacks host enzymes to mediate PA lactylation, and expands the molecular regulatory network of influenza virus infection.

Competing Interest Statement

The authors have declared no competing interest.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.07.10.737663v1

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Isolation and characterization of a clade 2.3.4.4b genotype #D1.1 #H5N1 virus from dairy #cattle in #Wisconsin

 

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.

Source: 

Link: https://journals.asm.org/doi/10.1128/jvi.00761-26

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#Cattle and #human #organoids reveal 2.3.4.4b #H5N1 cross-species #transmission potential and #neuraminidase-specific neutralizing #antibodies in humans

 

Abstract

The unexpected circulation of clade 2.3.4.4b H5N1 influenza viruses in dairy cattle and the transmission to diverse mammalian species poses a pandemic risk. We sought to explore cattle and human respiratory susceptibility to the 2.3.4.4b H5N1 virus. We establish long-term expandable cattle airway and mammary organoids. The 2.3.4.4b H5N1 virus exhibits high replicative fitness in cattle mammary organoids, recapitulating its remarkable mammary tropism. The virus also replicates robustly in cattle airway organoids, suggesting an underrecognized respiratory component in ongoing outbreaks. Interestingly, human airway and nasal organoids are highly susceptible to the 2.3.4.4b H5N1 virus. Yet, a novel organoid-based neutralization assay reveals that N1 antibodies in human sera had cross-neutralizing activity against the 2.3.4.4b H5N1 and ancestral H5N1-VN1194 viruses. The cross-neutralization, exclusively manifested in the organoid-based assay, is enhanced after seasonal influenza vaccination and diminished after depleting N1-specific antibodies. Therefore, cross-neutralizing N1 antibodies are likely limiting zoonotic infection by H5N1 viruses in humans.

Source: 

Link: https://www.nature.com/articles/s41467-026-74345-w

____

#Assessment of #influenza virus and #coronavirus #tropism, #replication competence and disease severity in ex vivo and in vitro cultures of the #human respiratory tract

ABSTRACT

The emergence of animal influenza viruses circulating in poultry and human populations poses a significant public health threat, yet current risk assessment tools that connect surveillance data to human transmission risk and disease severity are lacking. To address this, we employed a semi-quantitative approach to analyze virus tropism and replication competence, conducting risk assessments of influenza and coronavirus adaptation to human transmission in an ex vivo model, and evaluating virus-induced impairment of alveolar fluid clearance (AFC) in vitro as a correlation of disease severity. Our results showed that seasonal influenza A H1N1, H3N2, influenza B, MERS-CoV, and SARS-CoV exhibited productive viral replication and tissue infection in bronchial tissues, whereas wild bird surveillance isolates such as H5N3 and H7N1 showed minimal replication when compared to pandemic H1N1 and highly pathogenic avian influenza (HPAI) H5N1. Notably, differential lung viral replication and tissue tropism were detected for H5N6 and H9N2. HPAI H5N1, H7N9, MERS-CoV, and SARS-CoV caused more severe AFC impairment than seasonal H1N1, H3N2, and influenza B viruses, correlating with their clinical severity. Overall, these findings revealed an important association between viral tropism and human transmissibility in ex vivo explants, as well as the impairment of AFC in vitro, which aligns with the clinical manifestations of disease severity across different viral strains.

Source: 

Link: https://www.microbiologyresearch.org/content/journal/jgv/10.1099/jgv.0.002281

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A #bovine #H5N1 virus efficiently replicates in differentiated #human #nasal epithelial #cells

 

