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Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay
Dissertation (MSc (Microbiology))--University of Pretoria, 2008.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2013
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| _version_ | 1867613501420208128 |
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| access_status_str | Open Access |
| author2 | Paweska, Janusz Tadeusz |
| author_browse | Paweska, Janusz Tadeusz |
| author_facet | Paweska, Janusz Tadeusz |
| collection | Thesis |
| dc_rights_str_mv | © University of Pretoria 2008E994/ |
| description | Dissertation (MSc (Microbiology))--University of Pretoria, 2008. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/25469 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:37:09.154Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/25469 Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay Paweska, Janusz Tadeusz Nel, Louis Hendrik Markotter, Wanda Venter, Marieka elizabethb@nicd.ac.za Botha, Elizabeth Magdelena South Africa (SA) West Nile virus (WNV) Infections Molecular UCTD Dissertation (MSc (Microbiology))--University of Pretoria, 2008. West Nile virus (WNV) belongs to the Flaviviridae family, a virus family of which many members are known as human pathogens. WNV has a worldwide distribution and strains that cluster in lineage II is endemic to sub-Saharan Africa. The complete nucleotide sequence of four lineage II West Nile virus strains, isolated in South Africa from patients with mild or severe WNV infections, were determined. Using a murine model, these strains had been shown to produce either highly or less neuroinvasive infections and induced similar genes to corresponding highly or less neuroinvasive lineage I strains. Nucleotide and amino acid sequence comparison between highly and less pathogenic lineage II strains demonstrated that the non-structural genes and in particular the gene coding for the NS5 proteins were the most variable. All the lineage II strains sequenced in this study were found to possess the E-protein glycosylation site previously postulated to be associated with virulence. Comparison of the signalase cleavage sites suggested that lineage II strains may be cleaved slightly more efficiently than lineage I strains in the C-prM junction, but less efficiently between prM and E genes. Relative to the highly neuroinvasive strains sequenced in this study major deletions were found in the 3’ noncoding region of 2 lineage II strains shown in previous studies to be either less- or not at all neuroinvasive. This is the first report of full genome sequences of highly neuroinvasive lineage II WNV strains. Currently available commercial WNV ELISA kits were developed with lineage I WNV strains and are expensive to use. For these reasons the development of a potential ELISA diagnostic assay based on the South African lineage II strain, H442, was envisaged. Such assay, if reliable and efficacious would be a useful tool towards WNV surveillance. The prM and E genes were selected to be expressed as recombinant antigens because of their co-expression nature and because the envelope protein is the principal target for neutralization. After cloning of the respective genes and verification of integrity, a mammalian expression system was utilized. Different mammalian cells and transfection media were tested and BHK 21 cells with SuperFect transfection medium were found to be best. Attempted expression of proteins was tested with immunofluorescent antibody testing as well as SDS-PAGE and Western blot analysis. Expression of recombinant WNV antigens were also tested in indirect and sandwich ELISA’s systems. It was however not possible to perform these two ELISA systems at a satisfactory level or clearly indicated if expression of proteins was successful. Microbiology and Plant Pathology MSc unrestricted 2013-09-06T21:44:26Z 2008-08-19 2013-09-06T21:44:26Z 2008-04-18 2008-08-19 2008-06-12 Dissertation Botha, EM 2008, Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay, MSc Dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/25469> 2008E994/gm * http://hdl.handle.net/2263/25469 http://upetd.up.ac.za/thesis/available/etd-06122008-130924/ en © University of Pretoria 2008E994/ application/pdf University of Pretoria |
| spellingShingle | South Africa (SA) West Nile virus (WNV) Infections Molecular UCTD Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay |
| title | Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay |
| title_full | Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay |
| title_fullStr | Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay |
| title_full_unstemmed | Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay |
| title_short | Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay |
| title_sort | molecular characterization of south african lineage ii west nile virus isolates and development of a diagnostic assay |
| topic | South Africa (SA) West Nile virus (WNV) Infections Molecular UCTD |
| url | http://hdl.handle.net/2263/25469 http://upetd.up.ac.za/thesis/available/etd-06122008-130924/ |