A Preliminary Study On The Anti-swine Influenza Virus Effect Of Mitochondrial Protein MAVS

Swine influenza(SI)is abbreviated swine flu.It is a kind of acute,highly contagious respiratory disease caused by swine influenza virus(SIV)in pigs.The clinical features are sudden,coughing,difficulty breathing,loss of appetite,fever and pregnant sow abortion.This disease has high incidence and low mortality,but will lead to decline in production when pig is infected.This brings not only serious economic losses to the pig industry,but also potential threat to the human public health.Innate immune response is the body's first line of defense defensing pathogenic microorganisms.It can make react differently to different pathogens' invading,activate specific signaling pathways and has very strict regulatory mechanisms.Currently there are two known signaling pathways in the body,Toll-like receptor(TLR)and RIG-?-like receptor(RLR).The middle signal transduction molecules mediated by them are different.The former are TRIF and MyD88 while the latter is MAVS.In the signal transduction's downstream,these two pathways gather into one place,activate a series of kinase,thereby activate IRF-3 and NF-?B.The activated IRF-3 and NF-?B get into the nucleus to start the expression of interferon-?,and complete the start of the innate immune response.After learning how swine influenza virus infects the host,knowing how the host is to inhibit viral replication and exert antiviral innate immune response has important significance.This study screened out the appropriate cell lines to research on swine influenza virus infecting cells.After expressing MAVS,the expressions of the related receptors to innate immune system,important adapter proteins and effector molecules of the infected cells at the mRNA level were detected.The MAVS protein's antiviral effect against SIV H3N2 subtype was investigated further.Experiment I SIV infections on three hose cell innate immune signaling pathwaysIn this experiment,African green monkey kidney cells(Vero cells),passaged chicken embryo fibroblasts(DF1)and human lung epithelial cells(A549 cells)were infected by influenza virus subtype H3N2.The proliferations of the influenza virus in these three kinds of cells at MOI = 1 were detected.Then the expression situations of the innate immune system-related receptor(TLR3/7,RIG-I/MDA5),important streets protein(MAVS,MyD88,IRF3/7)at the mRNA level when the virus reaches the peaks were detected.The result showed that Vero cells and A549 cells were more sensitive to influenza virus subtype H3N2.The virus titer reached a peak at 16h,and the potency of the virus was relatively higher.But due to Vero cells were IFN deficient cells,there were no significant differences between the infected cells' and the blank cells' related receptors and important adapter protein in the innate immune response,except for TLR7 and MDA5.A549 cells' related receptors,important adapter teins and effector molecules expressed significantly differentially between the inoculation group and the blank group.However DF1 cells were insensitive to swine flu virus subtype N3N2.Viral titers peaked at 6h,but the virus titers reduced by about 100 times compared to Vero cells and A549 cells.Experiment II Construction and expression of MAVS eukaryotic expression vectorTo construct MAVS eukaryotic expression vector of mitochondrial antiviral protein,MAVS primers with flag tags were designed in this study,which were used to amplify the gene of the target protein MAVS,connect pMD19-T vector to do sequence analyzing and connect the correct serial with eukaryotic expression vector pEF4/V5-His A,B,C.So then MAVS eukaryotic expression vector pEF-MAVS was constructed.Through double enzyme digestion experiment,the bands in accordance with the size of the target gene were identified.The expression situations of the target gene eukaryotic expression vector in A549 cells were detected with Western-blotting also.The result showed that there were specific bands according with the size of the target protein existing.Experiment III The preliminary study on the impcct of MAVS on SIVMitochondrial antiviral signaling protein(MAVS)as an adapter protein played an important role in the regulation of the host's innate immune signaling pathways process.The purpose of this study was to use SIV subtype H3N2 to stimulate cells after over expressing MAVS into A549 cells,and use real-time quantitative PCR analysis method to analyze the expressions' changes in mRNA levels of important receptors,adapter proteins and effector molecules in these two antiviral signaling pathways of TLR and RIG-?/MDA5.It was found that,MAVS can promote expression of a downstream factor,and further improve the body's resistance to viral infections.Western-blot to detect changes in protein levels MAVS,found that MAVS degradation occurs after the virus needs further study specific mechanisms.