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.

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.

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.

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.

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.

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.

Newly emerging or “re-emerging” influenza viruses have been regarded as a huge global threat to human public health. However, there are few reports of human deaths caused by newly emerging influenza viruses derived from pigs and poultry. Here, we described the clinical and virological features of a fatal encephalitis caused by a novel H3N2 reassortant virus generated from swine H3N2 and human H7N9 viruses. A 7-year-old boy was diagnosed with acute encephalitis in Yixing, China, in August 2022. Chest computed tomography (CT) showed mild pneumonia. Brain CT indicated acute encephalitis companied brain parenchyma swelling. Haematological examinations revealed a markedly elevation of lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, creatine kinase and cytokines. Pathogenic analysis confirmed that a novel H3N2 virus (A/Yixing/805/2022, YX805) was responsible for this case. Phylogenetic analysis showed that the surface protein-coding genes were originated from swine-origin H3N2 viruses, whereas the internal protein-coding genes were derived from human-origin H7N9 viruses. This virus triggers stronger cytokines storm than these genetically related H7N9 viruses and has a natural resistance to neuraminidase inhibitors. The YX805 virus is highly pathogenic to mice. Our study highlights the urgent need to enhance epidemiological surveys for reassortment events between swine and avian influenza virus by full genome sequencing.

Currently, there is increasing spillover of highly pathogenic H5N1 avian influenza virus (AIV) to mammals, raising a concern of pandemic threat about this virus. Although the function of PA protein of the influenza virus is well understood, the understanding of how phosphorylation regulates this protein and influenza viral life cycle is still limited. We previously identified PA S225 as the phosphorylation site in the highly pathogenic H5N1 AIV. In this study, we investigated the role of phosphorylation in regulating PA function and viral fitness through dephosphorylation (PA S225A) or continuous phosphorylation (PA S225E)-mimetic mutation of PA S225. Structure analysis revealed that PA S225A or PA S225E mutation had no obvious effect on the structure of PA protein. Replication assay in vitro showed that PA S225A phosphorylation-ablative mutation significantly inhibited virus replication both in mammalian and avian-derived cells, while PA S225E enhanced viral replication in these cells. Correspondingly, PA S225A dephosphorylation significantly attenuated viral replication and virulence in mice, while PA S225E enhanced these aspects in mice. Mechanistically, PA S225A mutation significantly decreased viral polymerase activity, disabled viral ribonucleoprotein complex (vRNP) assembly and attenuated PA nuclear accumulation. Altogether, our study directly suggested that phosphorylation of PA protein at site S225 enhances viral fitness of the highly pathogenic H5N1 virus in mammals by assuring effective vRNP activity, providing a framework for further study of phosphorylation events in influenza virus life cycle.

The novel H10N3 avian influenza virus (AIV) has infected four individuals since 2021 and caused severe respiratory damage, posing a significant threat to public health. However, its pathogenic mechanisms remain poorly understood. Our findings revealed that H10N3 infection induces severe lung damage and causes death in mice, even at low doses. The elevated levels of multiple pro-inflammatory factors in the bronchoalveolar lavage fluid were significantly increased during infection, displaying hallmarks of a cytokine storm. Transcriptome sequencing further revealed systematic activation of inflammation-related pathways, predicting that viral infection induces multiple forms of programmed cell death, including apoptosis, pyroptosis, and necroptosis. Protein-level validation showed that the activation of key cell death markers, including Caspase-3, GSDMD, and MLKL, significantly increased as the infection progressed, with their dynamic changes correlating strongly with the expression pattern of viral proteins. This study elucidates the central role of the synergistic effect between the cytokine storm and multiple cell death pathways in H10N3 pathogenesis. These findings not only advance our understanding of the pathogenic mechanisms of AIVs but also provide a critical theoretical basis for the development of targeted therapeutic strategies.

