Cloning, Expression Of Bovine And Ovine IFIT1 Genes And Their Antiviral Effects On Btv Proliferation

Bluetongue(BT),caused by bluetongue virus(BTV),is an infectious disease of ruminant that is transmitted by insect vectors.To date,Eleven serotypes including BTV-1~5,7,9,12,15,16 and 24,have been identified in China,and BTV-1 and BTV-16 are the two most prevalent serotypes in China.IFIT family,also also known as ISG56 family,is a class of interferon induced genes that includes 4 members of IFIT1,IFIT2,IFIT3 and IFIT5 genes.The gene expression levels of IFIT are usually low in the cells,but the expression of IFIT family genes increase significantly when cells are infected with viruses.IFIT family can participate in regulating innate immune response by recognition of viral nucleic acid or protein,thereby enhancing the antiviral effects of cells.However,currently little information is available about the IFIT family genes in cattle and sheep and whether they can inhibit BTV replication is unclear.In this study,bovine and ovine IFIT1 genes were cloned and sequenced,and the sequence analyses were performed by informatics softwares.The eukaryotic expression plasmids of bovine and ovine IFIT1 genes were constructed and their effects on BTV replication were evaluated in the transfected cells.(1)Cloning and sequence analysis of bovine and ovine IFIT1 genesThe RT-PCR primers were designed based on published IFIT1 gene sequences of other species in GenBank.Total RNAs were extracted from primary sheep testis(PST)and MDBK cells,respectively.RT-PCR was performed to obtain bovine and ovine IFIT1 genes,which were ligated with pMD 18-T vector and sequenced.The sequence analyses showed that bovine and ovine IFIT1 genes have more than 65% similarity with that of yaks,tibetan antelopes,goats,sheep,alpacas,camels,wild boar,the giant pandas,horses and human beings.The phylogenetic tree analysis showed the close relationships of IFIT1 genes from ruminants including cattle,sheep,camels,yaks,tibetan antelopes,goats,and alpacas,which were distinct from the orders Perissodactyla,Primates and other species.(2)Eukaryotic expression of bovine and ovine IFIT1 genes and their effects on BTV proliferationBovine and ovine IFIT1 genes were subcloned into expression vector pcDNA3.1(+)and recombinant expression plasmids pcDNA3.1-BO-IFIT1 and pcDNA3.1-OV-IFIT1 were constructed,which were transfected into BHK21 cells.Western-blot and immunofluresence assay showed that bovine and ovine IFIT1 proteins were successfully expressed in BHK21 cells.BHK21 cells expressing bovine and ovine IFIT1 were infected with BTV and then analysed by Western-blot,qRT-PCR,and plaque assay to examine the effect of IFIT1 on BTV proliferation.The results indicated that bovine and ovine IFIT1 were able to inhibit BTV replication,and virus titers and NS1 protein were reduced significantly.In addition,qRT-PCR analyses demonstrated that the expression levels of IFIT1 genes increased significantly in BTV-infected PST and MDBK cells,compared with uninfected cells.These results indicated that bovine and ovine IFIT1 play an important role in anti-BTV infection.