Background: Recently, Influenza A virus (IAV) has been shown to activate several programmed cell death pathways that play essential roles in host defense. Indeed, cell death caused by viral infection may be mediated by a mixed pattern of cell death instead of a certain single mode. Ferroptosis is a novel form of regulated cell death (RCD) that is mainly mediated by iron-dependent lipid peroxidation. Based on the proteomic data, we wondered whether IAV causes ferroptosis in host cells. Method: In this study, a quantitative proteomics approach based on an iTRAQ combined with LC–MS/MS was used to profile proteins expressed in A549 cells infected with H1N1 swine influenza virus (SIV). Meanwhile, we measured the intracellular iron content, reactive oxygen species (ROS) release and lipid peroxidation in response to SIV infection. Finally, a drug experiment was conducted to investigate the effects of ferroptosis on modulating SIV survival. Results: The bioinformatics analysis revealed several proteins closely relevant to iron homeostasis and transport, and the ferroptosis signaling pathway are highly enriched in response to SIV infection. In our experiment, aberrant expression of iron-binding proteins disrupted labile iron uptake and storage after SIV infection. Meanwhile, SIV infection inhibited system the Xc⁻/GPX4 axis resulting in GSH depletion and the accumulation of lipid peroxidation products. Notably, cell death caused by SIV as a result of iron-dependent lipid peroxidation can be partially rescued by ferroptosis inhibitor. Additionally, blockade of the ferroptotic pathway by ferrostatin-1 (Fer-1) treatment decreased viral titers and inflammatory response. Conclusions: This study revealed a new mode of cell death induced by IAV infection, and our findings might improve the understanding of the underlying mechanism involved in the interaction of virus and host cells.

The large-scale outbreaks of severe diarrhea caused by viruses have occurred in pigs since 2010, resulting in great damage to the pig industry. However, multiple infections have contributed to the outbreak of the disease and also resulted in great difficulties in diagnosis and control of the disease. Thus, a Luminex xTAG multiplex detection method, which was more sensitive and specific than general multiplex PCR method, was developed for the detection of 11 viral diarrhea pathogens, including PKoV, PAstV, PEDV, PSaV, PSV, PTV, PDCoV, TGEV, BVDV, PoRV, and PToV. To investigate the prevalence of diarrhea-associated viruses responsible for the outbreaks, a total of 753 porcine stool specimens collected from 9 pig farms in Shanghai during 2015–2018 were tested and the pathogen spectrums and co-infections were analyzed. As a result, PKoV, PAstV and PEDV were most commonly detected viruses in diarrheal pigs with the rate of 38.65% (291/753), 20.32% (153/753), and 15.54% (117/753), respectively. Furthermore, multiple infections were commonly seen, with positive rate of 28.42%. Infection pattern of the viral diarrhea pathogens in a specific farm was changing, and different farms had the various diarrhea infection patterns. A longitudinal investigation showed that PEDV was the key pathogen which was closely related to the death of diarrhea piglets. Other pathogens might play synergistic roles in the pathogenesis of diarrhea disease. Furthermore, the surveillance confirmed that variant enteropathogenic viruses were leading etiologic agents of porcine diarrhea, either mono-infection or co-infections of PKoV were common in pigs in Shanghai, but PEDV was still the key pathogen and multiple pathogens synergistically complicated the infection status, suggesting that controlling porcine diarrhea might be more complex than previously thought. The study provides a better understanding of diarrhea viruses in piglets, which will aid in better preventing and controlling epidemics of viral porcine diarrhea.

