Genetic diversity of subtype C HIV-1 env variants in peripheral blood mononuclear cell (PBMC) DNA from infected mother-child pairs : a comparison of Heteroduplex Mobility Assay (HMA) and Base Excision Sequence Scanning (BESS) methods

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dc.contributor.authorLoubser, A Sen_ZA
dc.date.accessioned2015-07-14T08:34:13Z
dc.date.available2015-07-14T08:34:13Z
dc.date.issued2004en_ZA
dc.descriptionIncludes bibliographical references.en_ZA
dc.description.abstractImmune system pressure on HIV-1 replication drives the antigenic changes seen over time. The monitoring of changes in viral sequences can provide important information on the nature of the immune response and the correlates of protection. Viral diversification may also occur due to other selective pressures such as cell availability and differences in viral fitness. Information on the genetic characteristics of HIV-1 variants present in the mother and her infected infant are useful data for establishing whether any common features exist between source infection and transmitted genotypes. This helps in the understanding of the mechanism of transmission and the selective pressures occurring during and following transmission. The overall aim of this study was to explore alternative methods other than DNA sequencing for the monitoring of genetic diversity in the third variable region (V3) of the HIV-1 env gene of integrated HIV-I variants in peripheral blood mononuclear cells (PBMC's) derived from infected mother-child pairs. Two methods for displaying DNA differences were compared: I-leteroduplex Mobility Assay (I-IMA) and Base Excision Sequence Scanning (BESS). These methods were validated using sequence data. Extracted PBMC DNA from infected mother-child pairs were used to amplify the V3 region by nested PCR. DNA fragments were cloned into plasmid vectors and analyzed by HMA and BESS to establish subtype and intrasample genetic diversity. In addition, a PCR-ELISA quantitation system was developed to measure copy numbers of integrated HIV-1 genomes in order to confirm whether a sufficient number of template molecules were present to be representative of the total viral quasispecies. In conclusion, this study compared two methods (HMA and BESS) as cost-effective alternatives to DNA sequencing for HIV-1 diversity studies. in addition, a novel application of the BESS assay was demonstrated. Diversity studies are reliant on estimation of adequate input of amplifiable copies. The PCR-ELISA quantitation system developed provided an efficient and specific method for determining DNA copy number.en_ZA
dc.identifier.apacitationLoubser, A. S. (2004). <i>Genetic diversity of subtype C HIV-1 env variants in peripheral blood mononuclear cell (PBMC) DNA from infected mother-child pairs : a comparison of Heteroduplex Mobility Assay (HMA) and Base Excision Sequence Scanning (BESS) methods</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Virology. Retrieved from http://hdl.handle.net/11427/13400en_ZA
dc.identifier.chicagocitationLoubser, A S. <i>"Genetic diversity of subtype C HIV-1 env variants in peripheral blood mononuclear cell (PBMC) DNA from infected mother-child pairs : a comparison of Heteroduplex Mobility Assay (HMA) and Base Excision Sequence Scanning (BESS) methods."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Virology, 2004. http://hdl.handle.net/11427/13400en_ZA
dc.identifier.citationLoubser, A. 2004. Genetic diversity of subtype C HIV-1 env variants in peripheral blood mononuclear cell (PBMC) DNA from infected mother-child pairs : a comparison of Heteroduplex Mobility Assay (HMA) and Base Excision Sequence Scanning (BESS) methods. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Loubser, A S AB - Immune system pressure on HIV-1 replication drives the antigenic changes seen over time. The monitoring of changes in viral sequences can provide important information on the nature of the immune response and the correlates of protection. Viral diversification may also occur due to other selective pressures such as cell availability and differences in viral fitness. Information on the genetic characteristics of HIV-1 variants present in the mother and her infected infant are useful data for establishing whether any common features exist between source infection and transmitted genotypes. This helps in the understanding of the mechanism of transmission and the selective pressures occurring during and following transmission. The overall aim of this study was to explore alternative methods other than DNA sequencing for the monitoring of genetic diversity in the third variable region (V3) of the HIV-1 env gene of integrated HIV-I variants in peripheral blood mononuclear cells (PBMC's) derived from infected mother-child pairs. Two methods for displaying DNA differences were compared: I-leteroduplex Mobility Assay (I-IMA) and Base Excision Sequence Scanning (BESS). These methods were validated using sequence data. Extracted PBMC DNA from infected mother-child pairs were used to amplify the V3 region by nested PCR. DNA fragments were cloned into plasmid vectors and analyzed by HMA and BESS to establish subtype and intrasample genetic diversity. In addition, a PCR-ELISA quantitation system was developed to measure copy numbers of integrated HIV-1 genomes in order to confirm whether a sufficient number of template molecules were present to be representative of the total viral quasispecies. In conclusion, this study compared two methods (HMA and BESS) as cost-effective alternatives to DNA sequencing for HIV-1 diversity studies. in addition, a novel application of the BESS assay was demonstrated. Diversity studies are reliant on estimation of adequate input of amplifiable copies. The PCR-ELISA quantitation system developed provided an efficient and specific method for determining DNA copy number. DA - 2004 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2004 T1 - Genetic diversity of subtype C HIV-1 env variants in peripheral blood mononuclear cell (PBMC) DNA from infected mother-child pairs : a comparison of Heteroduplex Mobility Assay (HMA) and Base Excision Sequence Scanning (BESS) methods TI - Genetic diversity of subtype C HIV-1 env variants in peripheral blood mononuclear cell (PBMC) DNA from infected mother-child pairs : a comparison of Heteroduplex Mobility Assay (HMA) and Base Excision Sequence Scanning (BESS) methods UR - http://hdl.handle.net/11427/13400 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/13400
dc.identifier.vancouvercitationLoubser AS. Genetic diversity of subtype C HIV-1 env variants in peripheral blood mononuclear cell (PBMC) DNA from infected mother-child pairs : a comparison of Heteroduplex Mobility Assay (HMA) and Base Excision Sequence Scanning (BESS) methods. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Virology, 2004 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/13400en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Virologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherVirologyen_ZA
dc.titleGenetic diversity of subtype C HIV-1 env variants in peripheral blood mononuclear cell (PBMC) DNA from infected mother-child pairs : a comparison of Heteroduplex Mobility Assay (HMA) and Base Excision Sequence Scanning (BESS) methodsen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMScen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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