Influenza viruses represent a significant threat to global public health. MicroRNAs (miRNAs), a class of small non-coding RNA molecules, play pivotal roles in regulating gene expression and have emerged as critical modulators of host-virus interactions. To investigate the functions of miRNAs during influenza virus infection, a miRNA library was constructed and sequenced using DF1 cells infected with the H9N2 influenza virus. From the differentially expressed miRNAs, we identified miR-92 as a key antiviral host factor. Mechanistically, miR-92 inhibits viral replication by targeting TNFRSF1B, which, in turn, enhances type I interferon signaling responses. Promoter region analysis demonstrated that the transcription factor OCT1 binds to the miR-92 promoter and positively regulates its transcriptional activity. Additionally, our study revealed that TNFRSF1B interacts with TNF receptor-associated factor 3 (TRAF3) and mediates TRAF3 degradation via the autophagolysosomal pathway. Specifically, TNFRSF1B facilitates the removal of K63-linked polyubiquitin chains from TRAF3. Collectively, these findings indicate that TNFRSF1B negatively regulates IFN-I responses through autophagolysosomal-mediated TRAF3 degradation, while miR-92 counteracts this inhibitory effect to exert antiviral activity. In summary, our research delineates a novel regulatory axis that modulates the interferon pathway and H9N2 influenza virus replication, providing new insights into host antiviral defense mechanisms and potential therapeutic targets.IMPORTANCEThe miR-17-92 cluster is a well-established key regulator of viral infection. However, the specific role of miR-92, an essential member of this cluster, in modulating avian influenza virus infection remains poorly defined. Here, we demonstrate that avian miR-92 exerts robust antiviral activity by directly inhibiting AIV replication. Critically, we report for the first time that the transcription factor OCT1 binds to the promoter region of miR-92 and transcriptionally regulates its expression. Mechanistically, miR-92 targets TNFRSF1B to enhance type I interferon production. Strikingly, TNFRSF1B mediates the degradation of TRAF3, thereby dampening IFN-I signaling. Together, our findings establish miR-92 as a pivotal antiviral effector during AIV infection. Beyond advancing our understanding of small RNA-mediated control of influenza virus replication, this work identifies miR-92 and its regulatory network as promising targets for the development of miRNA-based antiviral therapeutics.

Avian influenza virus is a globally prevalent pathogen in poultry populations, causing severe economic losses to the poultry industry worldwide. In the present study, we demonstrate that miR-449c generates two mature isoforms including miR-449c-3p and miR-449c-5p and exerts potent inhibitory effects on avian influenza virus replication. Mechanistically, we identified distinct antiviral pathways mediated by these two mature miR-449c isoforms. MiR-449c-5p enhances type I interferon responses by targeting the Suppressor of cytokine signaling 3 (SOCS3), thereby suppressing avian influenza virus replication. Meanwhile, miR-449c-3p inhibits viral replication through direct targeting and binding to the PB1 gene of avian influenza virus. Furthermore, we characterized the role of avian SOCS3 in avian influenza virus infection. We found that SOCS3 promotes avian influenza virus replication by dampening interferon responses, which is achieved through its interaction with and subsequent degradation of interferon regulatory factor 7 (IRF7), a key transcription factor that drives the expression of interferon stimulated genes (ISGs). Collectively, our findings uncover a novel dual-mode antiviral mechanism orchestrated by avian miR-449c, providing a theoretical foundation for the development of novel antiviral strategies.

Avian influenza virus (AIV) and infectious bronchitis virus (IBV) are major respiratory pathogens of poultry, and their co-circulation complicates disease control. Here, we developed a chimeric subunit vaccine, RBD-HA, in which the receptor-binding domain (RBD) of the QX-type IBV spike protein replaced the immunodominant head domain of H9N2 AIV haemagglutinin. Structural analyses showed that RBD-HA formed a stable trimeric assembly, supporting the use of the HA stalk as an antigen-presenting scaffold. In chickens, RBD-HA induced humoral responses against both viruses and protected against homologous and heterologous H9N2 AIV challenge. In parallel, RBD-HA induced IBV-reactive and neutralizing antibody responses and provided protection against QX-type IBV challenge. The vaccine also elicited cross-reactive neutralizing activity and reduced viral shedding and tissue damage after H6N6 AIV challenge. These findings provide proof of concept for a bivalent subunit vaccine targeting two major avian respiratory viruses.

