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Functional characterisation of African horse sickness virus non-structural protein NS3 by reverse genetics.
Dissertation (MSc (Genetics))--University of Pretoria, 2016.
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| Format: | Thesis |
| Language: | English |
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2026
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| _version_ | 1867613686891282432 |
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| access_status_str | Open Access |
| author2 | van Staden, V. |
| author_browse | van Staden, V. |
| author_facet | van Staden, V. |
| collection | Thesis |
| description | Dissertation (MSc (Genetics))--University of Pretoria, 2016. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/110089 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:40:05.686Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2026 |
| publishDateRange | 2026 |
| publishDateSort | 2026 |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/110089 Functional characterisation of African horse sickness virus non-structural protein NS3 by reverse genetics. van Staden, V. linda.ferreira2707@gmail.com Theron, Jacques Ferreira, Linda NS3/NS3A Transmembrane AHSV Reverse genetics Viroporin Dissertation (MSc (Genetics))--University of Pretoria, 2016. African horse sickness virus (AHSV) is a dsRNA virus of the Orbivirus genus and is the causative agent of the equid disease African horse sickness. Upon infection, AHSV produces seven structural proteins that form the virus particle, and five non-structural proteins with various supportive roles during virus replication. Previous studies on the non-structural protein NS3 of AHSV and the related bluetongue virus (BTV) indicated that NS3 acts as a pleiotropic protein with potential roles in cellular pathogenesis and in mediating virus trafficking and release from infected cells. Conserved features in both BTV and AHSV NS3 proteins include two in-frame start codons, two hydrophobic domains and cytoplasmic N- and C-terminal regions. Through the use of reverse genetics, most of these functional domains have been linked to different aspects of the BTV infection cycle, such as entry and release. However, the non-essential nature of NS3 to virus replication was also determined, with viable NS3/NS3A knockout mutants successfully generated. Limited work has been done for AHSV NS3/NS3A. Consequently questions regarding its role in the AHSV infection cycle, and possible differences in its functionality to that of the cognate BTV NS3/NS3A, remain unanswered. This project therefore aimed to improve our understanding of the role of NS3/NS3A in the final stages of AHSV morphogenesis. Through the use of a fully plasmid-based reverse genetics system, five different recombinant AHSV mutants, each containing a modified Seg-10 gene segment and therefore expressing an altered version of the NS3 protein, with one or two key functional domains disrupted, were generated. Preliminary phenotypic characterisations (virus titration assays, light and transmission electron microscopy) of the recombinants, and their comparison to the wild-type AHSV, were done. This showed clear differences between the respective virus strains with regards to their ability to induce cell death, their intracellular virus distribution, their membrane association and their potential virus release mechanisms. 6 The rescue of a recombinant virus with its second start codon disrupted, and its subsequent propagation in both mammalian and insect cells, confirmed that NS3A is not essential to AHSV replication. This mutation did not appear to affect the virus’s ability to induce cell death, with plaque formation and cytopathic effect (CPE) progression identical to that of the wild-type virus. Yet the absence of NS3A appeared to limit the ability of the virus to replicate in insect cells, with reduced virus titres observed. The reason for this still needs further investigation, with release of this recombinant from both cell types appearing unaffected by the introduced mutation. Recombinants with one or both of their NS3 transmembrane domains disrupted showed retarded CPE, and the inability to form plaques, with the infected mammalian cells remaining viable for longer. These mutant viruses showed no clear association with cellular membrane structures, and appeared to aggregate together in the cytoplasm to a much greater extent than observed for the wild-type virus. These results illustrate the necessity of NS3 (specifically the hydrophobic domains) in mediating the interaction of the virus with intracellular membranes, viral trafficking, virus release and cellular integrity and viability. This work paves the way for future studies focussing on elucidating the role of AHSV NS3 in mediating CPE and different morphogenesis events through its interaction with cellular membranous structures and host trafficking proteins. Genetics MSc (Genetics) 2026-05-15T17:26:16Z 2026-05-15T17:26:16Z 16/05/30 2016 Dissertation http://hdl.handle.net/2263/110089 en application/pdf |
| spellingShingle | NS3/NS3A Transmembrane AHSV Reverse genetics Viroporin Functional characterisation of African horse sickness virus non-structural protein NS3 by reverse genetics. |
| title | Functional characterisation of African horse sickness virus non-structural protein NS3 by reverse genetics. |
| title_full | Functional characterisation of African horse sickness virus non-structural protein NS3 by reverse genetics. |
| title_fullStr | Functional characterisation of African horse sickness virus non-structural protein NS3 by reverse genetics. |
| title_full_unstemmed | Functional characterisation of African horse sickness virus non-structural protein NS3 by reverse genetics. |
| title_short | Functional characterisation of African horse sickness virus non-structural protein NS3 by reverse genetics. |
| title_sort | functional characterisation of african horse sickness virus non structural protein ns3 by reverse genetics |
| topic | NS3/NS3A Transmembrane AHSV Reverse genetics Viroporin |
| url | http://hdl.handle.net/2263/110089 |