Cell Entry Of Classical Swine Fever Virus And The Role Of GBF1 And Rab2 In Its Multiplication

Classical swine fever(CSF),casued by classical swine fever virus(CSFV),is a highly contagious disease and poses an enormous threat to pig industry.CSF is listed as one of the notifiable swine diseases by the Office International Des Epizooties(OIE)and designated as one of the class A animal disease in China.Classical swine fever virus(CSFV)is a member of genus Pestivirus of the Flaviviridae family,which is a single-stranded positive-sense enveloped RNA virus.Series of research on CSFV pathogenesis has been carried out aroud the world and make some achievements.However,the mechanism of CSFV spreading,cell entry and replication are still not totally discovered.Therefore,this research made efforts to test the CSFV spread in vitro culture system,CSFV cell entry pathway and relationship between CSFV replication and Golgi-ER retrograde trafficking regulation protein GBF1 and Rab2 based on ST cell infection model.The results were shown as follow:1.CSFV proliferated with cell division.In addition,CSFV spread could not be inhibited with low concentration of neutralizing antibody.By adding low metling agarose and neutralizing antibody in the supernates,CSFV spread was greatly blocked,which demonstrated that CSFV spread by the cell free pathway other than cell-to-cell transmission in vitro culture system.Furthermore,CSFV spread with different concentration of neutralizing antibody were test and the results shown that CSFV spread could not be inhibited when the concentration of neutralizing antibody was lower than 1×105 ND50.Finally,CSFV could proliferated with cell division was found by analyzing the relationship between cell numbers of CSFV infected cells in plaques and the cell doubling time.2.CSFV entry into ST cell by clathrin-mediated endocytosis pathway.Real-time PCR and indirect immunofluorescence assay were used to analyze the cellular CSFV content at early stage after CSFV infection.Cellular CSFV was greatly reduced when the inhibitors chlorpromazine(disrupt the assembly of clathrin coat pits)and dynasore(inhibited dynamin GTPase activity)were added in the CSFV infection process.Furthermore,the shRNA aim at clathrin heavy chain(CHC)and dynamin-2 wereconstructed and transfected to ST cells.After selecting by puromycin,the CHC and dynamin-2 down-regulation cells were purified.Cellular CSFV were greatly decreased in the CHC and dyanmin-2 down-regulation cells compared with the ST cells and scrambled shRNA transfected cells.These experimental results provided that CSFV can entry into ST cells by clathrin-mediated endocytosis pathway.3.CSFV cell entry relied on early endosome and late endosome.NH4 Cl was used to retard the endosome acidification.After NH4 Cl treatment in the CSFV infection process,the cellular CSFV concentration was greatly decline.So,CSFV cell entry need acid induction.Moreover,the Rab5(marker of early endosome),Rab7 and Rab9(marker of late endosome)shRNA vectors were constructed and transfected into ST cell.After puromycin selecting,the Rab5,Rab7 and Rab9 down-regulation cells were selected for further test.After that,the cellular CSFV concentration at the early stage after CSFV infection in ST,Rab5,Rab7,Rab9,and scrambled shRNA trancfected cells were test and the results indicated that CSFV cell entry was inhibited when Rab5,Rab7 or Rab9 was down-regulated.These results demonstrated that Rab5,Rab7 and Rab9 were necessary for CSFV cell entry,and suggested that early endosome and late endosome were helpful in CSFV cell entry process.4.GBF1 and Rab2 regulated CSFV multiplication.Brefeldin A and golgicide A were used to inhibited activity of GBF1.CSFV multiplication was suppressed when the inhibitors were used in both RNA replication and viral particles generation by real-time PCR and viral titration.In addition,the GBF1,ARF1 and Rab2down-regulation cells were constructed.CSFV proliferation was blocked when GBF1 or Rab2 expression was down-regulated.However,CSFV proliferation dynamic on ARF1 down-regulation cells had no different with ST cells or scrambled shRNA transfected cell.Furthermore,CSFV proliferation was promoted by GBF1 and Rab2over-expression using a lentiviral system.The results showed that CSFV multiplication was mediated by GBF1 and Rab2 rather than ARF1.In conclusion,the CSFV spread pathway in vitro culture systerm,the CSFV cell entry pathway and its organelle and protein dependency in susceptible cells,relationships between CSFV proliferation and Golgi-ER retrograde regulation protein GBF1 and Rab2 were tested and observed in this study.The results offer us new data and theory for a better understanding of CSFV life cycle.