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Evaluation of inhibitory effect of shRNA constructs in bluetongue virus (BTV) Replication

KrishiKosh

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Title Evaluation of inhibitory effect of shRNA constructs in bluetongue virus (BTV) Replication
 
Creator P, Manju Elizabeth
 
Contributor S. M, Byregowda
 
Subject biological phenomena, diseases, genetic techniques, rna, viruses, proteins, genes, dna, replication, pcr
 
Description Bluetongue is an economically important disease for the farming community however vaccines effective for all 26 serotypes are unavailable. The present study was carried out for developing shRNA producing cassettes targeting genes expressing VP1, VP3 and VP6 proteins of BTV to evaluate the inhibition of virus replication. Two siRNAs were selected for conserved regions of each gene and corresponding shRNA producing cassettes were obtained on successful cloning in psiRNA vector. Endotoxin free plasmid DNAs of positive clones were used for transfection in BHK21 cell lines. Initial studies were carried out by transfection of the cells followed by infecting with four BTV serotypes [BTV1, 10, 16 and 23; (8h post transfection)] at 100TCID50 and 1000TCID50 virus and harvested at 36h post infection. Upon titration, inhibition of replication was observed in cells transfected with shRNA producing cassettes of VP1 gene and VP6 gene whereas inhibition was limited with VP3shRNA forming cassettes. For time course study the cells were transfected and infected with BTV1 (100TCID50) and harvested at 24, 48 and 72h p. i. Titration of samples indicated inhibition of replication at 24h p. i. but not after 36h p.i. Transfection was carried out with 12 combinations of shRNA producing cassettes and all of them were found to be effective even at 48h p.i. The Ct values on q RT PCR of the above sample (24h p. i., 48h p. i. combinations) indicated that inhibition was achieved by both constructs of VP1 gene and one construct of VP3 gene (VP3shRNA1) at 24h p. i., while all the combinations of shRNAs could produced inhibition of replication at 48h p.i. The results of the present study revealed that knocking down of VP1 and VP6 genes will be effective for the control of BTV multiplication and transfection with combinations of shRNA producing cassettes showed synergetic effect.
 
Date 2016-05-17T14:46:13Z
2016-05-17T14:46:13Z
2012-07-28
 
Type Thesis
 
Identifier Th-10238
http://krishikosh.egranth.ac.in/handle/1/66033
 
Language en
 
Format application/pdf
 
Publisher University of Agricultural Sciences, Bengaluru