Abstract

Highly pathogenic avian influenza (H5N1) viruses of clade 2.3.4.4b have caused significant losses in bird populations worldwide and repeatedly infected mammals, including humans, without sustained human to human transmission. Here we show that an H5N1 virus (H5N1Tex/24) isolated from bovine milk in Texas in 2024 replicates just as efficiently in differentiated human nasal epithelial cells as a pandemic H1N1 virus strain from 2009 (H1N1HH4/09), at both 37 °C and 33 °C. The adaptive mutations PB2 M631L and PA K497R promoted replication at 33 °C but had no effect on replication at 37 °C. An H5N1 virus (H5N1BE/22) isolated from a pelican in 2022, which lacked these mutations, replicated efficiently at 37 °C but poorly at 33 °C, and this limitation was not overcome by the introduction of the PB2 M631L and PA K497R mutations. The differentiated nasal epithelial cell cultures expressed receptors for both human and avian influenza viruses. Accordingly, no HA mutations associated with altered receptor specificity were detected. H5N1Tex/24 was able to effectively suppress the production of interferon-λ, yet remained sensitive to the antiviral effects of this cytokine. These findings suggest that H5N1Tex/24 possesses intrinsic traits supporting efficient replication in differentiated human upper airway cell cultures.

Source: npj Viruses, https://www.nature.com/npjviruses/

Link: https://www.nature.com/articles/s44298-026-00208-2

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Mechanistic and #antigenic boundaries of #Henipavirus and Parahenipavirus #glycoproteins

 

Abstract

Henipaviruses, in the Paramyxoviridae family, includes the highly virulent Nipah virus that causes reoccurring outbreaks of deadly disease. Recent discoveries of Henipavirus-like species, including the zoonotic Langya virus, have revealed much higher antigenic diversity than currently characterized and prompted the reorganization of these viruses into the Henipavirus and Parahenipavirus genera. Here, to explore the limits of structural and antigenic variation in both genera, collectively referred to as HNVs, we construct an expanded, diverse panel of HNV fusion and attachment glycoproteins from non-redundant HNV strains that better reflect global HNV diversity. We express and purify the fusion protein ectodomains and the attachment protein head domains and study their biochemical and biophysical properties. We perform immunization experiments in mice, eliciting antibodies reactive to multiple HNV fusion proteins. Cryo-electron microscopy structures elucidate molecular determinants of differential pre-fusion state stability and higher order contacts. A crystal structure of the Gamak virus attachment head domain reveals an additional domain appended to the conserved 6-bladed, β-propeller fold. Taken together, these studies expand the known structural and antigenic limits of the HNVs, reveal cross-reactive epitopes within both genera and provide foundational data for the development of broadly reactive countermeasures.

Source: 

Link: https://www.nature.com/articles/s41467-026-74212-8

____

#Antibodies to #influenza A virus #hemagglutinin and #neuraminidase limit egress and alter the physical properties of released virus particles

 

Abstract

Influenza A virus (IAV)-specific antibodies neutralize mature virions by inhibiting functional sites or, in some cases, by promoting virion aggregation. Many antibodies also act on infected cells to reduce virion yields, but the underlying mechanisms and effects on the physical properties and function of released virus particles remain incompletely characterized. Here we use flow virometry to acquire high-sensitivity yield and size measurements of virus particles released in the presence of antibodies. Combined with digital-droplet PCR and electron microscopy, this approach enables comprehensive characterization of antibody-induced changes in particle genome-content and morphology. We show that antibodies rapidly and dynamically alter released particle distributions, reducing yields and inducing the production of larger particles. Both effects result in part from aggregation induced by crosslinking of viral antigen on the infected-cell surface and inhibition of viral NA. However, yield reduction is not fully explained by aggregation, and a subset of induced larger particles are elongated virions. Finally, particles formed in the presence of HA stem-binding antibody, which does not inhibit attachment of mature virions, show reduced attachment in subsequent rounds of infection. Altogether, we uncover an unappreciated mechanism by which antibodies interfere with viral infection that occurs only during budding and release. Our work highlights the necessity of studying how the immune response shapes virus populations in the context of active infection processes.

Competing Interest Statement

T.I. is involved on a patent related to the flow virometry methodology: PCT/US2022/042125, status pending. The authors declare no other competing interests.

