African horsesickness virus (AHSV) belongs to the Orbivirus genus within the family Reoviridae. AHSV is transmitted to vertebrates by Culicoides midges and causes an acute disease in horses with a high mortality rate. The virion consists of an outer layer composed of proteins VP2 and VP5, which surround an icosahedral core containing two major proteins, VP3 and VP7, three minor proteins, VP1, VP4 and VP6, and ten segments of double-stranded (ds)RNA. The VP7 protein is not only important in maintaining the structural integrity of the virus particles, but has been reported to play a key role in Culicoides cell entry. The phenomenon of RNA interference (RNAi), which can be used to selectively silence homologous genes post-transcriptionally, has revolutionized approaches to study gene function and it is also projected as a potential tool to inhibit virus replication. In mammalian cells, RNAi can be triggered by the direct introduction of 21-23 nucleotide duplexes of small interfering RNA (siRNA) that specifically and potently inhibits endogenous and heterogeneous gene expression. Consequently, the aims of the investigation were to develop RNAi assays whereby the expression of the AHSV-9 VP7 gene could be suppressed in Spodoptera frugiperda insect cells and in mammalian BHK-21 cell culture, as well as to determine whether gene-specific siRNAs may prevent AHSV-9 infection in cell culture. To investigate RNAi-mediated silencing of an enhanced green fluorescent protein (eGFP) in S.frugiperda cells, the bacmid expression system was used. Transfection of the insect cells with an eGFP-specific siRNA prior to infection with the recombinant bacmid, inhibited eGFP protein expression by 75%, as quantified by fluorometry. Although the results suggest that RNAi could potentially be used as tool to study the function of an expressed transgene in insect cells, the lack of complete inhibition, coupled with the highly cytolytic nature of the bacmid, may complicate interpretation of the gene interference results. To investigate whether siRNAs targeting the AHSV-9 VP7 mRNA is able to silence VP7 protein expression, two siRNAs were designed that targeted different regions on the VP7 mRNA. siVP7-336 and siVP7-441, directed at nucleotides 336-356 and 441-461 on the VP7 coding strand, respectively, were chemically synthesized. The effect of these siRNAs on VP7 protein expression was evaluated by cotransfection of BHK-21 cells with the respective siRNAs and the VP7 expression plasmid pCMVVP7- eGFP. The results indicated that siVP7-336 and siVP7-441 inhibited VP7-eGFP expression by 88% and 75%, respectively. BHK-21 cells were subsequently transfected with the respective siRNAs in separate experiments followed by viral infection. The VP7 mRNA quantities were measured by quantitative reverse transcription PCR and the effect of the siRNAs on viral replication was evaluated by plaque assays. Of the two siRNAs, siVP7-336 was found to be the most effective inhibitor of VP7 transcription and suppressed VP7 mRNA by 93%. The exposure of BHK-21 cells to the VP7 genespecific siRNA, siVP7-336, also led to a 84% reduction in progeny virions, as measured by a plaque assay. Taken together, the results demonstrate that siRNA-mediated gene silencing is an efficient approach for reducing the level of VP7 transcripts and proteins and for inhibiting virus propagation.
Dissertation (MSc (Microbiology))--University of Pretoria, 2008.