Introduction: Swine influenza viruses (SIVs) pose significant economic losses to the pig industry and are a burden on global public health systems. The increasing complexity of the distribution and evolution of different serotypes of influenza strains in swine herds escalates the potential for the emergence of novel pandemic viruses, so it is essential to develop new vaccines based on swine influenza.Methods: Here, we constructed a self-assembling ferritin nanoparticle vaccine based on the hemagglutinin (HA) extracellular domain of swine influenza A (H1N1) virus using insect baculovirus expression vector system (IBEVS), and after two immunizations, the immunogenicities and protective efficacies of the HA-Ferritin nanoparticle vaccine against the swine influenza virus H1N1 strain in mice and piglets were evaluated.Results: Our results demonstrated that HA-Ferritin nanoparticle vaccine induced more efficient immunity than traditional swine influenza vaccines. Vaccination with the HA-Ferritin nanoparticle vaccine elicited robust hemagglutinin inhibition titers and antigen-specific IgG antibodies and increased cytokine levels in serum. MF59 adjuvant can significantly promote the humoral immunity of HA-Ferritin nanoparticle vaccine. Furthermore, challenge tests showed that HA-Ferritin nanoparticle vaccine conferred full protection against lethal challenge with H1N1 virus and significantly decreased the severity of virus-associated lung lesions after challenge in both BALB/c mice and piglets.Conclusion: Taken together, these results indicate that the hemagglutinin extracellular-based ferritin nanoparticle vaccine may be a promising vaccine candidate against SIVs infection.

The influenza A virus non-structural protein 1 (NS1) is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. In the avian influenza virus or human influenza virus, specific amino acids of NS1 have been shown to be important for the virus to antagonize host antiviral defenses and promote viral replication. However, little research has been reported regarding the swine influenza virus (SIV) NS1 protein. To study the effects of the key amino acids of NS1, we rescued NS1 mutants (S42P, D92E, and S42P/D92E) of the A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability and cytokine production as well as the intracellular localization in cultured cells. We found that the S42P and D92E mutation displayed no changes on NS1 nuclear localization. The S42P (but not D92E) mutation suppressed protein synthesis and reduced virus growth properties, and there was an inability to antagonize host cell interferon production and IRF3 activation, which led to high levels of IFN-α and IFN-β production. We conclude that the S42 residue of the NS1 of the A/swine/Shanghai/3/2014(H1N1) strain is the key amino acid in regulating the host IFN response by blocking the activation of IRF3 and thus facilitates virus replication.

Background: Nonstructural protein 1 (NS1) is a virulence factor encoded by influenza A virus (IAV) that is expressed in the nucleus and cytoplasm of host cells during the earliest stages of infection. NS1 is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. However, to date, the phosphorylation sites of NS1 have not been identified, and the relationship between phosphorylation and protein function has not been thoroughly elucidated. Method: In this study, potential phosphorylation sites in the swine influenza virus (SIV) NS1 protein were bioinformatically predicted and determined by Phos-tag SDS-PAGE analysis. To study the role of NS1 phosphorylation sites, we rescued NS1 mutants (Y73F and S83A) of A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability, cytokine production as well as the intracellular localization in cultured cells. Additionally, we used small interfering RNA (siRNA) assay to explore whether changes in the type I IFN response with dephosphorylation at positions 73 and 83 were mediated by the RIG-I pathway. Results: We checked 18 predicted sites in 30 SIV NS1 genes to exclude strain-specific sites, covering H1N1, H1N2 and H3N2 subtypes and identified two phosphorylation sites Y73 and S83 in the H1N1 SIV protein by Phos-tag SDS-PAGE analysis. We found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and reduced the ability of NS1 to antagonize IFN-β expression but had no effect on nuclear localization. Knockdown of RIG-I dramatically impaired the induction of IFN-β and ISG56 in NS1 Y73F or S83A mutant-infected cells, indicating that RIG-I plays a role in the IFN-β response upon rSIV NS1 Y73F and rSIV NS1 S83A infection. Conclusion: We first identified two functional phosphorylation sites in the H1N1 SIV protein: Y73 and S83. We found that dephosphorylation at positions 73 and 83 of the NS1 protein affected the antiviral state in the host cells, partly through the RIG-I pathway.