Funder Information Declared

Intramural Research Program of the National Institutes of Health (NIH), ZIAAI001385

G. Harold & Leila Y. Mathers Foundation, https://ror.org/02a7hjv13

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.06.15.729605v1

____

Cryo-EM structures of #Měnglà virus GP reveal combined #Ebola- and #Marburg-like epitope masking strategies for #antibody evasion

 

Abstract

Ebola virus (EBOV) and Marburg virus (MARV) are highly lethal filoviruses that cause severe hemorrhagic fever in humans. A recently identified bat-borne filovirus, Měnglà virus (MLAV), uses the same NPC1 receptor as EBOV and MARV, raising concerns about its potential cross-species transmission. Here, we report cryo-EM structures of the MLAV surface glycoprotein (GP) in its unbound form and in complex with the MARV-neutralizing antibody MR191. MLAV GP exhibits distinctive structural features in the Wing and heptad repeat 1D (HR1D) regions, retains a visible Cap structure even after protease treatment, and contains a MARV GP-like α2 helix. MR191, a broadly neutralizing marburgvirus antibody that targets the conserved NPC1 receptor-binding pocket in MLAV GP, nonetheless exhibits impaired neutralizing activity, likely due to shielding by the MLAV Cap. In addition, the MLAV mucin-like domain, α2 helix, and HR1A region hinder binding by representative broadly neutralizing ebolavirus antibodies targeting the GP-waist, including 6D6, CA45, ADI-15878, and ADI-15946. Together, these results provide the first structural insights into MLAV GP and identify immune evasion driven by structural and sequence divergence as a major challenge for pan-filovirus antibody development.

Source: 

Link: https://www.pnas.org/doi/abs/10.1073/pnas.2529436123?af=R

____

The #canine respiratory #epithelium is a permissive #ecosystem for #influenza interspecies #transmission and emergence

 

Abstract

The outcome of virus spillover ranges from dead-end infections to pandemics and is underpinned by host-pathogen interactions as well as evolutionary and epidemiological processes. The emergence of novel influenza A viruses (IAVs) has been associated with reassortment events involving multiple species, highlighting the importance of reservoir and intermediate hosts in viral emergence. Highly pathogenic H5N1 IAVs of the 2.3.4.4b genotype have caused a panzootic affecting a broad range of mammals. The role of dogs -arguably the most popular companion animal and a natural host of IAVs- in the ecology of IAVs under this new zooepidemiological scenario is unknown. To address this, we characterised the glycome of the dog respiratory epithelium, infected canine tracheal explants with multiple IAVs (including canine H3N2 and H3N8, equine H3N8, avian H3N8 and H5N1, swine H1N1, human H1N1 and H3N2, and bovine H5N1 viruses), and determined their cellular tropism. We show that the respiratory tract of dogs presents abundant sialylated glycans known to act as IAV receptors. Further, most IAVs (including 2.3.4.4b viruses) infected and replicated in dog tracheas, targeting mainly ciliated cells. Serological testing showed evidence of influenza spillover infections in dogs from the UK. Overall, our results show that the canine respiratory tract can provide a suitable environment for the generation of new IAVs. Given the multi-host contact networks of dogs in nature, they could act as recipients, bridging hosts, and/or mixing vessels for multiple IAV lineages, playing a central role in the ecology of influenza emergence.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

Medical Research Council, https://ror.org/03x94j517, MR/Y03368X/1, MC_UU_0034/2, MC_UU_0034/3

Biotechnology and Biological Sciences Research Council, BB/Y007093/1, BB/Y007298/1, BBS/E/PI/230001A, BBS/E/PI/230002A, BBS/E/PI/230002B, BBS/E/PI/230001C

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.06.04.730051v1

____

#SARS-CoV-2 #Omicron BA.2.86 and JN.1 expand #tropism in #human proximal #intestinal epithelium

 

