Study On Pathway Used By Peste Des Petits Ruminants Virus N75-1 Strain To Enter Host Cells

Peste des Petits Ruminants(PPR)is a highly contagious disease that infects small ruminants such as sheep and goats caused by the Peste des Petits Ruminants Virus(PPRV).It is the First Class contagion in China.In recent years,most of PPRV research focuses on protein function and vaccine preparation,but there are few reports about pathways of PPRV invasion of host cells.The aim of this study was to explore the infection characteristics of PPRV to Goat endometrial epithelial cells(EEC)and the pathway of virus into host cells,so as to provide the relevant theoretical and scientific basis for studying the infection mechanism of the virus to host cells.The results of the study are as follows:1.The proliferation of PPRV on goat endometrial epithelial cells was obtained.After PPRV was inoculated into EEC cells,thecytopathic effect(CPE))was observed after the passage of time.After 24 h to 48 h,the cells had no obvious morphological changes.At 72 h,the cells were found to have typical CPE,such as cells together,the formation of syncytia,refractive intensity becomes strong.At 96 h,the cells appear broken,death phenomenon.After inoculation of PPRV N75-1 strain in EEC,the time of entry of the virus into the cell was determined by laser confocal observation within 1-2 h.By collecting the cellular supernatant at 1 h,3 h,6 h,9 h,12 h,24 h,TCID50 test showed that the virus titer reached the highest level at 24 h after infection.By collecting the cell samples at 0 h,3 h,6 h,9 h,12 h,24 h,Western Blot showed that the expression of N protein was the highest at 12 h after infection and decreased at 24 h,and the proliferation of PPRV on EEC cells was obtained.2.PPRV penetrating EEC cells can not pass through the clathrin-mediated endocytosis pathway.Chlorpromazine(CPZ)is a clathrin endocytosis inhibitor that blocks the clathrin-mediated endocytosis pathway.First,EEC cells were treated with CPZ,and post infection,the number of PPRV entering the cells was not significantly different from that of the control by laser confocal microscopy.Western Blot and TCID50 experiments showed no significant changes in the expression of N protein,the virus titer did not change significantly.Second,we found that the expression of N protein and the virus titer of PPRV did not change significantly by transfecting si RNA of clathrin into EEC cells,and then by Western Blot and TCID50.The above experimental results show that PPRV penetrating EEC cells can not pass through the clathrin-mediated endocytosis pathway.3.PPRV can penetrate EEC cells through Caveola-mediated endocytosis pathway.Methyl-β-cyclodextrin(MβCD)is a cholesterol extract,nystatin is a cholesterol chelator,and the Caveola endocytosis pathway depends on cholesterol,so they can block the membrane Recessed endocytosis pathway.First,EEC cells were treated with two drugs,and after 12 h of inoculation,we observed that the PPRV entering the cells was significantly reduced relative to the control.By transfection of Caveola’s structural protein Caveolin-1 siRNA in cells and Western Blot and TCID50 experiments,we can see that the expression of N protein was significantly reduced after transfection,the virus titer titer was also significantly reduced.The above experimental results show that PPRV can penetrate EEC cells through Caveola-mediated endocytosis pathway.In conclusion,this study determined that PPRV invasion of EEC did not rely on clathrin-mediated endocytosis pathway,and relied on caveola-mediated endocytosis pathway by specific chemical inhibitor blocking and RNA interference.The results provide a theoretical basis for understanding the pathogenesis of the virus and finding new drug therapy targets.