Host long non-coding RNAs (lncRNAs) are emerging as critical regulators of influenza A virus (IAV) pathogenesis. Still, the functional landscape of avian host lncRNAs remains largely unexplored. In this study, we identified a novel H9N2induced transcript lncRNA, lncGVRP1, which serves as a conserved positive regulator of IAV replication across H9N2, H1N1, and H3N2 subtypes. In addition, we discovered that lncGVRP1 acts as a functional lncRNA containing a hidden open reading frame (ORF). This ORF encodes a novel 74-amino acid micropeptide, named GVRP1-ORF. Functional rescue experiments demonstrated that the enhancement of viral activity by the lncGVRP1 is strictly dependent on the peptide it encodes. Overexpression of the GVRP1-ORF recapitulated the pro-viral effect achieved by overexpressing the lncRNA. In contrast, an ORF-deleted mutant entirely failed to promote viral replication. Mechanistically, lncGVRP1 facilitates viral propagation by significantly suppressing the host type I interferon (IFN) response and downstream interferon-stimulated genes (ISGs). Furthermore, transcriptome-wide analysis indicated that lncGVRP1 modulates critical cellular machineries, including FoxO signaling and lysosomal trafficking. Collectively, our findings reveal a unique mechanism in which a lncRNA-derived micro-peptide hijacks host immunity to support viral persistence. Moreover, this micro-peptide may serve as a potential therapeutic target against influenza infection.

Circular RNAs (circRNAs) represent a class of covalently closed non-coding RNA molecules that exert vital regulatory effects on host-pathogen interplay. The H9N2 subtype of avian influenza virus (AIV) is a widespread pathogen on a global scale, inflicting considerable economic damage to the poultry sector and harboring potential risks of cross-species transmission to humans. Despite growing evidence suggesting that non-coding RNAs can modulate the replication of influenza viruses, the expression patterns and functions of avian-derived circRNAs during H9N2 AIV infection have been largely unclear. Here, we conducted a systematic investigation into the expression dynamics of circRNAs in DF1 cells infected with H9N2 AIV by high-throughput RNA sequencing technology. A total of 139 differentially expressed circRNAs were identified, with 58 exhibiting upregulation and 81 showing downregulation relative to non-infected control cells. Of note, a circRNA originating from exon 2 of the insulin receptor (INSR) gene displayed consistent upregulation during viral infection. Functional assays verified its contributing role in the replication of H9N2 AIV. Specifically, siRNA-mediated knockdown of circ-INSR significantly suppressed H9N2 AIV replication. This study is the first to identify circ-INSR as a host factor contributing to influenza virus replication. Our results provide a basis for developing circRNA-based strategies against H9N2 avian influenza virus.

Background: Influenza A virus (IAV) remains a major global health threat, and antiviral resistance underscores the need for innovative host-directed therapies. Circular RNAs (circRNAs) are emerging regulators of viral infection, but their functional roles and therapeutic potential in IAV infection remain poorly understood. Methods: Whole-transcriptome sequencing was performed to identify IAV-regulated circRNAs. The role of circCRK in viral replication was evaluated by gain- and loss-of-function approaches in vitro and by lipid nanoparticle (LNP)-mediated circCRK silencing in IAV-infected BALB/c mice. RNA pull-down, RNA immunoprecipitation, dual-luciferase reporter, rescue assays, and analyses of ERK/MAPK signaling and viral ribonucleoprotein (vRNP) trafficking were performed to elucidate the underlying mechanism. Findings: circCRK was significantly upregulated following IAV infection and promoted viral replication. Mechanistically, circCRK functioned as a competing endogenous RNA by sponging miR-516b-5p, thereby relieving repression of RAS1556, a transcript derived from RASGRP2. Activation of the circCRK/miR-516b-5p/RAS1556 axis enhanced ERK/MAPK signaling and facilitated vRNP nuclear export. Importantly, LNP-mediated circCRK silencing reduced viral burden, alleviated disease severity, and improved survival in infected mice. Interpretation: These findings identify circCRK as a host factor that promotes IAV replication through regulation of the miR-516b-5p/RAS1556 axis and reveal circCRK targeting as a promising host-directed antiviral strategy.

