Intermediate filaments (IFs) have long been regarded as a static scaffold responsible for maintaining cellular structure and integrity. However, recent studies have revealed that IFs, particularly vimentin and keratin, exert a profound and versatile influence on viral infection. In this narrative review, we summarize how these IFs influence multiple stages of the viral life cycle, including attachment/entry, replication, intracellular trafficking, assembly, and egress. We further discuss their contributions to cell-to-cell spread, host immune regulation, and oncogenic processes. Collectively, these findings illustrate how viruses exploit or remodel the IF network to facilitate propagation, and highlight IF - virus interfaces as potential targets for antiviral intervention.

Co-infection with avian influenza virus (AIV) is common in poultry populations, primarily due to the diversity of viral subtypes and the practice of mixed-species farming. However, the replication dynamics of different AIV subtypes within distinct avian host-species remain unclear. In this study, we isolated and identified H5N1 and H5N8 AIVs from a naturally co-infected duck and characterized their biological characteristic regarding co-infection under both in vitro and in vivo conditions. Phylogenetic analysis revealed the HA genes of both H5 subtype AIVs belonged to clade 2.3.2.1d, sharing identical segments excluding differences in the M and NA genes. The H5N1 virus exhibited significantly higher infectivity and replication than the H5N8 virus in chicken embryo fibroblasts (CEF) and duck embryo fibroblasts (DEF). Serial passages of co-infection in vitro revealed that H5N1 virus became dominant in the first passage in both CEF and DEF cells. In vivo, both viruses caused high mortality in SPF chickens, while nonlethal in SPF ducks. In co-infected SPF chickens, H5N1 virus exhibited a pronounced replication advantage and higher viral loads. However, compared with single infections, co-infection delayed death time and reduced replication capacity. Conversely, in co-infected SPF ducks, H5N8 virus exhibited a significant replication advantage; however, compared to single infections, the replication capacity of H5N8 virus was reduced while H5N1 virus was maintained. These findings suggest that host-species influence the replication fitness and dominance of AIVs during co-infection, highlighting the importance of enhanced epidemiological surveillance and reducing mixed-species farming to minimize reassortment and evolution of high-risk AIVs.

Currently, the continuous evolution of influenza A virus (IAV) still presents a major threat to human and animal health. Our sequence analysis reveals high variation at site 94 of the PA/PA-X gene in recent H5N1 and H7N9 IAV strains. To be noted, I94V mutation has become prevalent in recent human H7N9 viruses. However, the specific role of PA I94V mutation in regulating viral pathogenicity of H7N9 virus both in mammals and avian species is currently unknown. We first demonstrated that PA/PA-X I94V mutation increases the pathogenicity and viral fitness in mice while attenuating viral virulence in chickens. To determine the contribution of PA-I94V and PA-X-I94V in regulating the host adaptation of the H7N9 virus, we then systematically compared the phenotypes of the PA-X-deficient virus and the PA-X-deficient virus with PA I94V mutation. Notably, when deleted PA-X expression, the PA I94V mutation has no obvious effect on viral fitness in mice and chickens, suggesting the direct role of PA-X I94V in modulating viral virulence of H7N9 virus. Mechanistically, PA-X I94V mutation significantly increases viral polymerase activity and viral replication in mammalian cells. In addition, PA-X I94V mutation also modulates the host shutoff activity of PA-X and exerts an obvious role in inhibiting antiviral and cell death-related response. Collectively, our study advances our understanding of how the adaptive site in PA-X regulates host response and viral fitness in mice and chickens, offering deeper insights into the complex pathogenesis of influenza A virus.

