Preliminary Study On The Anti-PEDV Effect Of Porcine ZCCHC3 Protein

Porcine epidemic diarrhea virus(Porcine epidemic diarrhea virus,PEDV)is an infectious pathogen that seriously affects the pig industry,causing high mortality in pig farms and severe vomiting,diarrhea and dehydration in sick pigs.The innate immune response of the host is the first line of defense against the infection of pathogenic microorganisms,and PEDV can inhibit the innate immune response after invading the host cells.Therefore,we should deeply understand the interaction between virus and host,screen out effective antiviral proteins,and clarify their role in antiviral,so as to facilitate the treatment,prevention and control of the disease.CCHC type zinc finger protein 3(CCHC-type zinc finger protein 3 ZCCHC3)refers to a class of proteins with multiple zinc finger domains at the C terminal,which is expressed in all cells and tissues of organisms.At present,the antiviral effect of human ZCCHC3 protein has been studied.Related studies have shown that ZCCHC3 can inhibit the replication of a variety of viruses,including RNA viruses VSV,EMCV and SEV,and DNA viruses including HSV-1 and VACV.However,the value-added effect of porcine ZCCHC3-encoded proteins on PEDV is not clear.In this study,a real-time fluorescence quantitative RT-q PCR method for detecting PEDV content was established,and this method was used to detect the changes of virus content in Vero cells at different time points.Through the construction of eukaryotic expression vector p EGFP-ZCCHC3,liposome transient transfection of Vero cells,let it transient overexpression in the cells,using 12-well plate for cell culture,and explore its transfection conditions.The cells overexpressing ZCCHC3 protein were infected with PEDV,and the content of PEDV in the cells was detected by Western blot and RT-q PCR.In order to further study the effect of ZCCHC3 protein on the adsorption and internalization of PEDV,the cells overexpressing ZCCHC3 protein were infected by PEDV and incubated at 4 ? for 2 hours.the virus content on the cells was detected by RT-q PCR method to study its role in the stage of virus adsorption.After the cells were infected by the same method,the cells were incubated at 37 ? for 2 hours,and the virus content in the cells was detected by RT-q PCR to study its role in the stage of virus internalization.Finally,the contents of IFN-?,ISG56,IL6,TNF-? and CXCL10 in the cells overexpressing ZCCHC3 protein were detected by RT-q PCR method to study whether they can regulate virus replication by regulating the action of related antiviral factors.The results show that the PEDV real-time fluorescence quantitative RT-q PCR detection method has good specificity,sensitivity and stability.The fluorescence microscope showed that the transfection efficiency was the highest when the transfection reagent was 2 ? L / well,the eukaryotic expression vector p EGFP-ZCCHC3 was 1.5 ? g / well,and the transient transfection of Vero cells was 24 hours.Compared with the control group,there was no significant difference in the amount of PEDV adsorption,but significantly inhibited the internalization of PEDV.These data showed that overexpression of ZCCHC3 did not prevent the adsorption process of the virus,but could prevent the internalization of the virus.RT-q PCR was used to detect the transcriptional level of PEDV in ZCCHC3 transfected cells at different time points after infection.,Western blotting was used to detect the expression of viral N protein.The results showed that the content of viral RNA decreased significantly from 6h to 36 H in ZCCHC3 overexpressed cells.Similarly,Western blotting results showed that ZCCHC3 significantly inhibited the expression of PEDV N protein at 36 H,that is,ZCCHC3 could inhibit the process of virus replication.RT-q PCR method was used to detect the content of antiviral factors involved in innate immune response after virus infection.The results showed that ZCCHC3 could up-regulate the contents of IFN-?,ISG56,IL6,TNF-? and CXCL10.To sum up,this study revealed for the first time that porcine ZCCHC3 protein can inhibit PEDV replication,while studies have shown that ZCCHC3 can achieve antiviral effect by preventing virus internalization,and porcine ZCCHC3 can also participate in antiviral response by up-regulating the content of IFN-? and other cytokines.This study is the first time to link ZCCHC3 with PEDV replication,which provides theoretical support for enriching the antiviral gene spectrum,further elucidating the mechanism of PEDV infection and developing new antiviral drugs.