Abstract

Omicron SARS-CoV-2 has diversified into multiple sub-lineages, complicating assessment of their intrinsic phenotypes due to background population immunity. We compare replication and biological characteristics of variants from BA.1 to JN.1 using human bronchial and lung explants, airway organoids, colon cells, and proximal intestinal enteroids. XBB.1.5 and EG.5.1 achieve higher replication titres in respiratory tissues than BA.2.86 and JN.1, indicating enhanced respiratory fitness. EG.5.1 displays dual cell-entry pathways and greater replication in alveolar epithelial cells, supporting increased lung tropism and pathogenicity. In contrast, BA.2.86 and JN.1 rely on TMPRSS2-mediated entry in airways. Notably, BA.2.86 and JN.1 replicate more efficiently than EG.5.1 in proximal intestinal enteroids in an ACE2- and TMPRSS2-dependent manner, but not in colon cells. JN.1 exhibits elevated intestinal tropism with limited proinflammatory cytokine induction, suggesting potential for faecal transmission. Here we show XBB.1.5 and EG.5.1 greater transmissibility and severity potential whereas BA.2.86 and JN.1 exhibit enhanced intestinal adaptation.

Source: 

Link: https://www.nature.com/articles/s41467-026-74111-y

____

G4 #Eurasian avian-like #H1N1 swine #influenza viruses exhibit enhanced #pathogenicity potential in mice and #pigs

 

Abstract

Currently circulating swine influenza viruses (SIVs) mainly include H1N1, H1N2, and H3N2 subtypes. In this study, two G4 genotype Eurasian avian-like (EA) H1N1 SIVs were isolated from 556 samples collected between 2023 and 2026. A systematic analysis was conducted on the two EA H1N1 isolates (FYD30 and YZF69) to assess their pandemic potential. The hemagglutinin (HA) proteins of both H1N1 viruses possessed residues 225E and 228S, indicating enhanced affinity for human-like alpha-2,6-linked sialic acid receptors, which was confirmed by receptor-binding assays. Polymerase activity tests demonstrated that the two SIVs exhibited significantly higher activity in mammalian cells, relative to avian cells, which is consistent with the efficient replication in mammalian cells. Challenge experiments revealed that both H1N1 caused significant pathogenicity in mice and pigs, with YZF69 exhibited higher virulence than FYD30. The higher virulence of YZF69 may be attributed to its molecular features, including the NP Q357K mutation, and an additional glycosylation site in HA. In conclusion, currently circulating EA H1N1 SIVs have acquired key molecular signatures of mammalian adaptation, exhibit enhanced virulence in mammals, and continue to undergo extensive reassortment driven by international swine trade. These findings highlight the potential pandemic risk of SIVs and underscore the urgent need for strengthened surveillance.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.05.12.724537v1

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A single PA-X #mutation in #bovine-origin #H5N1 #influenza virus reduces #pathogenicity in mice

 

Abstract

Dairy cows have emerged as a reservoir for human infection with highly pathogenic avian influenza (HPAI) H5N1. At the bovine-human interface, H5N1 strains may acquire adaptive mutations that influence their zoonotic potential. Sequence analysis identified a K142E substitution (bovine to human) in the PA and PA-X proteins, with the potential to affect both polymerase activity and host shutoff. Here, we used a loss-of-function approach to investigate how the bovine substitution (E142K) in PA/PA-X impacts viral replication, host shutoff activity, and pathogenicity in the human H5N1 background. Viral growth kinetics demonstrated that the virus containing the E142K substitution is attenuated, with reduced replication compared to wild-type (WT) virus. Consistently, PA-X-mediated host shutoff activity was reduced, resulting in increased induction of interferon (IFN) responses relative to WT. In vivo, mice infected with the E142K mutant virus survived, whereas infection with the WT virus was uniformly lethal. Despite comparable viral titers and inflammation score in mouse lungs, cytokine and chemokine profiling revealed distinct immune responses, with reduced CCL2 and increased CCL5 and IFN-γ in mice infected with the E142K mutant virus compared to mice infected with the WT virus. These findings indicate that increased virulence of the human-adapted strain is driven by a PA-X mutation that modulates inflammatory responses, producing distinct immune signatures linked to host survival or viral lethality rather than changes in polymerase activity by PA. Collectively, these results highlight PA-X as a key determinant of pathogenicity of H5N1 and a potential target for the rational design of antiviral strategies.

