Investigation of the molecular epidemiology of HIV-1 in Khayelitsha, Cape Town, using serotyping and genotyping techniques
Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2005.
There are currently an estimated 5.3 million people infected with human immunodeficiency virus / acquired immunodeficiency syndrome (HIV/AIDS) in South Africa. HIV-1 group M Subtype C is currently responsible for the majority of HIV infections in sub-Saharan Africa (56% worldwide). The Khayelitsha informal settlement, located 30 km outside Cape Town, has one of the highest HIV prevalence rates in the Western Cape. The objective of this study was to investigate the molecular epidemiology of HIV-1 in Khayelitsha using serotyping and genotyping techniques. Patient samples were received from the Matthew Goniwe general health clinic located at site C in Khayelitsha. Serotyping was performed through a competitive enzymelinked immunosorbent assay (cPEIA). RNA was isolated from patient plasma and a two step RT-PCR amplification of the gag p24, env gp41 IDR, env gp120 V3 and pol genome regions performed. Sequences obtained were used for detailed sequence and phylogenetic analysis. Neighbour-joining and maximum likelihood phylogenetic trees were drawn to assess the relationship between the Khayelitsha sequences obtained and a set of reference sequences obtained from the Los Alamos National Library (LANL) HIV database (http://www.hiv.lanl.gov/). Through serotyping and genotyping the majority of HIV strains were characterised as HIV-1 group M subtype C. One sample (1154) was characterised as a possible C / D recombinant strain. In 9 other samples HIV-1 recombination cannot be excluded, as only one of the gene regions investigated could be amplified and characterised in these samples. The gag p24 genome region was found to be more conserved than the env gp41 IDR, with the env gp41 IDR more conserved than the env gp120 V3. The variability of the env gp120 V3 region indicates that patients might be dually infected with variant HIV-1 subtype C strains or quasispecies. Conserved regions identified in the Khayelitsha sequences can induce CD4+ T-cell responses and are important antibody recognition target sites. These conserved regions can play a key role in the development of an effective HIV-1 immunogen reactive against all HIV-1 subtypes. The majority of subtype C viruses were predicted to use CCR5 as their major chemokine co-receptor. The pol sequences analysed indicate that mutations associated with minor resistance to Protease Inhibitors (PIs) might be present in the Khayelitsha community. The identification of resistant mutations is vital for people receiving antiretroviral treatment (ART). It can influence the success of their treatment and delay the onset of AIDS. Serotyping is a quick characterisation method, but not always accurate. With genotyping detailed molecular analysis can be performed. However, with genotyping the success of amplification often depends on viral load. In Southern Africa a subtype C candidate vaccine appears to be the best option for future vaccine considerations. The sporadic detection of non-subtype C and recombinant subtype C viruses remains a concern and will thus have to be closely monitored. Phylogenetic analysis can help to classify and monitor the spread and evolution of these viruses.