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During infection, avian influenza virus (AIV) triggers endoplasmic reticulum (ER) stress, a well-established phenomenon in previous research. The Golgi apparatus, situated downstream of the ER and crucial for protein trafficking, may be impacted by AIV infection. However, it remains unclear whether this induces Golgi apparatus stress (GAS) and its implications for AIV replication. We investigated the morphological changes in the Golgi apparatus and identified GAS response pathways following infection with the H5 subtype AIV strain A/Mallard/Huadong/S/2005. The results showed that AIV infection induced significant swelling and fragmentation of the Golgi apparatus in A549 cells, indicating the presence of GAS. Among the analyzed GAS response pathways, TFE3 was significantly activated during AIV infection, while HSP47 was activated early in the infection process, and CREB3-ARF4 remained inactive. The blockade of the TFE3 pathway effectively inhibited AIV replication in A549 cells and attenuated AIV virulence in mice. Additionally, activation of the TFE3 pathway promoted endosome acidification and upregulated transcription levels of glycosylation enzymes, facilitating AIV replication. These findings highlight the crucial role of the TFE3 pathway in mediating GAS response during AIV infection, shedding light on its significance in viral replication.

Influenza poses a severe threat to global health. Despite the whole inactivated virus (WIV)‐based nasal vaccine being a promising strategy for influenza protection, the mucosal barrier is still a bottleneck of the nasal vaccine. Here, a catalytic mucosal adjuvant strategy for an influenza WIV nasal vaccine based on chitosan (CS) functionalized iron oxide nanozyme (IONzyme) is developed. The results reveal that CS‐IONzyme increases antigen adhesion to nasal mucosa by 30‐fold compared to H1N1 WIV alone. Next, CS‐IONzyme facilitates H1N1 WIV to enhance CCL20‐driven submucosal dendritic cell (DC) recruitment and transepithelial dendrite(TED) formation for viral uptake via the toll‐like receptor(TLR) 2/4‐dependent pathway. Moreover, IONzyme with enhanced peroxidase (POD)‐like activity by CS modification catalyzes a reactive oxygen species (ROS)‐dependent DC maturation, which further enhances the migration of H1N1 WIV‐loaded DCs into the draining lymph nodes for antigen presentation. Finally, CS‐IONzyme‐based nasal vaccine triggers an 8.9‐fold increase of IgA‐mucosal adaptive immunity in mice, which provides a 100% protection against influenza, while only a 30% protection by H1N1 WIV alone. This work provides an antiviral alternative for designing nasal vaccines based on IONzyme to combat influenza infection. A CS‐IONzyme‐based influenza mucosal vaccine with dual targeting of both nasal mucosa and submucosal DCs, leading to a robust immunoprotection against influenza virus is designed. Utilizing an excellent POD‐like activity, a catalytic immune‐adjuvant CS‐IONzyme against the bottleneck of mucosal vaccines is successfully developed by both fully mobilizing submucosal DCs to form TEDs for antigen uptake and strongly activating DC maturation.

Influenza virus with numerous subtypes and frequent variation limits the development of high‐efficacy and broad‐spectrum antiviral strategy. Here, a novel multi‐antiviral metastable iron sulfides (mFeS) against various influenza A/B subtype viruses is developed. This work finds that mFeS induces high levels of lipid peroxidation and •OH free radicals in the conservative viral envelope, which depends on Fe2+. This phenomenon, termed as a viral ferroptosis, results in the loss of viral infectibility and pathogenicity in vitro and in vivo, respectively. Furthermore, the decoction of mFeS (Dc(mFeS)) inhibits cellular ferroptosis‐dependent intracellular viral replication by correcting the virus‐induced reprogrammed sulfur metabolism, a conserved cellular metabolism. Notably, personal protective equipment (PPE) that is loaded with mFeS provides good antiviral protection. Aerosol administration of mFeS combined with the decoction (mFeS&Dc) has a potential therapeutic effect against H1N1 lethal infection in mice. Collectively, mFeS represents an antiviral alternative with broad‐spectrum activity against intracellular and extracellular influenza virus. mFeS induces the viral ferroptosis by enhancing the lipid peroxidation level, which results in a failed viral infection. In addition, Dc(mFeS) suppresses the cellular ferroptosis‐dependent intracellular replication of influenza virus via restoring influenza virus‐induced imbalance of sulfur metabolism, and the supply of intracellular S0. Comprehensively, mFeS&Dc through aerosol administration shows a potential preventive therapy against H1N1 virus infection in mice.