ABSTRACT Influenza A viruses (IAVs) pose an ongoing threat to humans and other species because of their zoonotic potential. Accumulating evidence has demonstrated that certain long non-coding RNAs (lncRNAs) exhibit differential expression during viral infection and modulate diverse facets of viral pathogenesis. As key regulatory RNAs, lncRNAs participate in fundamental physiological processes and disease progression via a wide array of functional interactions with DNA, RNA, and proteins. Here, we identified ckATP1A1-AS1 as an antiviral host lncRNA that is induced by IAV infection. Functional analyses demonstrated that ckATP1A1-AS1 overexpression restricted infection by multiple IAV subtypes, whereas ckATP1A1-AS1 knockdown enhanced viral replication. Mechanistically, during IAV infection, the transcription factor JUN transcriptionally activates ckATP1A1-AS1, which further enhances the expression of interferon-β and key interferon-stimulated genes, thereby positively regulating type I interferon immune responses. Furthermore, ckATP1A1-AS1 interacts directly with the viral nucleoprotein, competitively disrupting its binding to importin α5, impairing its oligomerization, and blocking the nuclear import of viral ribonucleoprotein complexes. Consequently, ckATP1A1-AS1 suppresses viral ribonucleoprotein assembly and reduces viral polymerase activity. These findings establish ckATP1A1-AS1 as a key antiviral lncRNA that restricts IAV replication by coordinating innate immune signaling and directly targeting several steps in the viral replication cycle. IMPORTANCE Accumulating evidence indicates that host long non-coding RNAs (lncRNAs) play important roles in regulating virus–host interactions during influenza A virus (IAV) infection. However, the functions and mechanisms of action of most IAV-associated lncRNAs remain unclear. This study identifies the novel chicken antisense lncRNA ckATP1A1-AS1 as a key antiviral factor with a unique dual mechanism: it is transcriptionally activated by transcription factor JUN and, in turn, upregulates the expression of interferon-β and key interferon-stimulated genes to positively regulate the type I interferon immune response. It directly interacts with viral nucleoprotein, competitively disrupting the binding of nucleoprotein to importin α5 and impairing nucleoprotein oligomerization, thereby suppressing viral ribonucleoprotein assembly and reducing viral polymerase activity.

IntroductionCircular RNAs (circRNAs) are covalently closed endogenous RNAs that regulate gene expression at the post transcriptional level and have been implicated in antiviral immunity. However, their functional roles and regulatory mechanisms during influenza A virus (IAV) infection remain incompletely defined.MethodscircZFYVE1 expression was profiled in A549 cells infected with the WSN strain of IAV using RT qPCR under time course and dose response conditions. The mechanistic function of circZFYVE1 was investigated through miRNA target prediction, luciferase reporter assays, and functional assays assessing its role as a competing endogenous RNA (ceRNA) and its impact on innate antiviral signaling.ResultscircZFYVE1 expression was induced in a time and dose dependent manner following WSN infection. Mechanistically, circZFYVE1 acts as a ceRNA by sponging hsa miR 4435, thereby relieving miRNA mediated suppression of the processing body associated protein LSM14A and enhancing innate antiviral signaling during infection.DiscussionThese findings define a circRNA–miRNA–mRNA regulatory axis that links IAV infection to innate antiviral responses, providing new insights into circRNA mediated host defenses. The circZFYVE1/LSM14A pathway represents a potential target for future studies aimed at modulating endogenous antiviral immunity.

Infectious bronchitis (IB) virus (IBV) remains a major pathogen threatening the poultry industry. Its rapid mutation and recombination continuously generate variants that disseminate worldwide. Between May 2024 and February 2025, 49 field strains were isolated from chickens vaccinated with live-attenuated IBV vaccines (H120, 4/91, or QXL87) in four Chinese provinces (Jiangsu, Anhui, Shandong, and Guangdong). Based on full-length S1 gene sequencing, all isolates were classified into genotype GI, including lineages GI-13 and GI-19. Phylogenetic analysis revealed that GI-19 (QX-type) comprised as much as 83.67%, with the nucleotide homology of the S1 gene to QXL87 varying from 93.4% to 99.8%. Recombination analysis indicated that the S1 genes of three isolates incorporate QXL87 and 4/91 genetic material, possibly arising from recombination between the QX-type and 4/91 vaccine strains. Virulence assessment in 1-day-old specific-pathogen-free (SPF) chickens demonstrated that four phylogenetically distant QX-type strains and one recombinant strain (with QXL87 as the major parent) induced varying degrees of tissue damage and mortality. Cross-neutralization assays demonstrated reduced antigenic relatedness between the circulating isolates and QXL87 vaccine strain. Structural mapping analysis further indicated that three amino acid mutations within the N-terminal domain (NTD) and two amino acid mutations in the C-terminal domain (CTD) of the S1 subunit alter its overall conformation, potentially leading to antigenic variation and facilitating immune evasion. Overall, these findings offer timely insights into the epidemiology and virulence heterogeneity of QX-IBV, providing valuable references for optimizing vaccine selection and development, as well as for preventing and controlling the disease.