The H7N9 subtype avian influenza virus (AIV) is currently the subtype with the highest number of human infection cases, with a mortality rate of nearly 40 %,40 %, posing a serious threat to public health. We have previously reported that two avian H7N9 isolates (A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013) exhibit distinct pathogenicity in mice, in which the polymerase proteins cooperatively increased pathogenicity in mice. Still, the enhancement did not reach the level of JTC11. To further investigate the genetic basis of the virulence difference, we constructed a series of mutant viruses using reverse genetics. We found that the combination of NA-N322S or NA-G389D mutations with PB2-E627K was not sufficient to increase the pathogenicity of H7N9 in mice, although it was able to enhance the neuraminidase activity of the virus. However, the NA-N322S and NA-G389D mutations combined with PB2-E627K significantly enhanced H7N9 pathogenicity. In addition, these combined mutations enhanced neuraminidase enzyme activity, thereby enhancing viral replication, inflammatory cytokine expression, and lung damage, ultimately increasing pathogenicity in mice. In conclusion, this study reveals that the virulence in H7N9 is a polygenic trait and identifies new virulence-associated residues (NAN322S+G389D combined with PB2-E627K). These findings not only enhance our comprehension of the molecular mechanisms underlying AIVs pathogenicity in mammals, but also provide early warning information for preventing cross-species transmission of the H7N9 virus and for potential future pandemics.

Newcastle disease virus (NDV) and H9N2 avian influenza virus (AIV) represent significant pathogenic risks to the poultry industry, leading to considerable economic losses. Vaccination is a widely used preventive measure against these pathogens, yet the lack of a live bivalent vaccine targeting NDV and H9N2 AIV imposes a heavy vaccination burden. Previously, we constructed a genotype-matched chimeric NDV vector, LX-OAI4S, in which the genotype I NDV backbone was replaced with the ectodomain of haemagglutinin-neuraminidase (HN) and modified using the attenuated F gene from the genotype VII vaccine strain A-VII. Based on the LX-OAI4S vector, we successfully generated three H9N2 recombinant viruses: LX-OAI4S-NPU-HA, LX-OAI4S-MU-HA, and LX-OAI4S-HNU-HA. These recombinants incorporated the H9N2 HA gene, flanked by untranslated regions (UTRs) from the NP, M, or HN gene of the NDV LX strain, inserted between the P and M genes of LX-OAI4S. The vaccine candidate LX-OAI4S-NPU-HA induced a more robust immune response in chickens against H9N2 AIV and NDV than the other two recombinants. This response effectively protects against virus shedding and lethal virus challenge. Furthermore, spray vaccination with LX-OAI4S-NPU-HA showed protective efficacy against H9N2 AIV and NDV. This study offers a promising strategy for comprehensive protection in regions threatened by H9N2 AIV and NDV.

Gasdermin D (GSDMD), an effector molecule of cell pyroptosis, is known to be activated in various cells during inflammation. However, the patterns of GSDMD activation in immune regulatory cells such as myeloid-derived suppressor cells (MDSCs) remain unclear. In this study, we found that neutrophils in colorectal cancer (CRC) tissues exhibited reduced GSDMD transcription, as evidenced by a single-cell RNA sequencing result. Consistent with this, cleaved GSDMD expression is negatively correlated with S100A8 in CRC tissues. Additionally, CD15+CD14-LOX1+ cells (G-MDSCs) from the peripheral blood of CRC patients exhibited a significant reduction in GSDMD activation. Mice with ubiquitous GSDMD deficiency bred in a clean environment exhibited a notable increase in G-MDSCs. These GSDMD-/- MDSCs enhanced immunosuppressive activity by both inhibiting effector T-cell activity and promoting regulatory T-cell induction. This enhancement was also observed in GSDMDflox/flox-S100A8Cre mice, in which GSDMD was specifically deleted in MDSCs. The tumor-promoting effects in the GSDMD-/- and GSDMDflox/flox-S100A8Cre mice were abrogated following MDSC depletion, as shown by the use of an anti-DR5 antibody. In the absence of GSDMD, G-MDSCs showed reduced inflammasome activation and decreased production of IL-1β and IL-18. Furthermore, a significant reduction in interferon-related factor 8/7 (IRF8/7) was observed in GSDMD-/- G-MDSCs via bulk RNA sequencing analysis. After treatment with LPS/nigericin, these cells maintained mitochondrial integrity, thus impairing the mtDNA release and the downstream cGAS/STING/TBK1/IRF8/7 signaling axis activation. Reduced IRF8/7 levels were responsible for increased differentiation of GSDMD-/- G-MDSCs. Finally, treatment with a GSDMD recombinant lentivirus injected into in situ tumors significantly inhibited tumor growth and reduced G-MDSC levels, suggesting that a GSDMD-based vaccine could simultaneously exert anti-carcinoma and anti-MDSC effects.