Innate immunity serves as the first barrier against virus infection. DEAD (Glu-Asp-Ala-Glu) box RNA helicases, originally considered to be involved in RNA processing and RNA unwinding, have been shown to play an important role in antiviral innate immunity. DEAD (Glu-Asp-Ala-Glu) box RNA helicases have been proven to contribute to antiviral innate immunity. The DDX21 RNA helicase was identified as a nuclear protein involved in rRNA processing and RNA unwinding. DDX21 was also proven to be the scaffold protein in the complex of DDX1-DDX21-DHX36, which senses double-strand RNA and initiates downstream innate immunity. Here, we identified that DDX21 undergoes caspase-dependent cleavage after virus infection and treatment with RNA/DNA ligands, especially for RNA virus and ligands. Caspase-3/6 cleaves DDX21 at D126 and promotes its translocation from the nucleus to the cytoplasm in response to virus infection. The cytoplasmic cleaved DDX21 negatively regulates the interferon beta (IFN-β) signaling pathway by suppressing the formation of the DDX1-DDX21-DHX36 complex. Thus, our data identify DDX21 as a regulator of immune balance and most importantly uncover a potential role of DDX21 cleavage in the innate immune response to virus. IMPORTANCE Innate immunity serves as the first barrier against virus infection. DEAD (Glu-Asp-Ala-Glu) box RNA helicases, originally considered to be involved in RNA processing and RNA unwinding, have been shown to play an important role in antiviral innate immunity. The precise regulation of innate immunity is critical for the host because the aberrant production of cytokines leads to unexpected pathological consequences. Here, we identified that DDX21 was cleaved at D126 by virus infection and treatment with RNA/DNA ligands via the caspase-3/6-dependent pathway. The cytoplasmic cleaved DDX21 negatively regulates the IFN-β signaling pathway by suppressing the formation of the DDX1-DDX21-DHX36 complex. In sum, our data identify DDX21 as a regulator of immune balance and most importantly uncover a potential role of DDX21 cleavage in the innate immune response to virus.

Newcastle disease (ND) is an acute, febrile, highly contagious disease caused by the virulent Newcastle disease virus (vNDV). The disease causes serious economic losses to the poultry industry. However, the metabolic changes caused by vNDV infection remain unclear. The objective of this study was to determine the metabolomic profiling after infection with vNDV. DF-1 cells infected with the vNDV strain Herts/33 and the lungs from Herts/33-infected specific pathogen-free (SPF) chickens were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) in combination with multivariate statistical analysis. A total of 305 metabolites were found to have changed significantly after Herts/33 infection, and most of them belong to the amino acid and nucleotide metabolic pathway. It is suggested that the increased pools of amino acids and nucleotides may benefit viral protein synthesis and genome amplification to promote NDV infection. Similar results were also confirmed in vivo. Identification of these metabolites will provide information to further understand the mechanism of vNDV replication and pathogenesis.

The fusogenically activated F and HN proteins of virulent NDV induce complete autophagic flux in DF-1 and A549 cells. However, the effect of both glycoproteins on mitochondria remains elusive. Here, we found that F and HN cooperation increases mitochondrial biogenesis but does not cause the mitochondria damage. We observed that both glycoproteins change the morphological characteristics and spatial distribution of intracellular mitochondria. F and HN cooperate cooperatively to induce ER stress and UPR mt . Our preliminary data suggested that F and HN cooperatively disturb mitochondrial fusion–fission homeostasis to enhance mitochondrial biogenesis, and eventually meet the energy demand of syncytium formation.

A stem glycosylation site of hemagglutinin (HA) is important to the stability of the HA trimmer. A previous study shows that the stem 10/11 overlap glycosylation site of the H5 subtype avian influenza virus may influence the cleavage of HA, whereas the exact site and its effect on virulence remain unclear. In this study, site-directed mutagenesis was used to generate single or double mutant rSY-Δ10(10NNAT), rSY-Δ11(10NNSA), and rSY-Δ10/11(10NNAA) of the overlapping glycosylation site (10NNST) on the HA of A/Mallard/Huadong/S/2005(SY). By using Western blot analysis, we show that both rSY-Δ11 and rSY-Δ10/11 mutant viruses had significant delay on HA cleavage and a reduced HA molecular mass compared to the wild-type virus rSY, while the rSY-Δ10 mutant virus exhibited a similar HA molecular mass to that of the wild-type virus rSY. Interestingly, both rSY-Δ11 and rSY-Δ10/11 mutant viruses reverted their glycosylation sites at 11N after passage, indicating that 11N is a true and critical glycosylation site. Compared to the wild-type virus rSY, rSY-Δ11 and rSY-Δ10/11 mutant viruses had decreased growth rates, reduced thermo- and pH-stability, decreased pathogenicity, and limited systemic spread. Therefore, our study suggests that the 11N glycosylation site plays a key role in HA cleavage, structural stability and pathogenicity in H5 subtype avian influenza virus.