The antigen variability of the infectious bronchitis virus (IBV) has hindered vaccine effectiveness and perpetuated its epidemic. We engineered a rapid attenuation method for IBV variants. The strategy involves creating the rH-CPDF7 backbone by recoding a segment of the H120 nonstructural protein (NSP) genome via codon pair deoptimization (CPD), facilitating S gene integration from IBV variants via transformation-associated recombination (TAR) cloning. These recombinant strains exhibited even lower pathogenicity, indicating the effectiveness of CPDF7 in reducing virulence. Importantly, the rH-CPDF7 backbone demonstrated versatility, being applicable to the development of attenuated strains for IBV variants, including the QX-type, TW-type, and GVI-type strains (different genotypes). In conclusion, our method allows for the rapid development of attenuated strains by integrating the S gene of IBV variants into the rH-CPDF7 backbone. These recombinant strains can elicit a strong immune response and provide effective protection against homologous challenges. This strategy is crucial for developing live-attenuated vaccines against emerging IBV strains.

Non-coding RNAs are crucial orchestrators in the intricate dance between viruses and host cells, among which the expression and function of enhancer RNAs (eRNAs) during influenza virus infection remain largely unexplored. This study utilized whole transcriptome high-throughput sequencing to investigate the molecular mechanisms underpinning the species-specific regulation of influenza virus replication by the miR-302 cluster-IRFs-IRF1AS axis both in vivo and in vitro. Mechanistically, the CTNNB1-induced miR-302 cluster targeted various interferon regulatory factors (mainly IRF1 and IRF2) with varying affinities and silencing efficiencies, except for miR-302e and miR-302f. Furthermore, miR-302 cluster-IRFs triggered the induction of interferon-induced hub genes and hub lncRNAs defined through weighted gene co-expression network analysis. Importantly, the intricate interplay between IRFs, direct targets of the miR-302 cluster, and IRF1AS, an indirect target, in terms of gene loci and transcriptional regulation reveals a crosstalk in the miR-302 cluster-IRFs-IRF1AS axis. That is, on the one hand, IRF1 and IRF7 bind to the promoter of IRF1AS to promote the transcription of eRNAs. On the other hand, IRF1AS functions as an enhancer cluster that orchestrates and cis-regulates the transcription of IRF1, thereby rapidly amplifying the antiviral immune response initiated by miR-302 cluster-IRFs. In conclusion, we have unveiled a novel regulatory network governed by the miR-302 cluster-IRFs-IRF1AS, offering fresh perspectives on immune regulatory mechanisms.IMPORTANCENon-coding RNAs play a crucial role in regulating the three-dimensional structure of chromatin. They influence gene expression through various mechanisms and thereby contribute to the onset and progression of influenza A virus pathogenicity. Our comprehensive whole transcriptome sequencing analysis reveals a novel finding: the species-specific regulation of influenza virus replication by the miR-302 cluster-IRFs-IRF1AS axis. Our findings indicate that the miR-302 cluster-IRFs axis facilitates the transcription of key hub genes and hub lncRNAs, most of which significantly inhibit influenza virus replication. Notably, the downstream IRF1AS assembles into an enhancer cluster, orchestrating and cis-regulating the transcription of IRF1 to activate the interferon system. This investigation enhances our understanding of the regulatory network underlying viral infections and offers novel insights into immune regulatory mechanisms.

