African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family, has a 10-segment double-stranded (ds)RNA genome enclosed within a double capsid. In addition to seven structural proteins (VP1-VP7), four non-structural proteins (NS1, NS2 and NS3/NS3A) are synthesized in infected cells and are involved in virus morphogenesis. Due to the lack of a reverse genetic system for orbiviruses, analyses regarding AHSV gene function have been limited to the characterization of individual virus proteins following their expression in heterologous expression systems. The phenomenon of RNA interference (RNAi), has, however, revolutionized approaches to study the function of individual genes. RNAi is an evolutionary conserved mechanism by which RNA duplexes, known as short interfering RNA (siRNA), can reduce gene expression through enzymatic cleavage of complementary mRNA. In addition to synthetic siRNA, RNAi can also be induced in mammalian cells by plasmid and viral vector systems that express short hairpin RNAs (shRNAs) that are subsequently processed to siRNAs by the cellular machinery. Consequently, the aim of this investigation was to establish a plasmid DNA vector-based RNAi assay whereby the expression of the AHSV-9 NS2 gene could be suppressed in BHK-21 cells. Complementary oligonucleotides corresponding to selected AHSV-9 NS2 gene sequences were chemically synthesized, annealed and cloned into the pSUPER shRNA delivery vector, downstream of the RNA polymerase III H1 promoter. The vector-expressed shRNAs targeted regions within the NS2 gene corresponding to nucleotides 211 to 230 (shRNA-211), 377 to 396 (shRNA-377) and 958 to 977 (shRNA-958), respectively. To determine whether the NS2-directed shRNAs was capable of silencing NS2 protein expression, BHK-21 cells were co-transfected with the respective pSUPER shRNA delivery vectors and a NS2 reporter plasmid, pCMV-NS2-eGFP. Fluorescence microscopy indicated that NS2-eGFP expression was makerdly reduced in these cells compared to cells transfected with the reporter plasmid only, and fluorometry analysis indicated that the level of inhibition mediated by the shRNAs were in excess of 80%. The potential of the NS2-directed shRNAs to reduce the level of NS2 transcripts in AHSV-9 infected BHK-21 cells was also investigated by transfection of the BHK-21 cells with the respective pSUPER shRNA delivery vectors, followed by virus infection. Results obtained by means of semi-quantitative real-time reverse transcriptase-polymerase chain reactions indicated that shRNA-377 interfered the most efficiently with NS2 mRNA expression, and the greatest silencing effect was observed at 24 h post-infection. During the course of this investigation it was also attempted to establish a BHK-21 cell line that stably expressed the NS2-directed shRNA-377. For this purpose, a recombinant pSUPER.Retro.Puro retroviral vector was constructed and following transfection of BHK-21 cells, stable transfectants were selected by growth in the presence of puromycin. Results indicated that although the derived cell line suppressed AHSV-9 NS2 mRNA expression, the plasmid DNA was maintained extrachromosomally. Overall, the results of this investigation have provided evidence that AHSV-9 NS2 gene expression can be suppressed in mammalian cells by vector-derived shRNAs.