Pathological events in a wide range of diseases, from severe infections to sterile inflammatory disorders. PMR serves as the terminal step that releases large quantities of damage-associated molecular patterns (DAMPs)—intracellular molecules that act as danger signals once outside the cell. These DAMPs can trigger strong inflammatory responses, and in many cases, may precipitate a cytokine storm, a hyperactive immune reaction that often amplifies tissue injury beyond the initial insult. For decades, scientists generally believed that PMR resulted from membrane pore-forming cell death, such as pyroptosis or necroptosis, which causes osmotic imbalance and passive membrane swelling. However, in recent years, it was discovered that Nerve Injury-Induced Protein 1 (NINJ1) mediates PMR through active oligomerization. This review first uses pathogen infection as a classical model to explore how multiple cell death pathways converge on plasma membrane rupture (PMR). Subsequently, we elaborate on the structure and function of NINJ1 as a core executor of PMR. Finally, we broaden our perspective from infection to other non-infectious but equally PMR-driven major diseases, and systematically evaluate the commonalities and prospects of NINJ1-targeted therapeutic strategies across different pathological scenarios. It is hoped that this will provide new insights for future researchers in this field.

ABSTRACT Long non-coding RNAs (LncRNAs) play pivotal regulatory roles in various biological processes, notably in immune regulation and viral infection. We previously identified the broad anti-influenza activity for LncRNA#61. Here, we further investigate the mechanism underlying its antiviral effect, both in vitro and in vivo. Using a lipid-nanoparticle-based delivery strategy, LncRNA#61 was successfully delivered into mice and effectively attenuated the replication and virulence of the highly pathogenic H5N1 influenza virus. Integrative transcriptomic analysis revealed that forced expression of LncRNA#61 markedly activated lipid metabolism, cell death, and Ragulator-Rag-mTORC1 pathways. Quantitative reverse transcription PCR analysis and a targeting metabolic assay further confirmed that LncRNA#61 is actively involved in regulating these pathways. Subsequent functional studies demonstrated that LncRNA#61 consistently enhances GSDMD-mediated pyroptosis both in murine LET-1 and canine MDCK cells. Notably, such pyroptosis was found to restrict H5N1 influenza virus replication. Intriguingly, ectopic expression of viral PA-X protein enhanced antiviral activity of LncRNA#61 both in vitro and in vivo. Mechanistically, PA-X interacts with LncRNA#61 and promotes LncRNA#61-mediated pyroptosis via a RagA-dependent reactive oxygen species pathway. Collectively, we here propose a novel model in which viral and host factors cooperate to activate a pro-death antiviral pathway. Our findings not only advance the fundamental knowledge of virus–host interactions but also cross-link cell death, innate immunity, and metabolic regulation, pinpointing novel therapeutic targets against influenza.IMPORTANCEA current priority in anti-influenza research is developing broad-spectrum, host-directed therapeutics with low resistance risk. Here, we reveal that LncRNA#61-induced pyroptosis exerts an antiviral effect by restricting H5N1 virus replication both in vitro and in vivo, highlighting a novel cooperative virus–host interaction that enhances antiviral immunity. Key contributions include the following: (i) identifying pyroptosis as a direct executioner mechanism that restricts H5N1 virus infection; (ii) revealing the unexpected role of forced expression of viral PA-X in augmenting antiviral activity of host LncRNA#61; and (iii) deciphering that LncRNA#61 interacts with PA-X and synergistically promotes GSDMD-mediated pyroptosis through a RagA‑ROS signaling cascade. Collectively, our work elucidates a novel antiviral mechanism wherein host LncRNA and viral protein co-opt the RagA-ROS-GSDMD axis to drive pyroptosis and inhibit viral replication. This discovery innovatively establishes a novel connection among viral pathogenesis, host cell death, and cellular metabolism, offering a fresh, integrative perspective for future studies on host-directed antiviral strategies.

Background Newcastle disease virus (NDV), a significant avian pathogen and promising oncolytic agent, relies on host metabolic pathways for replication. However, the metabolic alterations induced by NDV, particularly the connections at the gene and protein levels, remain poorly characterized. Results This study employed integrated transcriptomic, proteomic, and non-targeted metabolomic analyses to delineate the global metabolic changes in NDV-infected A549 cells. We identified 8,101 differentially expressed genes (DEGs), 1,587 differentially expressed proteins (DEPs), and 257 differentially expressed metabolites (DEMs) associated with organelle function, innate immunity, and metabolism. Crucially, our multi-omics approach revealed that NDV significantly remodels glycerophospholipid metabolism. NDV depleted Lysophosphatidylcholine (LPC) and Lysophosphatidylethanolamine (LPE), as well as specific phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species, while increasing phosphatidylserine (PS) at the late stage of infection. Strikingly, exogenous supplementation of unsaturated fatty acids, choline, phosphorylcholine, ethanolamine, phosphatidylethanolamine, and inositol markedly enhanced NDV replication. Concomitantly, NDV infection upregulated the transcriptional levels of key enzymes involved in glycerophospholipid biosynthesis. Conclusions This study demonstrates for the first time that NDV actively reprograms host glycerophospholipid metabolism to facilitate viral replication. This study uncovers a novel mechanism of NDV-host interaction and provides crucial insights for oncolytic strategies targeting this metabolic vulnerability.

