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H5 subtype avian influenza viruses can cause serious diseases in poultry and wild birds. On rare occasions, viruses can cause cross-species transmission to mammalian species, including humans, pigs, horses, canines, seals, and minks.

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.

Novel H5N8 highly pathogenic avian influenza viruses (HPAIVs) bearing the clade 2.3.4.4b HA gene have been widely spread through wild migratory birds since 2020. One H5N8 HPAIV (A/Wild bird/Cixi/Cixi02/2020; here after Cixi02) was isolated from migratory birds in Zhejiang Province, Eastern China in 25 November 2020. However, its pathogenicity in avian and mammal remains unknown. Hemagglutinin gene genetic analysis indicated that Cixi02 virus belonged to the branch II of H5 clade 2.3.4.4b originated from Iraq in May 2020. Cixi02 virus showed a binding affinity to both SA α-2, 3-galactose (Gal) and SA α-2, 6 Gal receptors, good pH stability, thermostability, and replication ability in both avian and mammal cells. The poultry pathogenicity indicated that Cixi02 virus was lethal to chickens. Moreover, the mammalian pathogenicity showed that the 50% mouse lethal dose (MLD

H9N2 subtype low pathogenicity avian influenza virus (AIV) poses a potential zoonotic risk. PA-X, a novel protein generated by PA gene ribosomal frameshift, is considered to be the virulence factor of H9N2 subtype AIVs. Our study found that rTX possessing PA-X protein enhanced the mammalian pathogenicity of H9N2 subtype AIVs compared with PA-X-deficient virus (rTX-FS). Furthermore, PA-X protein inhibited H9N2 subtype AIVs to infect dendritic cells (DCs), but not nonimmune cells (MDCK cells). Meanwhile, PA-X protein suppressed the phenotypic expression (CD80, CD86, CD40 and MHCII), early activation marker (CD69) and pro-inflammatory cytokines (IL-6 and TNF-α), whereas increased anti-inflammatory cytokine (IL-10) in DCs. After intranasally viral infection in mice, we found that PA-X protein of H9N2 subtype AIVs reduced CD11b

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. Methods: We evaluated the antiviral activities of our IONzymes using a hemagglutination assay, together with a 50% tissue culture infectious doses (TCID50) 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. Results: 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. Conclusion: 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.

SummaryA 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.

Genetic reassortments between highly pathogenic avian influenza (HPAI) H5 subtype viruses with different neuraminidase (NA) subtypes have increased in prevalence since 2010 in wild birds and poultry from China. The HA gene slightly evolved from clade 2.3.4 to clade 2.3.4.4, raising the question of whether novel clade 2.3.4.4 HA broke the balance with N1 but is matched well with NAx to drive viral epidemics. To clarify the role of compatibility between HA and NA on the prevalence of H5Nx subtypes, we constructed 10 recombinant viruses in which the clade 2.3.4 or clade 2.3.4.4 HA genes were matched with different NA (N1, N2 and N8) genes and evaluated viral characteristics and pathogenicity. Combinations between clade 2.3.4 HA and N1 or between clade 2.3.4.4 HA and NAx, but not between clade 2.3.4.4 HA and N1, or between clade 2.3.4 HA and NAx, promoted viral growth, NA activity, thermostability, low-pH stability and pathogenicity in chicken and mice. These findings suggest that both clade 2.3.4 HA/N1 and clade 2.3.4.4 HA/NAx displayed a better match, which could promote the increased prevalence of clade 2.3.4 H5N1 AIV (prior to 2010) and clade 2.3.4.4 H5Nx AIV (since 2010) in China, respectively.

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., Graphical abstract, Highlights • Oncolytic viruses NDV caused tumor cells death through ferroptosis • NDV-induced ferroptosis acts through nutrient deprivation by suppression of System Xc− • P53 activation is required for NDV-induced ferroptosis initiation • Ferritinophagy induced by NDV promotes ferroptosis through release of ferrous iron, Biological sciences; Virology; Cell biology; Cancer

International audience; AbstractA 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.

Hemagglutinin (HA) glycosylation of avian influenza virus (AIV) effects differently depending on the variation of glycosylation position and numbers. The natural mutation on the glycosylation sites of the AIV HA head occurs frequently. Our previous study shows that deletion of 158 or 169 glycosylation site on the HA head of the H5 subtype AIV strain rS-144-/158+/169+ increases the viral virulence in mammals; however, the mechanism remains unknown. In this study, several AIVs with different deletions at HA head glycosylation sites 144, 158 or 169 were tested for their biological characteristics to clarify the possible mechanism. We found that rS-144-/158-/169+ and rS-144-/158+/169- viruses induced higher levels of inflammatory cytokines than rS-144-/158+/169+ did in the infected cells, but the TCID50 , EID50 and MDT of the viruses showed no difference. Moreover, we found that rS-144-/158-/169+ and rS-144-/158+/169- viruses induced higher levels of endoplasmic reticulum (ER) stress in the cells. Inhibition of inositol-requiring enzyme 1α (IRE1α) phosphorylation reduced the inflammation induced by AIV infection. Furthermore, we found that rS-144-/158-/169+ virus activated the c-Jun N-terminal kinase (JNK), X-box binding protein 1 (XBP1), and nuclear factor-κB pathways by activating IRE1α phosphorylation under ER stress, whereas the rS-144-/158+/169- virus activated only the JNK pathway by altering IRE1α phosphorylation. In vivo analysis of Kira6 intervention further confirmed that ER stress played a key role in higher virulence for HA head 158 or 169 site de-glycosylation AIV. Our findings reveal that deletion of additional HA head glycosylation sites 158 or 169 enhanced the AIV virulence via activating of strong ER stress and inflammation.

International audience; AbstractThe 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.

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.

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 widespread avian pathogen Mycoplasma gallisepticum is a causative agent of respiratory disease. The wall-less prokaryotes lack some tricarboxylic acid cycle enzymes, therefore, the glycolysis metabolic pathway is of great importance to these organisms. Pyruvate kinase (PK) is one of the key enzymes of the glycolytic pathway, and its immunological characteristics in Mycoplasma are not well known. In this study, the M. gallisepticum pyruvate kinase fusion protein (PykF) was expressed in a pET system. The full-length of the gene was subcloned into the expression vector pET28a(+) to construct the pET28a-rMGPykF plasmid, which was then transformed into Escherichia coli strain BL21 (DE3) cells. The expression of the 62 kDa recombinant protein of rMGPykF in E. coli strain BL21 (DE3) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGPykF exhibited PK catalytic activity, which could reflect the conversion of NADH to NAD(+). Mouse anti-PykF antibodies were generated by immunization of mice with rMGPykF. Immunoblot and immunoelectron microscopy assays identified PykF as an immunogenic protein expressed on the surface of M. gallisepticum cells. Bactericidal assay showed that anti-rMGPykF antiserum killed 70.55% of M. gallisepticum cells, suggesting the protective potential of PykF. Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than 39.31% inhibition of adhesion in the presence of anti-rMGPykF antiserum, suggesting that PykF of M. gallisepticum participates in bacterial adhesion to DF-1 cells.

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.