Establishment Of An Assay Based On Pseudotype Virus For Localization Of Porcine Epidemic Diarrhea Virus S1Protein In The Infected Cells

Porcine epidemic diarrhea virus (PEDV) belongs to the family Coronaviridae, which primarily cause infection of the upper respiratory and gastronintestinal tract of hosts. The main membrane protein of PEDV is S protein which is confirmed that it has many antigen epitope areas. However, the understanding and knowledge regarding S protein mediated PEDV entry into cells are limited. Herein, S1protein which contains the main antigen epitope areas, we investigate its function in the target cell infection by constructing the pseudotype virus.In this research, PEDV propogated in Vero E6cells were used as immune antigen to make PEDV polyclonal antibody. The bioactivites of this antibody was evaluated by ELISA and indirect immunofluorescence and the results showed the antibody had good specifity for detecting PEDV.To further study the infection nature of PEDV, we chose the cell lines of swine small intestine epithelial cells (IECs) as the target cells which were separated from susceptible animal-porcine. Throughpropogating the PEDV in IECs for10passages, we examined the tenth passage and the results of reverse transcription PCR, indirect immunofluorescence and electron microscope showed PEDV could infect IEC cell line and passage stability. Herein, the IEC cell line was the susceptible cell line of PEDV.In order to research the PEDV S1protein function, we constructed the eukaryotic expression plasmid of S1and established the stable cell line expressed S1protein of PEDV in BHK-21cell line. The reverse transcription PCR and indirect immunofluorescence showed the successful construction. Then we used this stable cell line and the restructuring VSV system to prepare the pseudo viruses VSV△G*S1. The reverse transcription PCR and laser confocal microscopy showed the successful construction.In this study, to further investigate the function of S1protein in VSV△G*S1infected IEC cells, wedynamic positioning the S1protein in infected cells. The laser confocal microscopy showed the S1protein played a key role in PEDV adsorption and entrance. Furthermore, our research provided the effective materials and explored the role of S1protein in PEDV infection cell.