INHIBITION OF REPLICATION BY TARGETING SHRNAS AGAINST VP1 AND VP6 GENES OF BLUETONGUE VIRUS

Manju Elizabeth P.*, Byregowda S.M., Suryanarayana V.V.S., Ramanjinigowda P. H., Purushotham K.M.

ABSTRACT

Background: Bluetongue virus (BTV), a member of Orbivirus genus within the Reoviridae family causes a haemorrhagic disease mainly in sheep and is transmitted between its mammalian hosts by certain Culicoides spp. Until now 26 serotypes have been identified worldwide. Many vaccine strategies developed to control the disease are not yet completely successful because of their inability to offer cross protection. RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of homologous mRNA.RNA interference has been used to induce gene silencing in a large number of viruses but not attempted to BTV. Results: The shRNA producing cassettes targeting genes expressing VP1and VP6 proteins of BTV were obtained on successful cloning in psiRNA vector. Initial studies were carried out by transfecting the BHK21cells followed by infecting with four BTV serotypes [BTV1, 10, 16 and 23; (8h post infection)] at 100 TCID50 and 1000 TCID50 virus and harvested at 36h p.i. Upon titration, inhibition of replication was observed in cells transfected with shRNA producing cassettes of VP1 and VP6. The results of this study indicatThe results of this study indicatThe results of this study indicat The results of this study indicatThe results of this study indicat The results of this study indicat The results of this study indicatThe results of this study indicatThe results of this study indicatThe results of this study indicatThe results of this study indicat The results of this study indicatThe results of this study indicatThe results of this study indicatThe results of this study indicatThe results of this study indicat The results of this study indicatThe results of this study indicatThe results of this study indicatThe results of this study indicatThe results of this study indicatThe results of this study indicatThe results of this study indicat ed that ed that ed that ed that knocking down of VP1 gene (polymerase) and VP6 gene (helicase) of BTV is effective on inhibition of replication of BTV serotypes and the shRNA is effective upto 36h p.i. Conclusion: This study demonstrates that vector based shRNA methodology can effectively inhibit replication of bluetongue virus in BHK21 cells and can be used to control spread of BTV when used in vivo.

Keywords: RNAi, shRNA, BTV, TCID50