The Role And Mechanism Of Epidermal Growth Factor Receptor On Porcine Epidemic Diarrhea Virus Infection

Porcine epidemic diarrhea virus(PEDV)is the aetiological agent of porcine epidemic diarrhea(PED),which is characterized by acute and devastating enteric disease.As the name of it suggests,digestive malabsorption diarrhea is the main symptom.In addition,pigs infected with PEDV show various other clinical signs,like vomiting,weight loss and dehydration.All ages of pigs can be infected and the mortality rate of infected piglet is as high as 100%.PEDV infection has resulted in huge economic losses in pig industry worldwide.Although first reported over 40 years ago,the pathogenic mechanism is not clear yet.In our study,PEDV infection induces EGFR phosphorylation,indicating that PEDV infection activates EGFR.Previous studies showed that EGFR plays a vital role during some viruses’ infection and thus we investigated the role of EGFR during PEDV infection.Firstly,we measured whether EGFR activation affected PEDV infection by using a specific physical stimulus,EGF.The results showed that EGFR phosphorylation enhanced PEDV RNA levels and virus yields.Meanwhile,overexpressed EGFR facilitated PEDV replication which indicates EGFR is involved in PEDV infection.Then we exposed the cells to the specific inhibitors of EGFR,Erlotinib and Gefitinib,to measure the role of EGFR in PEDV infection.As showed by IFA,the number of PEDV-positive cells was significantly lower with the treatment of inhibitors.Treated cells decreased viral protein synthesis,RNA levels and virus titers in a concentration-dependent manner.To exclude the non-specific effect of the inhibitors,we used a specific small interfering RNA to knock down EGFR expression.After the treatment,we observed the expression of PEDV N protein reduced and EGFR knockdown also decreased RNA levels and virus loads.These data verified that EGFR plays an important role in PEDV infection.Therefore,we investigated the molecular mechanisms of the role of EGFR during PEDV infection.Firstly,we tested the effect of EGFR on IFN-I by using EGFR specific siRNA.The results showed that the mRNA levels of MxA,ISG15 and IFN-β was increased which suggested that inhibiton of EGFR expression restores antiviral responses of IFN to inhibit PEDV infection.In further analysis,the data revealed that inhibition of EGFR function displayed an increase of type I interferon related genes.To further investigate the regulatory mechanism of this protein,we found that STAT3,EGFR downstream cascade,was also activated upon PEDV infection that was similar to the case of EGFR,suggesting that PEDV infection activates EGFR and STAT3.Specific inhibition of STAT3 function by either inhibitor or siRNA augmented the antiviral activity of IFN-I.To further confirm the functional relevance of STAT3 to the PEDV infection,STAT3 was overexpressed by transient transfection assay.The results verified the positive effect of STAT3 overexpression on PEDV infection which further indicated STAT3 was involved in PEDV infection.Finally,data with STAT3 depletion in combination with EGF stimulation suggested EGFR activation-mediated antiviral activity suppression requires activation of STAT3 pathway to negatively regulate IFN-I response,which provides a novel therapeutic target for virus infection.In this study,we determined the role and mechanism of EGFR on PEDV infection for the first time.We observed that PEDV infection induced EGFR phosphorylation which negatively regulates the antiviral activity of IFN-I and STAT3 is involved in this process.Our findings might provide novel insights into PEDV infection and related pathogenesis.