Currently, the spread of H9N2 avian influenza virus (AIV) and avian infectious bronchitis virus (IBV) is one of the major predicaments facing the poultry industry. Virus-like particles (VLPs)-based vaccine, as one of the most promising alternative to traditional vaccines, provides new perspectives for the prevention of poultry diseases. Here, we generated a chimeric VLPs (VLPs) vaccine against both AIV and IBV by using baculovirus/insect cell expression system, and evaluated its efficacy in chickens. The VLPs is composed of three proteins: HA, M1, and rS. The HA and M1 proteins were derived from the H9N2 AIV A/chicken/Anhui/LH99/2017 (AH/99, H9N2), while rS was composed of the S1 subunit of the QX-type IBV CK/CH/JS/CZ211063 (CZ211063, GI-19) protein and the transmembrane domain (TM) and cytoplasmic tail domain (CTD) of the H9N2 AIV HA protein. Subcutaneous immunization with the VLPs vaccine induced a robust humoral immune responses, providing complete protection against H9N2 AIV in chickens. Furthermore, challenge experiments with QX-type IBV indicated that VLPs vaccine immunization significantly inhibited viral replication in the trachea, lung, and kidney, and suppressed viral shedding in the throat and cloaca. Additionally, histopathological analysis revealed that the VLPs vaccine effectively mitigated QX-type IBV-induced tissue damages in the respiratory and renal systems. Collectively, these results suggest that the VLPs vaccine developed in this study is a promising vaccine candidate for the avian influenza and infectious bronchitis control, and highlight the potential of VLP-based vaccines as a viable alternative to traditional egg-dependent vaccines in the prevention of poultry diseases.

Since the twentieth century, four influenza pandemics caused by IAV have killed millions of people worldwide. IAV infection could induce acute lung injury mediated by cytokine storms, which is an essential cause of death in critically ill patients. Consequently, it is crucial to explore the regulators and regulatory mechanisms of cytokine storms, which may provide potential drug targets and expand our understanding of acute lung injury. Previous studies have shown that JNK kinase is essential in promoting inflammatory responses during viral infections. In this study, we demonstrated that JNK kinase could regulate the IAV-induced cytokine storms by affecting the expression of pro-inflammatory and anti-inflammatory factors. Further studies revealed that inhibition of JNK kinase activity significantly downregulated the expression of the inflammatory amplifier TREM1. Besides, TREM1 knockdown could significantly inhibit the expression of pro-inflammatory factors. Furthermore, SP600125 is a specific inhibitor of JNK kinase. The results show that TREM1 overexpression reversed the effect of SP600125 treatment on the expression of pro-inflammatory factors. Together, we found that JNK kinase could activate the inflammatory amplifier TREM1 to promote inflammatory responses during influenza A virus infection. These findings may provide some inspiration for subsequent researchers to explore the regulatory mechanisms of cytokine storms induced by emerging viral infections.

Long non-coding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides(nt) with no protein-coding potential. Accumulating evidence indicates that the interaction between lncRNAs and influenza A virus (IAV) plays an crucial role in multiple biological processes, including host antiviral immunity and regulating viral replication. However, the mechanisms by which IAV resists antiviral immunity and enhances its replication by inhibiting lncRNA-mediated antiviral defense remain largely unexplored. In this study, we identified a lncRNA, designated LRIR (LncRNA Regulating IAV Replication), which is significantly downregulated upon IAV infection in A549 cells. Notably, LRIR knockdown significantly promotes IAV replication, whereas LRIR overexpression inhibits viral replication, suggesting that LRIR exerts its antiviral effect during IAV infection. Furthermore, the antiviral activity of LRIR primarily depends on the regions spanning from 258 to 381 nt and 38-97 nt. Mechanistically, LRIR was found to inhibit replication and transcription of the viral genome. Further studies indicated that LRIR suppresses IAV replication by downregulating the expression of transmembrane protease serine 2 (TMPRSS2). Collectively, our findings reveal that IAV infection suppresses LRIR expression, thereby weakening its negative regulation of TMPRSS2 and subsequently promoting viral replication. These results provide a theoretical foundation for the development of novel anti-IAV therapeutics.

Circular RNAs (circRNAs) represent a class of widespread and diverse covalently closed circular endogenous RNAs that play critical roles in regulating gene expression in mammals. However, the roles and regulatory mechanisms of circRNAs during influenza A virus (IAV) infection remain largely unexplored. In this study, we screened the circRNA transcription profiles of WSN-infected cells to identify circRNAs involved in viral replication and identified a novel differentially expressed circular RNA, circMYO9A. Mechanistically, circMYO9A acts as a competing endogenous RNA (ceRNA) for SERPINE1/PAI-1 by sponging miR-6059-3p, thereby increasing SERPINE1/PAI-1 expression, which restricts IAV haemagglutinin cleavage and subsequently reduces the infectivity of progeny viruses. Importantly, our findings demonstrate that circMYO9A significantly inhibits viral replication in the lungs of infected mice, potentially increasing their survival during IAV infection. These results demonstrate that circRNAs play crucial roles in inhibiting IAV replication and provide novel insights into potential therapeutic strategies involving circRNAs.

