African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, causes an acute disease in horses with a high mortality rate. AHSV encodes four nonstructural proteins (NS1, NS2, NS3/NS3A), whose functions in the viral life cycle are not fully understood. The NS1 protein is the most abundantly expressed viral protein during AHSV infection and forms tubular structures within the cell cytoplasm. No function has been ascribed to these tubules to date, although it has been suggested that they may play a role in cellular pathogenesis. Studies aimed at understanding the function of NS1 have been hampered by the lack of a suitable reverse genetics system for AHSV. However, the phenomenon of RNA interference (RNAi) has emerged as a powerful tool whereby the function of individual genes can be studied. In mammalian cells, RNAi can be triggered by exposing cells to double-stranded RNA either via exogenous delivery of chemically synthesized small interfering RNAs (siRNAs) or endogenous expression of short hairpin RNAs (shRNAs). Consequently, the aim of this investigation was to develop a plasmid DNA vector-based RNAi assay whereby expression of the AHSV-6 NS1 gene could be suppressed in BHK-21 cell culture with shRNAs directed to the NS1 gene. To investigate, complementary oligonucleotides corresponding to selected AHSV-6 NS1 gene sequences were chemically synthesized, annealed and cloned into the pSUPER shRNA delivery vector under control of the RNA polymerase III H1 promoter. The plasmid DNA vector-expressed shRNAs targeted sequences within the NS1 gene corresponding to nucleotides 710 to 728 (shNS1-710) and 1464 to 1482 (shNS1-1464), respectively. A NS1- eGFP chimeric gene was constructed and used towards establishing a simple assay whereby the gene silencing efficiency of different RNAi effector molecules could be evaluated by analysis of the protein level visually and quantitatively by fluorometry. The effect of the NS1- directed shRNAs on AHSV-6 NS1 protein expression was subsequently evaluated by cotransfection of BHK-21 cells with the respective recombinant pSUPER shRNA delivery vectors and the NS1 reporter plasmid pCMV-NS1-eGFP. The results indicated that shNS1- 710 and shNS1-1464 suppressed NS1-eGFP expression by 19% and 9%, respectively. The potential of the NS1-directed shRNAs to suppress NS1 mRNA expression was investigated by transfection of BHK-21 cells with the respective recombinant pSUPER shRNA delivery vectors, followed by transfection with the recombinant mammalian expression vector pCMVNS1 or infection with AHSV-6. Results obtained by semi-quantitative real-time PCR assays indicated that both NS1-directed shRNAs interfered with NS1 mRNA expression, albeit to different extents in the respective assays. Taken together, these results demonstrated that AHSV-6 NS1 gene expression can be suppressed in BHK-21 cells by plasmid DNA vectorderived shRNAs and suggests that this approach may, with further optimization, be useful in determining the function of the NS1 protein in virus-infected cells.