INTRODUCTION:The persistent circulation of H7N9 avian influenza viruses (AIVs) in poultry continues to threaten public health. While T cell-mediated immunity is known to enhance and promote cross-protective antiviral responses, exploiting avian T-cell immunity to improve vaccine efficacy remains challenging. Hemagglutinin (HA)-specific T-cell responses may serve as a key mechanism to broaden protection against antigenically diverse H7N9 strains. OBJECTIVES:This study aimed to determine whether HA-specific T-cell responses can enhance heterologous protection against H7N9 in poultry and whether T-cell epitope-supplemented vaccines can improve both humoral and cellular immunity. METHODS:Conserved HA epitopes recognized by chicken T cells were identified using immunological assays, resulting in two CD4 epitopes (P12, P15), one CD8 epitope (P4), and one dual-recognized epitope (P10). These peptides were pooled (PP) and incorporated into an H7N9 whole inactivated virus (WIV) vaccine formulated with a DDA/TDB nanoparticle adjuvant (H7N9 WIV + PP). Immune responses and protection were assessed following homologous and heterologous H7N9 virus challenge. RESULTS:The H7N9 WIV + PP vaccine elicited stronger humoral and cellular immune responses compared with WIV alone. Birds receiving H7N9 WIV + PP displayed significantly improved protection against antigenically divergent H7N9 challenge. Mechanistically, pooled epitope supplementation enhanced HA peptide-specific T-cell responses, which subsequently augmented HA-specific antibody production via peptide-specific CD4+ T-cell help. Concurrently, both CD4+ and CD8+ T cells and their effector molecules were mobilized, contributing to broad protective immunity. CONCLUSION:Incorporating conserved HA-derived T-cell epitopes into an H7N9 WIV vaccine enhances both humoral and cellular immunity and confers broader protection against heterologous H7N9 strains. This strategy provides a promising approach for developing broad-spectrum poultry vaccines targeting continuously evolving AIVs.

The H7N9 subtype of avian influenza virus (AIV) poses a significant and ongoing threat to public health. As a critical structural and functional component, the viral nucleoprotein (NP) is abundantly expressed during the early stages of AIV replication; however, its interactions with host proteins and their functional consequences remain largely uncharacterized. This study aimed to identify the NP-host interaction and elucidate the mechanisms by which these interactions modulate AIV replication. Here, we employed mass spectrometry and identified the DEAD-box helicase 6 (DDX6) as a novel NP-interacting partner, an association found to be regulated by an interferon-stimulated gene (ISG15). The NP-DDX6 interaction was robustly validated by co-immunoprecipitation, immunofluorescence co-localization, bimolecular fluorescence complementation, and molecular docking assays. Functional investigations revealed that DDX6 acts as a potent negative regulator of AIV replication. Mechanistically, DDX6 not only impaired the nuclear import of NP and suppressed viral polymerase activity, but also stimulated the production of interferon (IFN)-α/β. This IFN-I induction, in turn, triggers the expression of downstream antiviral effectors such as ISG15. Furthermore, we uncovered that DDX6 fine-tunes this pathway by playing a sophisticated dual regulatory role: it enhances the pool of free, antiviral ISG15 monomers while concurrently reducing ISGylation via two deubiquitinases (USP16/USP18). Collectively, these findings not only establish DDX6 as a crucial host factor with potent antiviral activity but also enrich our understanding of host-virus interaction networks.

