Evaluation Of The Suppressive Effect Of Pedv M Protein On Type Ⅰ Interferon Pathway

Porcine Epidemic Diarrhea（PED）is a swine contagious disease that causes huge economic losses to the pig industry.It has been reported in many regions in China since2010,and the disease is still spread in the immuned swine population in Sichuan province.To explore the prevalence status of the disease,this study conducted an epidemiological survey of PED in Sichuan,we collected 247 materials and detected 103 positive cases,the positive detection rate was 41.70%,which proved that PED is more prevalent in Sichuan,and homology analysis it indicates that the Sichuan strain is quite different from the vaccine strain,which may lead to insufficient immunity.In Porcine Epidemic Diarrhea Virus（PEDV）-infected cells,the viral dsRNAs activate Retinoic acid-inducible gene I（RIG-I）-like helicases receptors（RLRs）and induce host antiviral response.RIG-I is one of RLRs,the C-terminal Helicase of RIG-I is involved in causing the conformation change of viral RNA through its ATP enzyme activity,resulting the expose of the two linked N-terminal CARD recruitment domain,the exposed CARDs interact with the CARD region of VISA on mitochondria.This induces the release of TRAF3 and activates TBK1.The activated TBK1 causes the phosphorylation of IRF3 to form dimers and translocated into the nucleus activating the expression of type Ⅰ interferon（IFN）and the expression of various interferon stimulated genes（ISGs）and performing the antiviral innate immune response.Porcine epidemic diarrhea virus（PEDV）belongs to the family of Coronavirus alphavirus.Coronavirus M protein can inhibit the production of type Ⅰ IFN,however,whether the M protein of PEDV is involved in inhibition of interferon production has not been reported yet.In this study,we amplified the M gene of PEDV and constructed the eukaryotic expression plasmid of PEDV M gene（the eukaryotic expression plasmid pcDNA3.1-Myc-PEDV-M）,the expression of M protein was verified by Western bloting analysis.In the IFN-βpromoter dual luciferase assay,we determined that the PEDV-M significantly inhibited SeV-induced production of type Ⅰ IFN.To further confirm the inhibitive effect,we performed the dose-dependent assay,and confirmed that PEDV-M protein inhibits SeV-induced type Ⅰ IFN pathway signaling in a dose-dependent manner.In order to test the effect of PEDV-M protein on the expression of variuos ISGs in the downstream pathway of type Ⅰ IFN pathway,we performed Real-time PCR.The results showed that overexpression of PEDV-M significantly reduced the expression of ISG15,ISG54,ISG56,RIG-I and this inhibitive effect is dose-dependent.To investigate the expression of M protein on PEDV replication,we performed a tissue culture infective dose(TCID₅₀)assay.The results showed that the virus titer in the M overexpressing cells was significantly higher than that in the control group.Our results indicate that PEDV-M protein inhibits the activation of type Ⅰ IFN pathway signaling and promotes PEDV replication.