Effect Of PEDV Infection On Vero Cell Cycle And Its Mechanism

Porcine epidemic diarrhea(PED)is a strongly infectious pathogenic infectious disease caused by Coronaviridae coronavirus Porcine epidemic diarrhea virus(PEDV).The typical symptoms of this disease were swine watery diarrhea,vomiting and high piglet mortality at all ages,PED has been widely prevalent in our country since 2010,causing heavy losses to the pig industry.In this study,the effect of PEDV infection on cell cycle and related molecular mechanism were studied,and the effect of cycle arrest on the proliferation and pathogenicity of PEDV was analyzed in order to provide theoretical basis for elucidating the molecular pathogenic mechanism and prevention and control of PEDV.In this study,firstly,the interaction between PEDV and Vero cells was studied.The effect of PEDV on the viability of Vero cells was detected by MTT assay.The MOI of PEDV infection was determined at 0.5 MOI in this study and the collection times of Vero cells were 0h,6h,12 h,18h and 24 h post infection.The isolated and preserved Vero cells were separately infected with PEDV provided by our laboratory.Flow cytometry analysis was used to detect the effect of PEDV on Vero cell cycle,whose results showed that PEDV caused Vero cell cycle arrest at G0/G1 phase.To further confirm that the blockade of Vero cell cycle is caused by PEDV replication,qRT-PCR analysis of PEDV sgmRNA(N,M,ORF3)and TCID50 test were performed with PEDV-infected cells which showed that TCID50 and sgmRNA continued to increase during PEDV infection,suggesting that there were PEDV replication in PEDV-infected cells.And flow cytometry analysis performed with UV-PEDV-infected cells,PEDV-infected cells and mock cells showed UV-PEDV failed to cause cell cycle arrest at G0/G1 phase,suggesting that Vero cell cycle arrest is caused by PEDV replication at G0/G1 phase.To further investigate the effect of PEDV on Vero cell cycle,Western blot analysis was used to detect the expression of cyclin A,cyclin B1,cyclin D1,cyclin E,CDK2,CDK2 and CDK4.The results showed that the expressions of CDC2,CDK2,CDK4,CyclinA,CDK6,p53,p-p53(Ser15),p-p53(Ser20)and E protein up-regulated in PEDV-infected cells.To further analyze the correlation between PEDV-induced cell cycle arrest and p53 signaling,flow cytometry analysis was performed to analyze PEDV-infected cells with p53-specific inhibitor pretreatment,whose results showed that the inhibitor reversed the G0/G1 cycle arrest induced by PEDV.In the meantime,cyclin and cyclin dependent kinase in these cells were analyzed by Western blot which showed that the expression of p21,CDC2,CDK2,CDK4 and Cyclin were down-regulated while Cyclin E expression was up-regulated.To investigate the biological significance of cell cycle arrest in PEDV replication,Vero cells were treated with serum starvation,dithiothreitol and nocodazole to synchronize the cell cycle at G0/G1,G1/S and G2/M phase,respectively which was confirmed by flow cytometry and then infected with PEDV.At 18 h post infection,cell cycle progression of there cells were analyzed by flow cytometry and PEDV sgmRNA(N,M,ORF3)by qRTPCR.The flow cytometry results showed that more than 80% cells were synchronized at G0/G1 phase after 48 hours of serum starvation,70% cells at G1/S phase after dithiothreitol treatment,75% cells at G2/M phase after nocodazole treatmentinfection and PEDV infection could cause G0/G1 phase arrest in all these cells.TCID50 and qRT-PCR results showed that G0/G1 phase cells were more favorable for PEDV replication than G1/S phase,G2/M phase and unsynchronized cells.In summary,this study revealed that PEDV caused Vero cell cycle arrest at G0/G1 phase through p53 pathway and further confirmed that this cell cycle arrest could benefit PEDV replication,and explained the molecular basis of PEDV replication at cellular level,providing new ideas for improving PEDV replication and adaptive culture in vitro.