H7N9 subtype avian influenza virus (AIV) is a great threat for poultry industry in China. Serological assays that differentiate infected and vaccinated animals (DIVA) can facilitate H7N9 virus monitoring in vaccinated poultry. A serological method based on a peptide for DIVA diagnosis of H7N9 subtype AIV was previously established. However, complex manufacturing techniques are required for production of chemically-synthesized peptides, casting the affordability and accessibility issues of such assays in poultry farms. Here, to develop an H7N9 DIVA assay suitable for poultry use, a recombinant DIVA peptide fusion protein (DPFP) was expressed in E. coli and its potential as a diagnostic antigen for H7N9 DIVA was assessed. The DPFP composed of the E. coli thioredoxin A, the H7N9 DIVA peptide and His affinity tag was efficiently expressed in a soluble form in E. coli. The DPFP was recognized by H7N9 virus infection serum rather than by H7N9 vaccination serum as determined using immunoblotting. In addition, enzyme-linked immunosorbent assay based on the DPFP was established and optimized, which can well differentiate H7N9 virus infected and vaccinated chickens. OD450 of 0.5 was defined as the cut-off value for H7N9 DIVA diagnosis. Our study indicates that prokaryotic expression system can be used as a promising platform for preparation of diagnostic antigen for discriminating H7N9 virus infected and vaccinated chickens. The method established herein can be employed as an effective and affordable approach for diagnosis and control of H7N9 avian influenza in poultry.

The H9N2 subtype avian influenza virus (AIV) hemagglutinin (HA) protein is a major immunogen in which HA1 is a genetic variant and HA2 is relatively conserved. Identifying broad-spectrum antigen epitopes targeting HA1 is crucial for vaccine design and detection. Based on the phylogenetic and serological analyses, we identified 2 antigenic groups and 3 representative viruses: A/chicken/Jiangsu/JY040218C/2019, A/pigeon/Jiangsu/JY020616/2019, and A/chicken/Jiangsu/WX090312/2018. An overlapping peptide library was synthesized using HA1 amino acid sequences of the viruses as templates. Through peptide scanning of the sera against different strains of H9N2 subtype AIV, we identified peptides from 4 regions (H9-2/3, H9-20/21, H9-26, and H9-29/30/31) that demonstrated broad-spectrum reactivity. Immunological assay results demonstrated that H9-21 (219RIFKPLIGPRPLVNGLMGRI239), H9-26 (269SGESHGRILKTDLKMGSCTV289), and H9-30 (309YAFGNCPKYI GVKSLKLAVG329) effectively induced antibody generation and conferred partial protective efficacy against the parent virus JY040218C. The results of lymphocyte proliferation and ELISpot assays indicated that peptides H9-15 (159MRWLTQKNNAYPTQDAQYTN179), H9-22 (229PLVNGLMGRINYYWSVLKP G249), and H9-23 (239NYYWSVLKPGQTLRIKSDGN259) could effectively stimulate the expression of interferon-gamma in peripheral blood lymphocytes of chickens immunized against different strains of H9N2 AIV. Collectively, 5 novel cell epitopes H9-15, H9-22, H9-23, H9-26, and H9-30, including the best B cell epitope H9-26 and the best T cells epitope H9-22, were identified that could be targeted for vaccine design or detection approaches against H9N2 AIVs.

Host cells combat avian influenza virus (AIV) infection by targeting viral polymerase PB2 for degradation, yet how the virus counteracts this remains elusive. In this study, we analyze the host proteins interacting with H5 AIV PB2 and identify USP39 as a deubiquitinase with dual functions in viral replication. Catalytically, USP39 directly deubiquitinates PB2 at lysine 660 (K660), preventing its degradation and sustaining polymerase activity. In parallel, independently of enzymatic activity, USP39 promotes PB2-PB1 association, facilitating formation of ribonucleoprotein (RNP) complexes. These complementary functions amplify viral RNA synthesis, dampen host antiviral responses, and drive efficient viral replication. Consistently, a PB2 K660R substitution enhances viral replication in vitro and increases pathogenicity in mice. Our findings reveal a mechanism by which AIV hijacks USP39 to circumvent host ubiquitination, facilitate RNP biogenesis, and identify USP39 as a promising therapeutic target for broadly effective antivirals against pandemic-prone H5 viruses.

