Analysis Of MRNA/miRNA Profile Of Vero E6 Cells Infected With PEDV And The Mechanism Of The Influence Of Abl2 On Virus Replication

Porcine epidemic diarrhea virus（PEDV）belonging to the alpha Coronaviridae family,can cause acute and highly contagious intestinal infectious disease in piglets.The infection of PEDV leads to dynamic changes in the expression levels of mRNA and miRNA of host cells,as well as forming a complex network interacting with virus.Comprehensive analysis of the regulation of differentially expressed host factors in the process of replication of PEDV helps to completely grasp the changes in cellular components,biological functions,and signaling pathways,understand the pathogenic mechanism of PEDV and screen for the factors that influence virus invasion and regulate viral replication.Vero E6 cell line is used for isolation and serial passage of PEDV.In this study,Vero E6 cells were infected with PEDV attenuated strain CV777 and virulent strain LNct2 then subjected to sequence mRNA and miRNA profile.The CV777 infected group screened 33 differentially expressed genes and LNct2 infected group screened 1854 differentially expressed genes,compared with the control group.Two groups shared 16 differential expressed genes.Sequencing results of miRNA showed that the CV777 infected group had 23 differentially expressed miRNAs while the LNct2 group screened 70differentially expressed miRNAs,compared with the control group.Two groups shared 10 differentially expressed miRNA.Bioinformatics analysis of the differentially expressed host factors showed that the differentially expressed genes were mainly enriched in the regulation of intracellular signal transduction,cell metabolism,apoptosis and other pathways.The interferon stimulating factors APOBEC3,OASL,MX2,IFITM3 were down-regulated,suggesting that PEDV escaped from host innate immune response by antagonizing the expression of antiviral molecules of Interferon.In addition,PEDV infection activated MAPK signaling pathway and enhanced virus replication.Further research found that the up-regulated host factor Abl2 could influence virus replication.Abl2 is a non-receptor tyrosine kinase that regulates various processes of cell cycle.Through western blot and TCID₅₀ test,it was found that the expression of PEDV N protein and viral titers decreased significantly after silencing the expression of Abl2.The application of Abl2 inhibitors imatinb,GNF2and GNF5 all inhibited viral replication in dose-dependent manner,suggesting that Abl2 was involved in the process of viral replication.Adding inhibitors at different times of virus infection proved that Abl2 influenced PEDV replication mainly during the viral invasion stage,without affecting the virus adsorption process.After the addition of Abl2 inhibitors,the syncytia formed by virus-induced cells was significantly reduced detected by indirect immunofluorescence,and the number of syncytia was reduced as well.The experiment of Abl2 inhibitors added in cells after exogenously expressing PEDV S protein proved that Abl2 affected the formation of intercellular syncytia mediated by PEDV S protein.In addition,Abl2 inhibitors imatinb,GNF2,and GNF5 had a broad-spectrum inhibitory effect on swine enteric coronavirus.Inhibitors could significantly inhibit the infection of TGEV and PDCoV in ST cells by blocking the invasion of virus and cellular fusion.In order to study the regulation of 10 differentially expressed miRNAs caused by PEDV infection,the miRNA mimic/inhibitor experiment was used to screen the miRNAs that influenced PEDV replication.The results showed that miR-486-5p,miR-339-5p,miR-1271-5p could significantly inhibit PEDV replication,while miR-33a promoted viral replication.The target gene of miR-486-5p in the regulation of PEDV infection was SRSF3 detected by RT-qPCR,dual luciferase reporter system.The up regulation of SRSF3 promoted viral infection while silencing the expression of SRSF3 inhibited viral replication.In addition,SRSF3 did not affect virus replication through interaction with PEDV N protein.The mechanism of SRSF3 influencing PEDV infection needs to be further explored.The study constructed the mRNA/miRNA profile of PEDV infected Vero E6 cells,and found that Abl2 played the regulatory role in the invasion stage of PEDV.We also screened out different expressed miRNA that influenced PEDV replication,which could provide new drug targets of PEDV so as to provide new antiviral strategies.