H7N9 avian influenza virus (AIV) first emerged in February 2013 in China, and early isolates were all low pathogenic (LP). After circulation for a few years in live poultry markets of China, LP H7N9 AIVs evolved into a highly pathogenic (HP) form in late 2016. Deduced amino acid sequence analysis of hemagglutinin (HA) gene revealed that all HP H7N9 AIVs have obtained four-amino-acid insertion at position 339-342 (H7 numbering), making the cleavage site from a monobasic motif (LP AIVs) to a polybasic form (HP AIVs). Notably, the polybasic cleavage site motifs are diversified, of which PEVPKRKRTAR↓GLF motif is prevalent. To elucidate the reasons accounting for its dominance, recombinant H7N9 virus carrying PEVPKRKRTAR↓GLF (rJT157-2) motif was generated based on LP H7N9 virus A/chicken/Eastern China/JT157/2016 (JT157). Besides, another two viruses containing PEVPKGKRTAR↓GLF (rJT157-1) and PEIPKRKRTAR↓GLF (rJT157-3) cleavage site motifs were also constructed as comparisons. We found that rJT157-2 showed better biological characterizations in vitro including replication kinetics, plaque size, thermal and acid stability. In addition, animal experiments demonstrated that rJT157-2 was more pathogenic to both chickens and mice with higher virus titers and induced more severe changes in the lungs. These results suggested that HP H7N9 viruses carrying PEVPKRKRTAR↓GLF motif in the HA cleavage site were most likely adaptive mutants during the evolution of H7N9 AIVs.

The endemic status of goose parvovirus (GPV) continues to devastate the poultry industry in China. Novel GPV (NGPV) and Mutated GPV (MGPV) represent the predominant lineages. However, the comparative pathogenicity between these viruses remains poorly understood. Herein, we selected representative NGPV and MGPV strains as model viruses to assess their pathogenic potential both in vitro and in vivo. In vitro cellular and embryo assays demonstrated that both NGPV and MGPV were capable of replicating in DEF and GEF cells, leading to pronounced cytopathic effects. However, these viruses exhibited distinct levels of intra-embryonic replication capabilities. Furthermore, we conducted in vivo infection experiments and systematically evaluated the pathogenic differences between NGPV and MGPV by examining various indicators, including growth, clinical signs, gross pathology, skeletal development, viral load, and humoral response in the infected animals. The results showed that both NGPV and MGPV inhibited weight gain in goslings and ducklings, with NGPV exerting a more significant suppressive impact. MGPV induced classical gosling plague pathology in goslings, while NGPV led to short beak and dwarfism syndrome in ducklings, notably disrupting skeletal development. Moreover, MGPV and NGPV exhibited diverse host tropisms, with MGPV being more pathogenic to goslings and NGPV to ducklings. Both viruses elicited specific antibody responses, with MGPV being more effective in goslings and NGPV in ducklings. Additionally, MGPV exhibited stronger humoral response compared to NGPV. These findings enhance our understanding of the pathogenicity of prevalent GPV strains in waterfowl, offering a critical theoretical foundation for devising strategies to prevent GPV infections.

Since 2021, the novel H10N3 has caused four cases of human infection in China, the most recent of which occurred in December 2024, posing a potential threat to public health. Our previous studies indicated that several avian H10N3 strains are highly pathogenic in mice and can be transmitted between mammals via respiratory droplets without prior adaptation. By analyzing the genome sequence, we found that these H10N3 viruses carry the PB2-E627V mutation, which is becoming increasingly common in several subtypes of avian influenza viruses (AIV); however, its mechanism in mammalian adaptation remains unclear. Using a reverse genetics system, we investigated the role of PB2-E627V in the adaptation of H10N3 to mammals and poultry. Our findings demonstrate that the PB2-E627V mutation is critical for the high pathogenicity of novel H10N3 in mice and its ability to be transmitted through the air among mammals. Additionally, we found that the role of PB2-627 V in promoting AIV adaptation to mammals is comparable to that of PB2-627 K. More importantly, PB2-627 V appears to be equally suited to long-term persistence in poultry. Therefore, using PB2-627 V as a novel molecular marker to assess the epidemic potential of AIV is of great significance for preventing possible influenza pandemics in the future.