H9N2 subtype avian influenza virus (AIV) and avian pathogenic Escherichia coli (APEC) are prevalent pathogens in the poultry industry, and their co-infection results in synergistic pathogenicity in chickens and significant economic losses. However, the direct interactions between H9N2 AIV and APEC during infection remain unclear. We preincubated H9N2 AIV particles with APEC and found that H9N2 AIV could bind directly to the surface of APEC. The H9N2 AIV-APEC complex enhanced bacterial adhesion to chicken embryo fibroblasts and chicken macrophages (HD11), which could be inhibited by antiserum against H9N2 AIV. The chicken challenge experiment demonstrated that the complex caused significant reductions in body weight, increased bacterial loading in various organs, and resulted in more severe histopathological lesions than APEC alone. However, the complex did not affect virus replication and shedding but elicited a robust hemagglutination inhibition antibody response when compared to H9N2 AIV alone. Additionally, we found that a 3 h interaction between H9N2 AIV and APEC in the complex led to significant morphological changes in the viral envelope, ultimately resulting in the inhibition of virus replication in vivo. These findings suggest that the direct interaction between H9N2 AIV and APEC is more conducive to bacterial infection, providing new insights into the synergistic interaction between bacteria and influenza viruses during the early stages of co-infection with H9N2 AIV and APEC.IMPORTANCEH9N2 subtype avian influenza virus and avian pathogenic Escherichia coli are common pathogens in the poultry industry. Their co-infection causes more severe harm and leads to substantial economic losses for the industry, yet the direct interaction between them remained unclear previously. Studies have found that they can bind directly; the formed complex facilitates bacterial invasion of chicken hosts and results in more severe damage. This uncovers the key reason for the greater harm of co-infection, providing a new direction for the prevention and control of such diseases in the poultry industry.

Highly pathogenic avian influenza viruses (AIV) primarily circulate within poultry populations. However, continuous evolution and mutation accumulation drive antigenic drift and may enable the virus to evade host immunity and cross the species barrier. To identify residues associated with antigenic changes and virulence in the H5N1 virus under immune selection pressure, SPF chickens, SPF chicken embryos, and chicken embryo fibroblast cells were used as model to serially passage the SY (Re-5 like) virus in the presence of homologous chicken antiserum. Progeny viruses escaped the neutralizing capacity of the antiserum were sequenced. A total of twelve amino acid mutation sites were identified in the HA, PB2, and PB1 proteins. The results showed that in the HA of the H5N1 virus, both K205N and K205T mutation patterns resulted in a significant reduction in HI titers and microneutralization titers when tested with chicken antisera. The K32M and E69K mutations in PB2, along with the M246I mutation in PB1 could effectively attenuate viral pathogenicity in mice, whereas the S155N mutation in PB2 significantly enhanced it. Notably, under the immune pressure, the S155N mutation in PB2 delayed the emergence of K205N substitution in HA. This in vivo and in vitro method for selecting immune-escape mutants provides a valuable tool for predicting emerging antigenic variants and mammalian adaptive mutations, as well as elucidating the co-evolution dynamics between surface and internal genes in H5N1 viruses.