The H9N2 subtype avian influenza virus (AIV) continues to propagate and undergo evolution within China, thereby posing a significant threat to the poultry industry. This study encompassed the collection of 436 samples and swabs in East China over the period spanning 2018 to 2019, from which 31 strains of the H9N2 subtype viruses were isolated and purified. We revealed that the HA and NA genes of the 31 isolates categorized within the Y280 branch, while the PB2 and M genes were associated with the G1 branch, and the remaining genes aligned with the F/98 branch. Despite this alignment, antigenic mapping demonstrated differences between the 2018 and 2019 strains, with the early vaccine strains displaying low serological reactivity toward these isolates. Notably, the CK/SH/49/19 isolate exhibited lethality in mice, characterized by a PB2 E627V mutation and a HA deletion at amino acid position 217. Mechanistically, in vitro studies showed that the influenza virus CK/SH/49/19 carrying PB2 627V and HA 217​M mutations displayed enhanced replication capacity, attributed to the heightened activity of the polymerase with PB2 627V. Moreover, the absence of the amino acid at the HA 217 site obstructed viral adsorption and internalization, resulted in lower activation pH, and impeded the virus budding process. Critically, in vivo experiments revealed that CK/SH/49/19 (PB2 627V, HA 217Δ) triggered a robust activation of interferon response and interferon-stimulated genes. This study furnished a theoretical foundation for the scientific prevention and control strategies against H9N2 subtype avian influenza.

Prior research has indicated that the gut-lung-axis can be influenced by the intestinal microbiota, thereby impacting lung immunity. Rifaximin is a broad-spectrum antibacterial drug that can maintain the homeostasis of intestinal microflora. In this study, we established an influenza A virus (IAV)-infected mice model with or without rifaximin supplementation to investigate whether rifaximin could ameliorate lung injury induced by IAV and explore the molecular mechanism involved. Our results showed that IAV caused significant weight loss and disrupted the structure of the lung and intestine. The analysis results of 16S rRNA and metabolomics indicated a notable reduction in the levels of probiotics Lachnoclostridium, Ruminococcaceae_UCG-013, and tryptophan metabolites in the fecal samples of mice infected with IAV. In contrast, supplementation with 50 mg/kg rifaximin reversed these changes, including promoting the repair of the lung barrier and increasing the abundance of Muribaculum, Papillibacter and tryptophan-related metabolites content in the feces. Additionally, rifaximin treatment increased ILC3 cell numbers, IL-22 level, and the expression of RORγ and STAT-3 protein in the lung. Furthermore, our findings demonstrated that the administration of rifaximin can mitigate damage to the intestinal barrier while enhancing the expression of AHR, IDO-1, and tight junction proteins in the small intestine. Overall, our results provided that rifaximin alleviated the imbalance in gut microbiota homeostasis induced by IAV infection and promoted the production of tryptophan-related metabolites. Tryptophan functions as a signal to facilitate the activation and movement of ILC3 cells from the intestine to the lung through the AHR/STAT3/IL-22 pathway, thereby aiding in the restoration of the barrier. • Rifaximin ameliorated IAV infection-caused lung barrier injury and induced ILC3 cell activation. • Rifaximin alleviated IAV-induced gut dysbiosis and recovered tryptophan metabolism. • Tryptophan mediates rifaximin-induced ILC3 cell activation via the AHR/STAT3/IL-22 pathway.

