Mechanism Of Inhibition Of IFN-β Production In Early Stage Of PHEV Infection

Porcine hemagglutinating encephalomyelitis is an acute and highly contagious disease caused by Porcine hemagglutinating encephalomyelitis virus(PHEV).The disease has a high fatality rate for lactating piglets,It can cause the disease in adult pigs,which occurs and spreads in many countries.There are no effective vaccines and therapeutic drugs.Early prevention can effectively reduce the loss caused by the disease.Innate immunity is the first line of defense against virus invasion,and Interferon(IFN)is an important cytokine involved in the innate immune response.Preliminary studies found that the expression of type I IFN was not detected in the early stage of PHEV infection of the host.Although the production of IFN-β increased significantly in the later stage of infection,its immune effect on PHEV infection is still unclear,Therefore,this study explored the mechanism of IFN-β in the early stage of PHEV infection in order to provide new ideas for the understanding of the pathogenicity of PHEV and the pathogenicity of PHEV.To understand the relationship between IFN-β and PHEV,in this study,the inactivated virus was inoculated into RAW 264.7 cells and compared with the virulent virus.It was found that IFN-β m RNA was no longer produced after the virus was inactivated,but the production of IFN-β with toxic virus is normal.PHEV infected the cells which were treated with BX795 to block the expression of IFN-β,it was found that PHEV production increased in a dose-dependent manner with BX795 treatment.Treated the cells with Polymsinicl-polycytidylic acid(Poly(I:C))or add exogenous IFN-β to artificially increase the production of IFN-β,the results show that PHEV replication and proliferation are inhibited.The above research shows that PHEV is an IFN-sensitive coronavirus.To further clarify the immune response of IFN-β after PHEV infection,PHEV infected RAW 264.7 cells for different times.Enzyme-linked immunosorbent assay(ELISA)results showed that PHEV did not induce the cells to produce IFN-β in the early stage of infection(0-24 h),and PHEV replication and proliferation are normal at this time.The study also found that PHEV inhibit the phosphorylation of interferon regulation factor 3(IRF3)and the nucleus transfer of it,but nuclear factor-kappa B(NF-κB)and activating transcription factor-2(ATF-2)which are also transcription factors were not affected by PHEV infection.By researching the upstream signal molecules of IRF3 after PHEV infection,it was found that PHEV did not interfere with Retinoic acid-inducible gene I(RIG-I)and Melanoma differentiation-associated gene 5(MDA5),Mitochondrial antiviral signaling protein(MAVS)or TANK-binding kinase 1(TBK1)expression.After the cells were treated with Poly(I:C),PHEV was infected with the cells for 24 h,.It was found that PHEV could not decrease the production of IFN-βinduced by Poly(I:C),indicating that PHEV regulates IFN-β signal not through-by Tolllike receptors 3(TLR3)pathway.The above results imply that PHEV deactivates transcription factor IRF3 to limits IFN-β production.In order to explore the mechanism of antagonism of IFN-β immunity with PHEV,this study used cycloheximide to treat PHEV-infected cells.The results showed that as the drug inhibits viral protein synthesis,the production of IFN-β also increases.After constructing the eukaryotic expression vector of the viral protein,it was screened by the dual luciferase reporter gene system and found that the PHEV nuclear capsid protein can inhibit the activity of the IFN-β promoter,and it was proved that the N protein regulate the IFN pathway through IRF3 or IRF3 upstream signal molecules.The eukaryotic expression vector of each signal molecule of the IFN-β signaling pathway was constructed.After co-immunoprecipitation experiment,it was finally confirmed that MAVS can interacted with N protein.It was confirmed that MAVS is a potential target of viral protein to antagonizing IFN-β production.The above results indicate that PHEV N protein can interact with MAVS to inhibit IFN-β production.To sum up,this study reveals the mechanism of inhibiting IFN-β production in the early stage of PHEV infection.PHEV N protein interacts with MAVS to inhibit IRF3 activation and IFN-β production PHEV to evade innate immune redponse.The results of this study are not only conducive to the understanding of the pathogenicity of PHEV and the mechanism of evading host immunity,but also provide new ideas for vaccine design and drug target screening.