Competing Interest Statement

The authors have declared no competing interest.

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.05.09.724031v1

____

#Genetic and #biological characterization of #H9N2 avian #influenza viruses isolated from #swine in #China

 

Abstract

Background

H9N2 avian influenza virus (AIV) has been circulating in poultry in China for decades and are undergoing adaptation to mammals, posing potential pandemic risks. To investigate the prevalence of H9N2 AIVs in swine, we conducted surveillance in Shandong Province from 2021 to 2023.

Results

Two H9N2 influenza virus strains, A/swine/Shandong/417/2021(Sw/SD/417/21) and A/swine/Shandong/662/2022 (Sw/SD/662/22), were successfully isolated from swine and genetically characterized. Phylogenetic analyses showed that both isolates were reassortants containing gene segments from multiple H9N2 AIV lineages and closely related to currently circulating H9N2 AIV. Key molecular marker analysis revealed that both isolates carried mammalian-adaptive residues in the HA receptor-binding sites (183 N, 190 V, 226 L), a novel HA cleavage site variant (PSKSSRGL), PB2 mutations (A588V, E627V), and the M2 S31N substitution, suggesting potential adaptation to mammalian hosts and resistance to adamantane antivirals. Mice infection experiments demonstrated efficient viral replication in the respiratory tract, particularly in the lungs, but only mild histopathological changes were observed, with no significant weight loss or mortality, indicating low pathogenicity in mice. Serological surveillance of 3,172 swine serum samples showed a low prevalence of H9N2 influenza virus infection (0.44%), with positive samples sporadically distributed across regions and years.

Conclusion

In summary, although H9N2 AIV infection in swine is rare and generally mild, the presence of mammalian-adaptive markers and reassortant genomes highlights the potential risk of cross-species transmission and subclinical adaptation. Continuous avian–swine–human influenza surveillance is therefore essential to mitigate the potential threat posed by H9N2 AIV.

Source: 

Link: https://link.springer.com/article/10.1186/s12917-026-05501-z

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Variable #transmission efficiency of #mammalian origin #HPAI D1.1 #H5N1 strains in #ferrets

 

Abstract

Highly pathogenic avian influenza H5N1 2.3.4.4b genotype D1.1 lineage continues to predominate in the United States wild bird population and has spilled over into dairy cattle three independent times. To assess the transmission risk of this sublineage, we performed direct-contact transmission experiments for three distinct D1.1 strains in ferrets. Two of these strains were isolated from humans and one from a lethal cat infection. We found that only one human isolate (A/NV/10/2025) was able to transmit efficiently between ferrets. Compared to the other strains, this isolate harbored the mammalian adaptive PB2 D701N mutation, suggesting this mutation may be critical for D1.1 transmission as opposed to the PB2 E627K substitution present in the lethal cat isolate. Based on these data we conclude that the transmission fitness of D1.1 strains is modest but that special attention should be paid to emergence of adaptation at the PB2 701 position.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

National Institute of Allergy and Infectious Diseases, https://ror.org/043z4tv69, 75N93021C00015, 75N93021C00017

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.05.07.722809v1

____

Loss of nsp14-exonuclease activity impairs the #replication, proofreading, fitness, and #pathogenesis of #SARS-CoV-2

 