As the H9N2 subtype avian influenza virus (H9N2 AIV) evolves naturally, mutations in the hemagglutinin (HA) protein still occur, which involves some sites with glycosylations. It is widely established that glycosylation of the H9N2 AIV HA protein has a major impact on the antigenicity and pathogenicity of the virus. However, the biological implications of a particular glycosylation modification site (GMS) have not been well investigated. In this study, we generated viruses with different GMSs based on wild-type (WT) viruses. Antigenicity studies revealed that the presence of viruses with a 200G+/295G- mutation (with glycosylation at position 200 and deletion of glycosylation at position 295 in the HA protein) combined with a single GMS, such as 87G+, 127G+, 148G+, 178G+, or 265G+, could significantly affect the antigenicity of the virus. Pathogenicity assays revealed that the addition of GMS, such as 127G+, 188G+, 148G+, 178G+, or 54G+, decreased the virulence of the virus in mice, except for 87G+. The removal of GMS, such as 280G- or 295G-, increased the pathogenicity of the virus in mice. Further studies on pathogenicity revealed that 87G+/295G- could also enhance the pathogenicity of the virus. Finally, we selected the WT, WT-87G+, WT-295G-, and WT-87G+/295G- strains as our further research targets to investigate the detailed biological properties of the viruses. GMS, which can enhance viral pathogenicity, did not significantly affect replication or viral stability in vitro but significantly promoted the expression of proinflammatory factors to enhance inflammatory responses in mouse lungs. These findings further deepen our understanding of the influence of the glycosylation of the HA protein of H9N2 AIV on the pathogenicity and antigenicity of the virus in mice.

After viral infection, the virus relies on the host cell's complex metabolic and biosynthetic machinery for replication. However, the impact of avian influenza virus (AIV) on metabolites and gene expression in poultry cells remains unclear. To investigate this, we infected chicken embryo fibroblasts DF1 cells with H9N2 AIV at an MOI of 3. Our aim was to explore how H9N2 AIV alters DF1 cells metabolic pathways to facilitate its replication. We employed metabolomics and transcriptomics techniques to analyze changes in metabolite content and gene expression. Metabolomics analysis revealed a significant increase in glutathione-related metabolites, including reduced glutathione (GSH), oxidized glutathione (GSSG) and total glutathione (T-GSH) upon H9N2 AIV infection in DF1 cells. Elisa results confirmed elevated levels of GSH, GSSG, and T-GSH consistent with metabolomics findings, noting a pronounced increase in GSSG compared to GSH. Transcriptomics showed significant alterations in genes involved in glutathione synthesis and metabolism post-H9N2 infection. However, adding the glutathione synthesis inhibitor BSO exogenously significantly promoted H9N2 replication in DF1 cells. This was accompanied by increased mRNA levels of pro-inflammatory cytokines (IL-1β, IFN-γ) and decreased mRNA levels of anti-inflammatory cytokines (TGF-β, IL-13). BSO also reduced catalase (CAT) gene expression and inhibited its activity, leading to higher reactive oxygen species (ROS) and malondialdehyde (MDA) level in DF1 cells. qPCR results indicated decreased mRNA levels of Nrf2, NQO1, and HO-1 with BSO, ultimately increasing oxidative stress in DF1 cells. Therefore, the above results indicated that H9N2 AIV infection in DF1 cells activated the glutathione metabolic pathway to enhance the cell's self-defense mechanism against H9N2 replication. However, when GSH synthesis is inhibited within the cells, it leads to an elevated oxidative stress level, thereby promoting H9N2 replication within the cells through Nrf2/HO-1 pathway. This study provides a theoretical basis for future rational utilization of the glutathione metabolic pathway to prevent viral replication.

As a highly contagious acute respiratory disease, influenza A virus (A/WSN/1933) poses a huge threat to human health and public health. influenza A virus proliferation relies on glucose metabolism in host cells, yet the effects of influenza A virus on glucose metabolism and the underlying molecular mechanisms remain unclear. Here, we created models of WSN virus-infected mice and A549 cells, along with analyzing metabolomics and transcriptomics data, to investigate how WSN virus infection affects host cell glucose metabolism and specific mechanisms. Analysis of metabolites and gene expression showed that WSN virus infection triggers glycolysis in A549 cells, with notable upregulation of hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), hypoxia-inducible factor-1 alpha (HIF-1α), and elevated lactate levels. Additionally, it leads to mitochondrial impairment and heightened reactive oxygen species (ROS) generation. Elevated levels of glucose may enhance the replication of WSN virus, whereas inhibitors of glycolysis can reduce it. Enhancement of HIF-1α activation facilitated replication of WSN virus through stimulation of lactate synthesis, with the primary influence of glycolysis on WSN virus replication being mediated by ROS/HIF-1α signaling. Mice given HIF-1α inhibitor PTX-478 or glycolysis inhibitor 2-Deoxyglucose (2-DG) exhibited reduced lactate levels and decreased WSN virus replication, along with mitigated weight loss and lung damage. In summary, WSN virus-induced glycolysis has been demonstrated to enhance virus replication through the activation of the ROS/HIF-1α pathway, suggesting potential new targets for combating the virus.