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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.

PA-X protein arises from a ribosomal frameshift in the PA of influenza A virus (IAV). However, the immune regulatory effect of the PA-X protein of H1N1 viruses on the nasal mucosal system remains unclear. Here, a PA-X deficient H1N1 rPR8 viral strain (rPR8-△PAX) was generated and its pathogenicity was determined. The results showed that PA-X was a pro-virulence factor in mice. Furthermore, it reduced the ability of H1N1 viruses to infect dendritic cells (DCs), the regulator of the mucosal immune system, but not non-immune cells (DF-1 and Calu-3). Following intranasal infection of mice, CCL20, a chemokine that monitors the recruitment of submucosal DCs, was downregulated by PA-X, resulting in an inhibition of the recruitment of CD11b+ DCs to submucosa. It also attenuated the migration of CCR7+ DCs to cervical lymph nodes and inhibited DC maturation with low MHC II and CD40 expression. Moreover, PA-X suppressed the maturation of phenotypic markers (CD80, CD86, CD40, and MHC II) and the levels of secreted pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) while enhancing endocytosis and levels of anti-inflammatory IL-10 in vitro, suggesting an impaired maturation of DCs that the key step for the activation of downstream immune responses. These findings suggested that the PA-X protein played a critical role in escaping the immune response of nasal mucosal DCs for increasing the virulence of H1N1 viruses.

A differentiating infected from vaccinated animals (DIVA) vaccine is an ideal strategy for viral eradication in poultry. Here, according to the emerging highly pathogenic H7N9 avian influenza virus (AIV), a DIVA vaccine strain, named rGD4HALo-mH3-TX, was successfully developed, based on a substituted 12 peptide of H3 virus located at HA2. In order to meet with the safety requirement of vaccine production, the multi-basic amino acid located at the HA cleavage site was modified. Meanwhile, six inner viral genes from a H9N2 AIV TX strainwere introduced for increasing viral production. The rGD4HALo-mH3-TX strain displayed a similar reproductive ability with rGD4 and low pathogenicity in chickens, suggesting a good productivity and safety. In immuned chickens, rGD4HALo-mH3-TX induced a similar antibody level with rGD4 and provided 100% clinical protection and 90% shedding protection against highly pathogenic virus challenge. rGD4HALo-mH3-TX strain also produced a good cross-protection against low pathogenic AIV JD/17. Moreover, serological DIVA characteristics were evaluated by a successfully established competitive inhibition ELISA based on a 3G10 monoclonal antibody, and the result showed a strong reactivity with antisera of chickens vaccinated with H7 subtype strains but not rGD4HALo-mH3-TX. Collectedly, rGD4HALo-mH3-TX is a promising DIVA vaccine candidate against both high and low pathogenic H7N9 subtype AIV.

As obligate intracellular parasites, viruses rely completely on host metabolic machinery and hijack host nutrients for viral replication. Newcastle disease virus (NDV) causes acute, highly contagious avian disease and functions as an oncolytic agent. NDV efficiently replicates in both chicken and tumour cells. However, how NDV reprograms host cellular metabolism for its efficient replication is still ill-defined. We previously identified a significantly upregulated glutamate transporter gene, solute carrier family 1 member 3 (SLC1A3), during NDV infection via transcriptome analysis. To investigate the potential role of SLC1A3 during NDV infection, we first confirmed the marked upregulation of SLC1A3 in NDV-infected DF-1 or A549 cells through p53 and NF-κB pathways. Knockdown of SLC1A3 inhibited NDV infection. Western blot analysis further confirmed that glutamine, but not glutamate, asparagine, or aspartate, was required for NDV replication. Metabolic flux data showed that NDV promotes the decomposition of glutamine into the tricarboxylic acid cycle. Importantly, the level of glutamate and glutaminolysis were reduced by SLC1A3 knockdown, indicating that SLC1A3 propelled glutaminolysis for glutamate utilization and NDV replication in host cells. Taken together, our data identify that SLC1A3 serves as an important regulator for glutamine metabolism and is hijacked by NDV for its efficient replication during NDV infection. These results improve our understanding of the interaction between NDV and host cellular metabolism and lay the foundation for further investigation of efficient vaccines.

Abstract 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.

ABSTRACT: H9N2 subtype avian influenza (AI) is an infectious disease associated with immunosuppression in poultry. Here, the regulation function of PA-X protein was determined on the host innate immune response of H9N2-infected chicken bone marrow-derived DCs (chBM-DCs). Based on 2 mutated viruses expressing PA-X protein (rTX) or deficient PA-X protein (rTX-FS), and the established culture system of chBM-DCs, results showed PA-X protein inhibited viral replication in chBM-DCs but not in non-immune chicken cells (DF-1). Moreover, PA-X protein downregulated the expression of phenotypic markers (CD40, CD86, and MHCII) and proinflammatory cytokine (IL-12 and IL-1β) of chBM-DCs. The mixed lymphocyte reaction between chBM-DCs and chicken T cells showed PA-X protein significantly decreased H9N2-infected chBM-DCs to induce T cell proliferation, implying a suppression of the DC-induced downstream T cell response. Taken together, these findings indicated that PA-X protein is a key viral protein to help H9N2 subtype AIVs escape the innate immunity of chBM-DCs.

ABSTRACT For an investigation into the effects of glycosylation site modification on hemagglutinin (HA) on the biological characteristics of the H5N6 subtype avian influenza virus (AIV), the HA sequences of H5N6 AIVs from Global Initiative on Sharing All Influenza Data (GISAID) and the isolates in China were analyzed for genetic evolution and glycosylation site patterns. Eight recombinant H5N6 AIVs with different glycosylation site patterns were constructed, and their biological characteristics were determined. The results showed that H5N6 AIVs containing a 129-glycosylation site on HA are becoming prevalent strains in China. Acquisition of the 129-glycosylation site on the HA of H5N6 AIVs increased thermostability, decreased pH stability, and attenuated pathogenicity and contact transmission in chickens. Most importantly, H5N6 AIVs escaped the neutralization activity of the Re-8-like serum antibody. Our findings reveal that H5N6 AIVs containing the 129-glycosylation site affect antigenicity and have become prevalent strains in China. IMPORTANCE H5N6 avian influenza viruses (AIVs) were first reported in 2013 and have spread throughout many countries. In China, compulsory vaccine inoculation has been adopted to control H5 subtype avian influenza. However, the effect of vaccination on the antigenic drift of H5N6 AIVs remains unknown. Here, we found that H5N6 AIVs with the 129-glycosylation site on hemagglutinin were the dominant strains in poultry in China. The neutralization assay of the serum antibody against the H5 subtype vaccine Re-8 showed a significantly lower neutralization activity against H5N6 AIVs with the 129-glycosylation site compared to that against H5N6 AIVs without the 129-glycosylation site, indicating that the 129-glycosylation site may be a crucial molecular marker for immune evasion.

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. The Newcastle disease virus (NDV) can selectively replicate in tumor cells. We show that NDV-induced ferroptosis acts through p53-SLC7A11-GPX4 pathway. 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 the 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.

Abstract 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 UPRmt. 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.

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.

Abstract 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.

Abstract 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.

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.