Effect Of CD26/DPP4 On Replication Of Porcine Epidemic Diarrhea Virus In Vero Cells

Porcine epidemic diarrhea virus?PEDV?is a pathogen that causes Porcine epidemic diarrhea?PED?.PEDV infection causes severe diarrhea,vomiting,and dehydration,resulting in extremely high mortality rates.PEDV brings huge economic loss and harm to pig industry.In order to prevent and control the epidemic of virus,the most effective way is to vaccinate susceptible species,and the key to the development of effective vaccines lies in the study of virus infection process.The process of virus infecting host cells includes virus adsorption and binding to the receptors,virus uncoating and replication,assembly of nucleocapsid and viral genomes,dissolution of host cells and release of virus.Since there are many variants of PEDV and endemic strains which add the difficulties to control and prevent the epidemic of PEDV.The development of effective vaccines and antiviral drugs is great significance to the pig industry.CD26/DPP4 is a serine protease that is widely distributed in various tissues and cells and has a variety of biological functions.CD26/DPP4 is not only associated with diabetes and cardiovascular disease but also been discovered as the receptor of Middle East Respiratory Syndrome.The MERS-CoV and PEDV belong to Coronavirus.The studies have shown that the transmembrane protease serine 2?TMPRSS2?and Mosaic serine protease large-form?TMPRSS13/MSPL?which belong to the serine protease family played an important role in promoting the replication of PEDV in cells and promoting cell-cell fusion and virus-cell fusion in the infection of viruses^[1].CD26/DPP4 expressed in Vero cells,and Vero cells are the common cell line for PEDV culture.Therefore,in this experiment,sgRNA was designed in exon 2 of CD26/DPP4 using clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9?CRISPR/Cas9?technology.Single-stranded sgRNAs were annealed and polymerized into double-stranded gDNA.Then the gDNA was ligated with the pX330 vector.BbSI restriction was also required for pX330 vector before ligation.The CD26/DPP4 sgRNA expression vector was constructed and transfected into Vero cells by electroporation then obtain the monoclonal cells.By PCR,26 positive clones were obtained in this experiment.Four positive clones of Vero-KO-DPP4-1,Vero-KO-DPP4-12,Vero-KO-DPP4-34,Vero-KO-DPP4-58 and two heterozygous clones of Vero-KO-DPP4-24/25 were selected.Vero cells were used as a control to detect the expression level of CD26/DPP4 protein by Western Blot.It was found that the CD26/DPP4 protein expression level of positive clone cells was lower than that of Vero cells,and Vero-KO-DPP4-34 was significantly lower than Vero cells.Detection of virus copies of PEDV in Vero-KO-DPP4-34 cells was lower than in Vero cells,but the virus could still infect clonal cells,indicating that CD26/DPP4 may not be a solely receptor of the Vero cells to the virus.Through the detection of cell proliferation and apoptosis of cells,the cell proliferation ability of the four positive clonal cells was significantly lower than Vero cells,but only the apoptosis of Vero-KO-DPP4-34 cells was different with Vero cells.Therefore,this experiment aims to knock out the CD26/DPP4 gene of Vero cells by CRISPR/Cas9 technology,to detect the replication level of PEDV in cloned cells.Studying the effect of CD26/DPP4 on PEDV replication provides a theoretical basis for further exploration of the process of PEDV invasion of Vero cells.At the same time,it will provide new insights into the potential therapeutic targets of PEDV or provide new strategies to increase virus titer and expand virus production in vaccine production processes.