ABSTRACT

Coronaviruses (CoVs) replicate their RNA genomes with a higher degree of fidelity than other RNA viruses, a mechanism mediated by the proofreading and recombination activities of the exoribonuclease domain of replicase nonstructural protein 14 (nsp14-ExoN). Both murine hepatitis virus (MHV) and SARS-CoV tolerate nsp14-ExoN loss-of-function mutations (ExoN−) (D90A and E92A), but have impaired replication fidelity and pathogenesis; yet identical substitutions in MERS-CoV and SARS-CoV-2 have been reported to be lethal. Here, we report a saturation mutagenesis approach facilitating the recovery and analysis of several constellations of SARS-CoV-2 nsp14 ExoN-inactivating, loss-of-function substitutions, including the canonical D90A and E92A. Biochemical assays with purified WT or ExoN-nsp10-14 fusion proteins confirmed that active site substitutions abolished ExoN activity (ExoN−). SARS-CoV-2 ExoN− viruses exhibited impaired replication, RNA synthesis, and recombination, as well as decreased replication fidelity and loss of fitness in vitro. ExoN− viruses were significantly attenuated for replication in human primary airway epithelial cells and were attenuated for replication and pathogenesis in WT mice, as well as the highly susceptible K18 transgenic mice. In the absence of interferon signaling in vivo, SARS-CoV and SARS-CoV-2 ExoN− viral replication could be partially restored. These results demonstrate that SARS-CoV-2 ExoN− viruses are viable but highly impaired for replication, fitness, and fidelity in vitro, as well as innate immune antagonism and pathogenesis in vivo. Collectively, our results solidify the multiple critical roles of nsp14-ExoN across CoV genera and establish new approaches for rescuing and analyzing loss-of-function substitutions in studies of CoV replication, pathogenesis, and evolution.

Source: 

Link: https://journals.asm.org/doi/10.1128/mbio.00073-26

____

Opposing #cell type preferences for binding and #replication shape #influenza A virus infection in #human #airways

 

Abstract

Influenza A viruses (IAVs) pose a persistent threat to human health through seasonal epidemics and zoonotic spillover from avian reservoirs. As respiratory pathogens, they primarily target the airway epithelium. However, it remains unclear how host cell-specific barriers jointly shape viral tropism and replication in primary human airway cultures. Here, we show that avian IAVs can infect ciliated and secretory cells but preferentially bind to ciliated cells, consistent with higher abundance of their receptor alpha2,3-linked sialic acids, specifically sialyl Lewis X glycans, present on the apical surface of ciliated cells. Replication levels were comparable between secretory and ciliated cells for the avian strains, resulting in an overall preference for ciliated cells. In contrast, human IAVs also preferentially bind to ciliated cells but independently of alpha2,6-linked sialic acid abundance. Human IAVs replicate more efficiently than avian IAVs due to their ability to utilize human ANP32 proteins, but they also exhibit cell type-specific differences due to ANP32, allowing for higher viral RNA levels in secretory cells. Thus, preferential binding to ciliated cells coupled with enhanced replication in secretory cells equalizes overall infection levels across cell types for human IAVs. Together, our findings highlight the spatiotemporal complexity and interplay of IAV infection dynamics in the airway epithelium and redefine current models of influenza A virus tropism.

Competing Interest Statement

The authors have declared no competing interest.

Funder Information Declared

Swiss National Science Foundation, 310030_204166

UZH Candoc, FK-25-039

Source: 

Link: https://www.biorxiv.org/content/10.64898/2026.05.04.722582v1

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#Replication Efficiency of Contemporary Highly Pathogenic Avian #Influenza #H5N1 Virus Isolates in #Human #Nasal Epithelium Model

 

Abstract

Replication of influenza A virus in human nasal epithelium affects transmissibility and disease. We compared virus replication and immune responses in human nasal epithelium infected with seasonal and highly pathogenic avian influenza A(H5N1) viruses. Contemporary H5N1 viruses replicated better than the historical isolate; however, interferon response to B3.13 genotype viruses was dampened.

Source: 

Link: https://wwwnc.cdc.gov/eid/article/32/5/26-0053_article

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