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The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent
Many devastating zoonotic viruses such as West Nile and Rift Valley fever viruses are endemic to South Africa, affecting livestock and ultimately, through their arthropod vectors, also infecting humans. One such zoonotic virus that is of interest is Shuni virus (SHUV). SHUV belongs to the viral genu...
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2020
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| _version_ | 1867613665398620160 |
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| access_status_str | Open Access |
| author | Verbeek, Matthew James Robert |
| author2 | Hitzeroth, Inga |
| author_browse | Hitzeroth, Inga Verbeek, Matthew James Robert |
| author_facet | Hitzeroth, Inga Verbeek, Matthew James Robert |
| author_sort | Verbeek, Matthew James Robert |
| collection | Thesis |
| description | Many devastating zoonotic viruses such as West Nile and Rift Valley fever viruses are endemic to South Africa, affecting livestock and ultimately, through their arthropod vectors, also infecting humans. One such zoonotic virus that is of interest is Shuni virus (SHUV). SHUV belongs to the viral genus Orthobunyavirus, family Peribunyaviridae., and order Bunyavirales. Discovered in arthropods and humans in Nigeria, it was soon identified as a possible cause for cases of neurological disease in horses within South Africa. Studies have shown South African veterinarians who had come into contact with such cases tested positive for antibodies against the virus. Therefore, SHUV is being further investigated as a potential cause of neurological disease within humans and there is a need to develop appropriate quick and effective diagnostic reagents to allow for surveillance of the virus. The main focus for this study was the development of diagnostic reagents centred around the nucleocapsid (N) protein of the SHUV. The N proteins of closely related members of the order Bunyavirales have shown to be highly abundant in infection and cause an immune response in the infected hosts thus making it the ideal target. Using available SHUV genome sequences and data, the N protein gene was designed and synthesised to be expressed in both Escherichia coli and plant expression systems. The expression of the N protein in E. coli, followed by subsequent washing with BugBuster, led to a final mass of 5.1 mg of the SHUV N protein from a 1000 ml culture. This led to a SHUV N yield of 5.1 µg/ml of culture and was measured to make up 69.5% of the total soluble protein. The immunisation of rabbits with this recombinantly expressed SHUV N allowed for the development of polyclonal antibodies which were successfully used in immunoblot studies to detect plant produced SHUV N protein. Plants are an effective and possibly cheaper alternative production system to bacterial, mammalian, or insect cell cultures and thus the N protein was transiently expressed in N. benthamiana plants using Agrobacterium tumefaciens-mediated infiltration. The recombinant protein produced underwent purification using nickel affinity chromatography. This led to yields of 2.248 mg of SHUV N protein from 35 plants which gave a yield of 9.9 mg/kg of raw plant material. This purified plant produced N protein acted as an antigen for diagnostic assays such as ELISA, which was used to screen known SHUV infected sera. This led to mixed results due to the limited sera samples available. However, as a proof of concept, it has shown great potential and thus opens the door to a possible inexpensive dual-use assay for use in the diagnoses of both animal and human SHUV infection. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/31017 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:39:45.660Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Department of Molecular and Cell Biology |
| publisherStr | Department of Molecular and Cell Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/31017 The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent Verbeek, Matthew James Robert Hitzeroth, Inga Mbewana, Sandiswa Rybicki, Edward Molecular and Cell Biology Many devastating zoonotic viruses such as West Nile and Rift Valley fever viruses are endemic to South Africa, affecting livestock and ultimately, through their arthropod vectors, also infecting humans. One such zoonotic virus that is of interest is Shuni virus (SHUV). SHUV belongs to the viral genus Orthobunyavirus, family Peribunyaviridae., and order Bunyavirales. Discovered in arthropods and humans in Nigeria, it was soon identified as a possible cause for cases of neurological disease in horses within South Africa. Studies have shown South African veterinarians who had come into contact with such cases tested positive for antibodies against the virus. Therefore, SHUV is being further investigated as a potential cause of neurological disease within humans and there is a need to develop appropriate quick and effective diagnostic reagents to allow for surveillance of the virus. The main focus for this study was the development of diagnostic reagents centred around the nucleocapsid (N) protein of the SHUV. The N proteins of closely related members of the order Bunyavirales have shown to be highly abundant in infection and cause an immune response in the infected hosts thus making it the ideal target. Using available SHUV genome sequences and data, the N protein gene was designed and synthesised to be expressed in both Escherichia coli and plant expression systems. The expression of the N protein in E. coli, followed by subsequent washing with BugBuster, led to a final mass of 5.1 mg of the SHUV N protein from a 1000 ml culture. This led to a SHUV N yield of 5.1 µg/ml of culture and was measured to make up 69.5% of the total soluble protein. The immunisation of rabbits with this recombinantly expressed SHUV N allowed for the development of polyclonal antibodies which were successfully used in immunoblot studies to detect plant produced SHUV N protein. Plants are an effective and possibly cheaper alternative production system to bacterial, mammalian, or insect cell cultures and thus the N protein was transiently expressed in N. benthamiana plants using Agrobacterium tumefaciens-mediated infiltration. The recombinant protein produced underwent purification using nickel affinity chromatography. This led to yields of 2.248 mg of SHUV N protein from 35 plants which gave a yield of 9.9 mg/kg of raw plant material. This purified plant produced N protein acted as an antigen for diagnostic assays such as ELISA, which was used to screen known SHUV infected sera. This led to mixed results due to the limited sera samples available. However, as a proof of concept, it has shown great potential and thus opens the door to a possible inexpensive dual-use assay for use in the diagnoses of both animal and human SHUV infection. 2020-02-11T11:30:40Z 2020-02-11T11:30:40Z 2019 2020-01-28T13:01:41Z Master Thesis Masters MSc http://hdl.handle.net/11427/31017 eng application/pdf Department of Molecular and Cell Biology Faculty of Science |
| spellingShingle | Molecular and Cell Biology Verbeek, Matthew James Robert The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent |
| thesis_degree_str | Master's |
| title | The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent |
| title_full | The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent |
| title_fullStr | The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent |
| title_full_unstemmed | The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent |
| title_short | The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent |
| title_sort | expression of shuni virus nucleocapsid protein in nicotiana benthamiana for use as a diagnostic reagent |
| topic | Molecular and Cell Biology |
| url | http://hdl.handle.net/11427/31017 |
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