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. / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

African horsesickness (AHS), caused by African horsesickness virus (AHSV), is an infectious, non-contagious, insect-borne viral disease that affects members of the Equidae family. AHSV is a non-enveloped virus, consisting of 10 segments of double stranded RNA (dsRNA) encoding seven structural and four non-structural proteins. Infection of mammalian cell cultures with AHSV leads to severe cellular pathogenesis effects (CPE), whereas insect cells show no noticeable CPE. Differences are also apparent between different serotypes of AHSV with regards to viral production, viral release, membrane permeabilisation and CPE. In this study we investigated different aspects of the AHSV life cycle in cell culture. The first aim of this study was the development of a real-time RT-PCR assay to quantify and monitor dsRNA from AHSV-infected cells. The dsRNA was used to quantify viral production, as dsRNA (one copy of each segment) is found only within viral particles and is not free within the cytoplasm of infected cells, thus giving a true representation of the amount of virus. This was achieved by cloning genome segment 5, optimising the extraction and purification of dsRNA, optimising the cDNA synthesis reaction, as well as the establishment and standardisation of the real-time PCR reaction. The second part of the study investigated and compared viral production and viral release between three different serotypes of AHSV in either mammalian or insect cell lines. The amount of dsRNA, which represented cell associated virus from AHSV-3- and AHSV-4-infected BHK cells over a 48 hr time period, was monitored by real-time RT-PCR and revealed a second wave of dsRNA production. These findings possibly suggest that a second round of infection of released viruses is re-entering previously uninfected or infected cells to replicate further. AHSV production was monitored in KC cells and indicated no production of progeny virions. However, an improvement was obtained when AHSV was first passaged on KC cells before being used for infections. The results from this study are in agreement with the fact that for a particular virus to replicate efficiently in a specific cell line, it should first be adapted to those cells. The dsRNA was quantified from samples representing equivalent amounts of infectious virus (i.e. same titre values) of AHSV serotypes 2, 3 or 4. The amount of dsRNA was approximately four-fold higher from serotype 2 than from serotypes 3 and 4. When the percentage of viral entry into cells was analysed, the majority (approximately 90%) of virus from serotypes 3 and 4 entered the cells, whereas serotype 2 showed viral entry of only about 50%. These findings suggested that a large amount of virus from serotype 2 was non-infectious, while the majority of virus from serotypes 3 and 4 was infectious. However, serotype 2 was a great deal more cytotoxic to cells (e.g. earlier onset and severity of CPE) when compared to cells infected with either serotypes 3 or 4. / Dissertation (MSc)--University of Pretoria, 2010. / Genetics / unrestricted