| Bluetongue is an acute non-contagious disease caused by bluetongue virus(BTV)that seriously affects ruminants such as sheep,cattle and deer.The outbreaks in Europe have caused major economic losses in recent years.The bluetongue is listed as one of the notifiable animal diseases by the World Organization for Animal Health(OIE).Viperin is one of interferon-stimulated genes(ISGs)discovered in 2001,which could be induced by many kinds of viruses and even bacteria,and has been found that it has the functions of inhibiting virus replication,immune regulation and metabolic regulation.However,little information is available about bovine and ovine Viperin genes and whether they inhibit BTV replication is unknown.In this study,bovine and ovine Viperin genes were cloned and sequenced from primary sheep testes(PST)cells and Madin-darby bovine kidney(MDBK)cells,respectively.The sequence analyses were performed by bioinformatics softwares.The eukaryotic expression plasmids of bovine and ovine Viperin genes were constructed and their antiviral roles on BTV replication were evaluated in the transfected cells.In this study,the mRNA levels of Viperin genes in cultured cells and animals were firstly analyzed by real-time quantitative PCR.The results showed that all transcription levels of Viperin genes in BTVinfected PST,MDBK cells and sheep blood increased significantly,compared with the uninfected control.The primers were designed based on the predicted Viperin sequences deposited in GenBank,and the total RNA was extracted from BTV-infected PST and MDBK cells.RT-PCR was performed to amplify bovine and ovine Viperin genes,which were ligated into pMD18-T vector.The sequencing results showed that both open reading frames(ORF)of bovine and ovine Viperin gene consist of 1092 base pairs.The sequence analyses showed that the bovine and ovine Viperins were similar to these of other species,containing N-terminal,radical S-adenosyl methionine(SAM)and C-terminal domains.The leucine zipper and SAM domain are highly conserved among different species.Phylogenetic analyses revealed the close relationships of Viperin genes from ruminants including alpaca,wild yak and tibetan antelopes,which were distinct from other species.VP5 protein is an important component of the outer capsid of BTV,and has membrane permeation function,which mediates the release of virus particles from the endosomes into the cytoplasm.Here the recombinant VP5?41aa protein(rVP5?41aa)with the deletion of N-terminal 41 aa was expressed in E.coli and purified successfully by His-Tag purification resin.Polyclonal antibody was preparaed by immunizing New Zealand white rabbits with rVP5?41aa.Western blot results showed that the VP5?41aa antibody can react with VP5?41aa expressed in E.coli as well as the VP5 protein in BTV-infected cells.The antibodies can also recognize the VP5 protein with native conformation in BTV-infected cells by immunofluorescence assay.The antibody would be useful to evaluate the BTV replication and further study the function of VP5 protein.To explore the effects of Viperins on BTV replication,the bovine and ovine Viperin genes were subcloned into the eukaryotic expression vecter pcDNA3.1(+),and the recombinant expression plasmids were transfected into BHK-21 cells respectively to examine their antiviral effects on BTV replication.The results showed that both of Viperins were expressed successfully in BHK-21 cell.Compared with the BHK-21 cells transfected with pcDNA3.1(+),the expression of BTV structural protein VP5,VP6 and non-structural protein NS1 decreased significantly in BHK-21 cells that expressing bovine and ovine Viperin proteins.Real-time quantitative PCR results showed that overexpression of Viperin protein decreased the level of BTV NS3 mRNA effectively.Plaque assay further confirmed that the bovine and ovine Viperins reduced significantly the virus titers.These results clearly demonstrated that bovine and ovine Viperins inhibited the replication of BTV,indicating their important roles in anti-BTV infection. |
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