Study On Cell Cycle Blocking Induced By Porcine Epidemic Diarrhoea Virus

Porcine epidemic diarrhea virus (PEDV) is a virulent pathogenof pigs,which cause watery diarrhea, vomiting, weight loss, anorexia symptoms in pigs.After first outbreak at1971, PEDV has has erupted in many countries. This is aserious hazard for pig industry around the world. Since October2010, a newoutbreak began in China, resulted in significant economic losses so far.This study focus on PEDV proliferation and cell cycle blocking caused bythis virus, aiming to provide theoretical support for the disease pathogenesis andprevention of disease. Firstly, based on the N gene of PEDV-HLJ/2011strainisolated by our lab, we established a Taq Man-MGB fluorescence quantitativePCR method. With high specification and sensitivity, this method coulddiagnose clinical diarrheal samples, also could test the proliferation curve ofPEDV. Then we detected the proliferation curve of PEDV-HLJ/2011by CPEobservation and Real-time PCR. The results showed that this virus proliferatefastst within12-24h post infected. By using flow cytometry and Western Blot,we detectd the cell cycle blocking impacted by PEDV. We found that theinfected cell were blocked in G1/G0phase. To confirm this blocking are causedby activation of DNA damage pathways, we use Ch2.k, H2A.X phosphorylatedantibodies detected changes in the phosphorylation levels of these molecules,and the change cycle of the infected cells and viral proliferation after addingchemical agent to blocke this pathway. The result showed that bloking thispathway could inhibit cell cycle blocking and PEDV proliferation. This studycould provide a foundamental result for PEDV infection and proliferation in cell,which could be useful for its pathogenesis study.