Accumulating studies have identified the pivotal role of long non-coding RNAs (lncRNAs) in participating in host-virus interactions during virus infections. However, the regulatory roles of lncRNAs in influenza A virus (IAV) infection are still not fully elucidated. In this study, using high-throughput sequencing, we comprehensively compared the expression profiles of lncRNAs and mRNAs in mouse lungs infected either with the nonpathogenic parental (SDL124) H7N9 virus or its moderately pathogenic mouse-adapted (S8) variant. A total of 7636 significantly differentially expressed (SDE) lncRNAs were obtained in the S8-infected group compared to the mock group. As for the SDL124 group, 1042 SDE lncRNAs were identified. Subsequently, the mRNAs co-expressed with SDE lncRNAs were subjected to functional annotation and pathway enrichment analysis. The results indicated that the target mRNAs regulated by the S8 virus were mainly enriched in various immunological processes and exhibited a strong correlation with inflammatory-related signaling pathways. Moreover, 12 lncRNAs and 10 mRNAs co-expressed with SDE lncRNAs were selected and successfully verified by RT-qPCR. Among these lncRNAs, NONMMUG032982.2 and NONMMUG032328.2 exhibited strong antiviral activity against IAV. Additionally, these two lncRNAs were chosen for further in-depth bioinformatics analysis, including transcription factor prediction, coding capacity assessment, genomic location, construction of secondary structure, and prediction of potential interacting proteins. Taken together, these findings provide a cluster of lncRNAs probably associated with the virulence of IAV in mice and shed light on the anti-IAV effects of two functional lncRNAs, establishing a molecular foundation for further exploring the regulatory mechanisms of lncRNAs in IAV infection.

Non-neutralizing antibodies contribute to protection against the H7N9 subtype avian influenza virus through Fc effector functions. Antibody subclass plays a critical role in determining binding affinity to Fc receptors (FcR) and downstream Fc effector functions. In this study, to assess whether antibody subclass switching can enhance antibody protective efficacy, subclass of a non-neutralizing monoclonal antibody (mAb) against the hemagglutinin of H7N9 virus was switched from IgG1 to IgG2a, and their activity and protective efficacy were assessed. Subclass switching caused no significant changes in antigen binding, hemagglutination-inhibition and virus neutralizing activities of the antibody. The hybridoma-derived mAb conferred no protection against H7N9 virus infection. Interestingly, both IgG2a and IgG1 antibodies produced in CHO cells provided full protection against mortality caused by a sublethal H7N9 virus challenge. Upon lethal challenge, IgG2a antibody conferred 70% protection, whereas its IgG1 counterpart only provided 30% protection. However, both antibodies did not decrease virus loads in mouse lungs. Moreover, compared to the IgG1 antibody, affinity of the subclass-switched IgG2a antibody to FcR was significantly increased, as evidenced by enhanced binding of the antibody to recombinant murine FcγRI and to FcRs on murine macrophages. Our findings highlighted a benefit of subclass-engineering to antibody modification and development of therapeutic antibodies.

As the world's largest waterfowl producer, China faces economic losses from Goose Parvovirus (GPV), worsened by waterfowl circovirus co-infections that increase pathogenicity and immunosuppression. However, current surveillance systems for these viruses lack systematization. This study conducted a comprehensive genetic analysis of GPV and waterfowl circovirus to identify genomic characteristics and recombination events. Surveillance analysis of GPV revealed a significant host-associated genotypic divergence from 2018 to 2024 in China. Goose isolates were predominantly the Mutated GPV (MGPV, 87%), while duck isolates were mainly the duck-adapted Novel GPV (NGPV, 88%). This divergence was corroborated by a global analysis, which confirmed high intra-clade similarity but substantial overall genetic diversity. Whole-genome recombination analysis revealed one isolate as a unique NGPV recombinant, with an NGPV strain as the major parent and an Early GPV strain as the minor parent. Furthermore, our study identified a persistent, host-specific co-circulating GPV and waterfowl circovirus: MGPV/GoCV in geese and NGPV/DuCV in ducks. Phylogenetic analysis shows that waterfowl circovirus exhibits significant genetic diversity: GoCV has two lineages (GoCV-I prevalent in China, GoCV-II with cross-species transmission to European Anser anser), while DuCV has three genotypes with distinct geographical distributions and host ranges across Asia and North America. Genomic analysis corroborated these phylogenetic findings and indicated ongoing genetic variation and recombination as key drivers of waterfowl circovirus evolution. In conclusion, this study systematically elucidates the ongoing adaptive evolution and genetic plasticity of GPV and waterfowl circovirus, providing a scientific basis for targeted waterfowl disease prevention strategies.

