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

Novel vaccine strategies for the presentation of immunologically important epitopes to the immune system are continuously being developed. Two such systems include the particulate protein and live viral vector delivery systems. In his study the long-term objective is to explore the African horsesickness virus (AHSV) serotype 9 viral protein 7 (VP7) and the Lumpy skin disease (LSDV) viral vector as two different vaccine strategies, particularly in view of the development of an HIV-1 vaccine. Consequently two very specific objectives were outlined in this study. The first was to express the HIV¬1 subtype C strain Du 151 gp41 epitopes ALDSWK and RVLAIERYLKD on the surface of the AHSV-9 VP7 particulate protein crystalline structures. A longer-term aim is to synthesise large quantities of these chimeric VP7 crystals in order to assess the immune response against the inserted epitopes. Secondly, the efficiency of the LSDV bi-directional promoter pA7LA8R in expressing chimeric VP7 proteins was to be evaluated by utilising the late element of this promoter to determine expression levels. Nucleotide sequences encoding the ALDSWK and RVLAIERYLKD epitopes were amplified from the HIV-1 subtype C strain Du 151 gp160 gene utilising PCR. These sequences were cloned individually as well as in combination into a multiple cloning site (549-566bp) present in the AHSV-9 VP7 gene. Recombinant pFASTBAC vectors PFASTBAC-VP7-MT 177-RVLAIERYLKD, PFASTBAC-VP7-MT 177-ALDSWK AND PFASTBAC-VP7-MT-177-RVLAIERYLKD-ALDSWK were identified, sequenced and used in the generation of recombinant baculoviruses utilising the BAC-to-BAC™ Baculovirus expression system. Expression of all three chimeric proteins, VP7-ALDSWK, VP7-RVLAIERYLKD and VP7- RVLAIERYLKD-ALDSWK was detected in infected Sf9 insect cells utilising SDS-PAGE. Further investigations will involve high-level expression of these proteins, which in turn will allow their characterisation as well as solubility, scanning electron and immunogenicity studies. In order to evaluate the efficiency of the LSDV bi-directional promoter, the AHSV-9 VP7 gene was cloned under the control of the late element (pA7L) of this promoter. The recombinant pHSsgpt-VP7 transfer vector was subsequently transfected into lamb testis cells infected with wild type LSDV in order to generate recombinant LSDV-VP7. Several rounds of recombinant virus selection in the presence of mycophenolic acid resulted in the loss of the LSDV-VP7 recombinant. Due to this unforeseen result, the expression of the VP7 protein from the late element of the pA7LA8R bi¬directional promoter could not be quantified and the efficiency of this promoter was not determined. The loss of LSDV recombinants, which contain a gene under the control of the late promoter element pA7L, has occurred previously and is suspected to be because of the instability of these recombinants. Due to the difficulties inherent in working with the LSDV viral vector delivery system, it has subsequently been decided to explore an alternate poxviral vector system. The focus in this study is now being shifted onto the promising Modified Vaccinia Ankara (MVA) viral vector system. / Dissertation (MSc (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

African horse sickness (AHS) is a non-contagious viral disease transmitted by an arthropod vector and is endemic to sub-Saharan Africa. The disease affects all equine species, but is more severe in horses and other equid species not native to Africa. Vaccination is the only demonstrated means of its prevention. The horse-owning public provides much anecdotal evidence of prophylactic strategies, such as repellents, stabling, alternate hosts, traps, paraffin, blankets, smoke or fans. The present study investigated the relationship of these strategies to the incidence of AHS, and evaluated alternate hosts, wind speed and repellents on the activity of males and females of the different Culicoides species.. Cypermethrin and citronella-containing repellents repelled the most female midges. Sheep and cattle offer an alternate blood meal to gravid and nulliparous female midges. Fans are very effective in keeping midges away from horses. Methods are summarised for the horse owner to implement in addition to vaccination to prevent AHS. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

African horse sickness virus causes a non-contagious, infectious disease of equids. It is epizootic to sub-Saharan Africa and parts of the Middle East. The epizootics caused by the virus have caused widespread devastation amongst equids worldwide. Fortunately no epizootic has lasted more than 5 years outside of sub- Saharan Africa. It is vectored by species of Culicoides midges (Diptera: Ceratopogonidae) and most importantly by the two Avarita species of C. imicola Keiffer and C. bolitinos Meiswinkel. The literature pertaining to the study and research of the virus, the disease and the vectors is reviewed. Models allowing prediction of future possible outbreaks as well as details of control strategies and findings of researchers are presented and discussed. The virus needs a long term reservoir host in which to overwinter and various theories are discussed. Control measures in South Africa are suggested so that outbreaks of the disease can be reduced. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermarizburg, 2008.

African horse sickness (AHS) is a viral disease with high mortality rates, vectored by the Culicoides midge and affecting members of the Equidae family. AHS is endemic to South Africa, and, as a result, affects export and international competitiveness in equine trade, and impacts significantly on the South African racehorse and performance horse industries. AHS also has devastating consequences for rural and subsistence equine ownership. The protocol developed in this dissertation has the potential to serotype and confirm the AHS virus within a few hours at significantly less cost than current methods. It will ease the financial and time constraints of studying an outbreak in real time and has the potential to solve many of the unknown factors surrounding AHS, particularly and most importantly, the role that each serotype plays in outbreaks and the form of the disease contracted by horses. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.