Newcastle disease virus (NDV), a widespread poultry pathogen, spreads efficiently via the respiratory tract. However, the precise mechanism governing its spreading infection remains unclear. This study reveals that NDV-induced tight junction (TJ) injury is crucial for viral replication and spread. NDV infection significantly reduced TJ proteins OCLN and ZO-1 through multiple degradation pathways involving viral proteins, disrupting TJ integrity and promoting cell migration. Knockdown of OCLN and ZO-1 further enhanced viral replication and spread, underscoring their importance. Concurrently, NDV altered the distribution of OCLN and ZO-1, accompanied by cytoskeletal rearrangements of vimentin and F-actin. Notably, NDV triggered vimentin and F-actin rearrangement to form cage-like structures, benefiting TJ injury and viral replication. Critically, vimentin rearrangement was essential for the redistribution of OCLN, ZO-1, and F-actin, facilitating viral replication, spread, and inflammation. MLC/p-MLC activation was required for vimentin-mediated TJ injury, thereby promoting NDV replication and spread. Unlike avirulent strains, the virulent NDV promoted replication and spread through vimentin-mediated TJ injury, subsequently worsening lung damage in chickens. These findings elucidate how NDV rapidly disseminates and worsens lung damage, providing insights relevant to the pathogenesis and treatment of viral pneumonias, including those caused by coronaviruses and influenza viruses.

Since the national vaccination program was implemented with the H5/H7 bivalent vaccine in poultry in September 2017, the prevalence of H7N9 avian influenza viruses (AIVs) has been controlled effectively in China, and low pathogenic H7N9 viruses have disappeared nationwide. However, highly pathogenic H7N9 viruses still exist, causing sporadic outbreaks especially in some regions of northern China. During our routine surveillance in poultry in 2020, we isolated two strains of H7N9 subtype AIV from breeder layer farms in northern China. We found that these two chicken-origin H7N9 isolates were both highly pathogenic (HP) based on the sequence of the HA gene. Deduced amino acid sequences of the HA gene revealed that both strains had a four-amino-acid (KRTA) insertion at position 339-342 and an I335V mutation in the cleavage site to make the motif PEVPKRKRTAR↓GLF. Remarkably, both strains gained the F102V and N157D mutations (H3 numbering) in their HA genes, which have never been reported before. Solid-phase direct binding assay showed that these two isolates both had dual-receptor binding characteristics, while thermal and acid stability assays indicated that they were relatively stable in high-temperature or acidic conditions. In addition, the animal experiments demonstrated that both strains were highly pathogenic to chickens but low pathogenic to mice. These results suggested that the evolution of H7N9 subtype AIV is still continuing, and they pose a potential threat to poultry and public health. Thus, attentions should be paid to the importance of continual surveillance of the H7N9 AIVs.

Several viruses, including influenza A virus (IAV), encode viral factors to hijack cellular RNA biogenesis processes to direct the degradation of host mRNAs, termed “host shutoff.” Host shutoff enables viruses to simultaneously reduce antiviral responses and provides preferential access for viral mRNAs to cellular translation machinery. IAV PA-X is one of these factors that selectively shuts off the global host genes. However, the specific role of PA-X host shutoff activity in viral fitness of IAV remains poorly understood. Herein, we successfully mapped PA-X 100 V as a novel site important for host shutoff of the H7N9 and H5N1 viruses. By analysing the polymorphism of this residue in various subtype viruses, we found that PA-X 100 was highly variable in H7N9 viruses. Structural analysis revealed that 100 V was generally close to the PA-X endonuclease active site, which may account for its host shutoff activity. By generating the corresponding mutant viruses derived from the parental H7N9 virus and the PA-X-deficient H7N9 virus, we determined that PA-X 100 V significantly enhanced viral fitness in mice while diminishing viral virulence in chickens. Mechanistically, PA-X 100 V significantly increased viral polymerase activity and viral replication in mammalian cells. Furthermore, PA-X 100 V highly blunted the global host response in 293T cells, particularly restraining genes involved in energy metabolism and inflammatory response. Collectively, our data provided information about the intricate role of the PA-X host shutoff site in regulating the viral fitness of the H7N9 influenza virus, which furthers our understanding of the complicated pathogenesis of the influenza A virus.