H3 subtype avian influenza viruses (AIVs) are frequently detected in poultry and wild birds, however, systematic characterization of contemporary isolates remains limited. We aimed to investigate the genetic evolution, pathogenicity, and transmission characteristics of H3 subtype AIVs circulating in Eastern China. Seven H3 subtype AIVs isolated between 2014 and 2021, including five H3N2, one H3N3, and one H3N6 strain, were analyzed. Phylogenetic analysis showed that all isolates belonged to the Eurasian lineage. Evidence of extensive reassortment with other AIV subtypes, as well as adaptive mutations associated with pathogenicity, and cross-species transmission, particularly in H3N2 subtype AIVs, was identified. Notably, H3N2 subtype AIVs exhibited dual receptor-binding properties, recognizing both SA α-2,3-Gal and SA α-2,6-Gal receptors. Although all isolates demonstrated low pathogenicity in chickens, mice, and guinea pigs, variations in transmission efficiency were observed. The H3N2 strain A/Duck/Anhui/LY/2021 showed the highest capacity for cross-species and aerosol transmission among guinea pigs. Overall, these findings indicate that H3 subtype AIVs have the potential for cross-species transmission and highlight the importance of continued surveillance of H3 subtype AIVs circulating in nature.

Clade 2.3.4.4b H5Nx highly pathogenic avian influenza viruses (HPAIVs) have caused extensive outbreaks in poultry worldwide. H5 HPAIVs have caused sporadic but severe human infections in China, representing a persistent zoonotic threat. Here, we identified a duck-origin H5N6 HPAIV (A/Duck/Jiangsu/628/2022) through routine surveillance and assessed its biological characteristics and mammalian pathogenesis. Phylogenetic analysis revealed >98% nucleotide identity between strain 628 and the concurrent human H5N6 strain A/Yangzhou/125/2022. Molecular characterization identified multiple mammalian adaptation markers: hemagglutinin substitutions (S137A, T160A, T192I) associated with enhanced human receptor binding; neuraminidase mutations (I117T, D198N) linked to reduced neuraminidase inhibitor susceptibility; and polymerase complex changes (PB1-D622G, PA-K142Q) conferring increased mammalian cell replication. In vitro studies demonstrated that 628 virus replicated more efficiently in mammalian than in avian cells and exhibited dual receptor-binding specificity. Mouse pathogenicity assays revealed moderate virulence with progressive lung pathology. Critically, transmission experiments confirmed both direct contact and airborne transmission capabilities of 628 in guinea pigs. These findings demonstrate that circulating H5N6 viruses have acquired partial mammalian adaptation while retaining avian fitness, significantly elevating pandemic potential. Enhanced surveillance of wild bird populations, poultry farms, and live poultry markets is urgently needed to develop effective prevention and control strategies.

H5 subtype avian influenza virus (AIV) can infect both chickens and ducks, leading to substantial economic losses. Nevertheless, certain strains cause silent infections in ducks. In this study, a goose-origin clade 2.3.4.4h H5N6 AIV was isolated, which caused high mortality in mixed-gender white leghorn chickens but no deaths in mixed-gender mallard ducks. After independent serial in vitro passage in duck embryo fibroblasts (DEFs) and in vivo passage in specific-pathogen-free (SPF) ducks, the DEF-passage 10 (P10) virus induced markedly higher mortality rates and viral loads in SPF ducks compared to the DEF-P1 virus and the original parental virus prior to passage. Similarly, the in vivo-passaged P3 and P4 viruses exhibited significantly higher mortality rates than the P1 virus in SPF ducks, with 100% mortality and markedly increased viral titers in the organs. A whole-genome SNP analysis identified seven high-frequency mutations in the M1, NA and NS1 proteins. The NS1-F161L substitution virus exhibited significantly increased mortality rates, viral loads in multiple tissues, and a robustly induced innate immune response in ducks. Furthermore, dynamic evolutionary variations in the NS1 protein among global H5 avian influenza viruses revealed that the NS1-F161L substitution became dominant in clade 2.3.4.4b viruses in 2021 and subsequent years. Collectively, our findings demonstrate that host-driven adaptation can rapidly increase the pathogenicity of H5N6 AIVs in ducks and identify NS1-F161L as a critical virulence marker. These results offer novel insights relevant to the molecular surveillance, virulence prediction, and risk assessment of circulating H5 AIVs in waterfowl.