Medical Virology

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Browsing Medical Virology by browse.metadata.advisor "Cloete, Ruben"

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    Investigating the structural impact of hiv-1 integrase natural occurring polymorphisms and novel mutations identified among group m subtypes circulating in sub-Saharan Africa
    (Stellenbosch : Stellenbosch University, 2020-12) Mikasi, Sello Given; Jacobs, Graeme Brendon; Cloete, Ruben; Van Zyl, Gert Uves; Engelbrecht, Susan; Ikomey, George Mondinde; Kasang, Christa; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
    Introduction HIV/AIDS remains a major health concern worldwide, with sub-Saharan Africa (SSA) carrying the largest burden.HIV is characterised by extremely high genetic diversity, with all the major groups and subtypes circulating in SSA. Combination antiretroviral therapy (cART) have substantially reduced HIV related deaths, but this is counteracted by the development of HIVdrug resistance, caused by certain drug resistance-associated mutations (RAMS). Integrase (IN) strand transferase inhibitors (INSTIs), the newest class of antiretroviral drugs,has a high genetic barrier and can be used in individuals that previously exhibited resistance to other classes of drugs. The World Health Organisation (WHO) approved the use of Dolutegravir (DTG) as part of first-line cART. Methods This is a descriptive experimental design study, which aimed to identify IN natural occurring polymorphisms (NOP) among different HIV-1 group M subtypes and Drug resistance mutations within the HIV-1 pol gene fragment of INSTI naïve patients from South Africa (SA) and Cameroon (CR), using the Stanford University genotypic resistance interpretation algorithm. Structural computational methods that included; homology modelling, molecular docking, molecular dynamics simulations and interaction analysis was performed to understand the structural impact of mutations from diverse HIV-1 subtypes on DTG drug binding. ResultsWe observed low-level RAMs against INSTIs in SA (2.2%) and CR sequences (5.4%). Through Fisher’sexact test we noted that the two NOPs occurred: VI72I and R269K, with p-values ≤0. 05, were statistically enriched. The impact of having these mutations are yet to be fully understood. Through molecular modelling and stability predictions, we observed a destabilizing effect of the known G140S mutant on the HIV-1C IN protein structure and simulation analysis showed that it affected structural stability and flexibility of the protein structure. Interactions analysis of different drug binding conformations to different HIV-1 IN subtypes reported differences in the number of binding interactions to different HIV-1 IN subtypes, but we did not observe any significant differences in binding affinity for each INSTIs. This implies no significant alteration to the binding site in the wild type IN, which may not prevent INSTIs drug binding. In addition, all accessory mutations that resulted in a change in the number of interactions encompassing residues were found within the stable alpha-helix secondary structure element and not in close proximity to the drug active site.ConclusionThe study data indicate that RAMS against INSTIs remain low both in SA and in CR.Subtype C in SA and CRF02_AG in CR continues to be the driving force ofthe epidemic. We further reported on the impact of various NOPs on drug susceptibility. The analyses suggested that NOPs does not have an impact on IN protein structure and stability,and does not affect drug binding in the WT IN, but the known mutation G140S affect DTG binding. The study support recommendations made by the WHO to use DTG as part of salvage therapy in patients with RAM’s and accessory mutations. Data obtained from thisstudy can help to tailor effective treatment strategies in the African population, where diverse HIV subtypes circulate.
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    Multidisciplinary viral analyses in people living with HIV-1C and receiving second-line combination antiretroviral therapy (cART) in South Africa
    (Stellenbosch : Stellenbosch University, 2019-12) Obasa, Adetayo Emmanuel; Jacobs, Graeme Brendon; Neogi, Ujjwal; Kamalendra, Singh; Cloete, Ruben; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology
    ENGLISH ABSTRACT: The use of combination Antiretroviral Therapy (cART) has grown since its first introduction into the South African public sector. cART has significantly reduced the mortality rate caused by human immunodeficiency virus (HIV) in both high- and low-to-middle-income countries. The development of drug resistance has challenged the outcome of cART. This has led to the introduction of Integrase (IN) strand transfer inhibitors (InSTIs) as part of the first-line cART regimen. Due to their superior efficacy and high genetic barrier, this class of drugs was previously reserved as salvage therapy. The World Health Organization (WHO) supports InSTIs as first-line regimen non-nucleoside reverse transcriptase inhibitors (NNRTIs) particularly in regions where pre-treatment drug resistance to NNRTIs reaches 10%. Therefore, this study aimed to (i) to investigate the prevalence of InSTI mutations in treatment-naïve and treatment-experienced PLHIV using genotypic assays, which included Sanger sequencing, next-generation sequencing (NGS) and molecular modelling; (ii) analysed Long Terminal Repeats (LTR) to identify transcription factor binding sites. Chapter 2: Ninety-one (n = 91) treatment-naïve patients were obtained before the start of antiretroviral treatment in South Africa. Furthermore, we included 314 South African patient sequences obtained from the Los Alamos National Library database (www.lanl.gov). The IN gene ~ 900 base pairs [bps] was amplified and sequenced using conventional DNA Sanger sequencing. Homology structure was generated using the cryoEM structure of HIV-1B IN intasome (PDB file 5U1C) using ‘Prime’ of Schrodinger Suit. Chapter 3: Ninety-six (n = 96) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained for further analyses. We performed conventional DNA Sanger sequencing to analyse the complete pol gene (~3011bps) and sequences were analysed using the Stanford HIV drug resistance database to assess genotypic resistance associated mutations (RAMs). Chapter 4: Fifty-six (n = 56) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained. We performed a high-throughput (HT) sequence analyses on the complete pol gene using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV drug resistance database. Chapter 5 and 6: We performed in-silico analyses on diverse HIV-1 subtypes based on 8114 sequences. These included treatment naïve and downloaded sequences from the HIV Los Alamos National Library Database (www.lanl.gov). Homology derived molecular models of HIV-1 IN tetramers from different subtypes were generated using cryoEM structure of the HIV-1B IN intasome. Chapter 7: Fifty-six (n = 56) treatment-experienced patients receiving boosted protease inhibitors (bPIs) as part of their cART treatment regimen were obtained. We performed Sanger sequencing to analyse the LTR gene (~ 474 bps) followed by bioinformatics analyses to identify transcription factor binding sites.The data indicates that in South Africa, the prevalence of RAMs against InSTIs is low and InSTIs can be used as a potential viable salvage therapy option and/or first-line regimen. Molecular modelling was done for IN structural analyses, which revealed how naturally occurring polymorphisms might affect structural stabilities, viral DNA binding and drug-binding propensity. This study represents a true baseline InSTI resistance rate as the treatment-naïve patients were obtained before the cART introduction. We propose GRT for people living with HIV (PLHIV) before treatment initiation and we recommend continued InSTIs drug resistance monitoring when introduced on a larger scale in South African.