Cholesterol represents one of the key constituents of small,dynamic,sterol-and sphingolipid-enriched domains on the plasma membrane.It has been reported that many viruses depend on plasma membrane cholesterol for effi...Cholesterol represents one of the key constituents of small,dynamic,sterol-and sphingolipid-enriched domains on the plasma membrane.It has been reported that many viruses depend on plasma membrane cholesterol for efficient infection.In this study,the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus(PRRSV) infection of MARC-145 cells was investigated.Pretreatment of MARC-145 cells with methyl-β-cyclodextrin(MβCD),a drug used to deplete cholesterol from cellular membrane,significantly reduced PRRSV infection in a dose-dependent manner.This inhibition was partially reversed by supplementing exogenous cholesterol following MβCD treatment,suggesting that the inhibition of PRRSV infection was specifically mediated by removal of cellular cholesterol.Further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry,especially virus attachment and release.These results indicate that the presence of cholesterol in the cellular membrane is a key component of PRRSV infection.展开更多
Pseudorabies virus(PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK^(-)/gE^(-)/GP5^(+)expressing GP5 of porcine reproductive and respiratory ...Pseudorabies virus(PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK^(-)/gE^(-)/GP5^(+)expressing GP5 of porcine reproductive and respiratory syn-drome virus(PRRSV),based on the PRV genetically depleted vaccine strain TK^(-)/gE^(-)/LacZ^(+),scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene(the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK^(-)/gE^(-)/LacZ^(+),resulting in a PRV recombinant named TK^(-)/gE^(-)/GP5m^(+),which expressed the modified GP5m protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK^(-)/gE^(-)/GP5m^(+)and TK^(-)/gE^(-)/GP5^(+)expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies(the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK^(-)/gE^(-)/GP5m^(+)against PRRSV were higher than that induced by TK^(-)/gE^(-)/GP5^(+).Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.展开更多
基金supported by the National Natural Science Foundation of China (Grant No. 30770082)National Basic Research Program of China (Grant No. 2005CB523200)
文摘Cholesterol represents one of the key constituents of small,dynamic,sterol-and sphingolipid-enriched domains on the plasma membrane.It has been reported that many viruses depend on plasma membrane cholesterol for efficient infection.In this study,the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus(PRRSV) infection of MARC-145 cells was investigated.Pretreatment of MARC-145 cells with methyl-β-cyclodextrin(MβCD),a drug used to deplete cholesterol from cellular membrane,significantly reduced PRRSV infection in a dose-dependent manner.This inhibition was partially reversed by supplementing exogenous cholesterol following MβCD treatment,suggesting that the inhibition of PRRSV infection was specifically mediated by removal of cellular cholesterol.Further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry,especially virus attachment and release.These results indicate that the presence of cholesterol in the cellular membrane is a key component of PRRSV infection.
基金This work was supported by the National Natural Sciences Foundation of China(Grant No.30300257)the National Basic Research Program of China(No.2005CB523200)the Youth Scientist Project of Wuhan City(No.20025001041).
文摘Pseudorabies virus(PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK^(-)/gE^(-)/GP5^(+)expressing GP5 of porcine reproductive and respiratory syn-drome virus(PRRSV),based on the PRV genetically depleted vaccine strain TK^(-)/gE^(-)/LacZ^(+),scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene(the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK^(-)/gE^(-)/LacZ^(+),resulting in a PRV recombinant named TK^(-)/gE^(-)/GP5m^(+),which expressed the modified GP5m protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK^(-)/gE^(-)/GP5m^(+)and TK^(-)/gE^(-)/GP5^(+)expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies(the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK^(-)/gE^(-)/GP5m^(+)against PRRSV were higher than that induced by TK^(-)/gE^(-)/GP5^(+).Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.