Riemerella anatipestifer causes epizootics of infectious disease in poultry that result in serious economic losses to the duck industry. Our previous studies have shown that some strains of R. anatipestifer can form a biofilm, and this may explain the intriguing persistence of R. anatipestifer on duck farms post infection. In this study we used strain CH3, a strong producer of biofilm, to construct a library of random Tn4351 transposon mutants in order to investigate the genetic basis of biofilm formation by R. anatipestifer on abiotic surfaces. A total of 2,520 mutants were obtained and 39 of them showed a reduction in biofilm formation of 47%-98% using crystal violet staining. Genetic characterization of the mutants led to the identification of 33 genes. Of these, 29 genes are associated with information storage and processing, as well as basic cellular processes and metabolism; the function of the other four genes is currently unknown. In addition, a mutant strain BF19, in which biofilm formation was reduced by 98% following insertion of the Tn4351 transposon at the dihydrodipicolinate synthase (dhdps) gene, was complemented with a shuttle plasmid pCP-dhdps. The complemented mutant strain was restored to give 92.6% of the biofilm formation of the wild-type strain CH3, which indicates that the dhdp gene is associated with biofilm formation. It is inferred that such complementation applies also to other mutant strains. Furthermore, some biological characteristics of biofilm-defective mutants were investigated, indicating that the genes deleted in the mutant strains function in the biofilm formation of R. anatipestifer. Deletion of either gene will stall the biofilm formation at a specific stage thus preventing further biofilm development. In addition, the tested biofilm-defective mutants had different adherence capacity to Vero cells. This study will help us to understand the molecular mechanisms of biofilm development by R. anatipestifer and to study the pathogenesis of R. anatipestifer further.

Influenza poses a severe threat to human health in the world. However, developing a universal anti-viral strategy has remained challenging due to the presence of diverse subtypes as well as its high mutation rate, resulting in antigenic shift and drift. Here we developed an antiviral strategy using iron oxide nanozymes (IONzymes) to target the lipid envelope of the influenza virus. We evaluated the antiviral activities of our IONzymes using a hemagglutination assay, together with a 50% tissue culture infectious doses (TCID ) method. Lipid peroxidation of the viral envelope was analyzed using a maleic dialdehyde (MDA) assay and transmission electron microscopy (TEM). The neighboring viral proteins were detected by western blotting. We show that IONzymes induce envelope lipid peroxidation and destroy the integrity of neighboring proteins, including hemagglutinin, neuraminidase, and matrix protein 1, causing the inactivation of influenza A viruses (IAVs). Furthermore, we show that our IONzymes possess a broad-spectrum antiviral activity on 12 subtypes of IAVs (H1~H12). Lastly, we demonstrate that applying IONzymes to a facemask improves the ability of virus protection against 3 important subtypes that pose a threat to human, including H1N1, H5N1, and H7N9 subtype. Together, our results clearly demonstrate that IONzymes can catalyze lipid peroxidation of the viral lipid envelope to inactivate enveloped viruses and provide protection from viral transmission and infection.

Summary A number of new cell death processes have been discovered in recent years, including ferroptosis, which is characterized by the accumulation of lipid peroxidation products derived from iron metabolism. The evidence suggests that ferroptosis has a tumor-suppressor function. However, the mechanism by which ferroptosis mediates the response of tumor cells to oncolytic viruses remains poorly understood. Newcastle disease virus can selectively replicate in tumor cells. We show that NDV-induced ferroptosis acts through p53-SLC7A11-GPX4 pathway. The expression of tumor suppressor gene p53 increased after NDV infection, and the expressions of SLC7A11 and SLC3A2 were down-regulated, leading to the inhibition of glutathione synthesis and a decrease in glutathione peroxidase 4 expression. The chemical compound erastin, which induces ferroptosis, also down-regulated glutathione synthase expression and caused lipid peroxide accumulation and cell death. Meanwhile, the levels of intracellular reactive oxygen species and lipid peroxides increased in tumor cells. Ferritinophagy was induced by NDV promotion of ferroptosis through the release of ferrous iron and an enhanced Fenton reaction. Collectively, these observations demonstrated that NDV can kill tumor cells through ferroptosis. Our study provides novel insights into the mechanisms of NDV-induced ferroptosis and highlights the critical role of viruses in treating therapy-resistant cancers. Competing Interest Statement The authors have declared no competing interest. * ABBREVIATIONS NDV Newcastle disease virus NP Nucleocapsid protein NCOA4 nuclear receptor coactivator 4 FTH1 ferritin heavy chain 1 FTL ferritin light chain TFR1 transferrin receptor protein 1 qPCR quantitative polymerase chain reaction AMPK AMP-activated protein kinase PUFAs polyunsaturated fatty acids BAP1 BRCA1-associated protein 1 ACSL4 acyl-CoA synthetase long-chain family member 4 ALOX 12 12-lipoxygenase DCFDA 2′,7′-dichlorofluorescin diacetate TBHP tert-butyl hydroperoxide MDA malondialdehyde LDH lactate dehydrogenase ROS reactive oxygen species LPO lipid peroxidation GSH glutathione LIP-1 liproxstain-1 PFTα pifithrin-α Baf-A1 bafilomycin A.