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Browsing Medical Virology by browse.metadata.advisor "Engelbrecht, Susan"
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- ItemAnalysis of HIV-1 diversity and inflammatory markers in HIV-associated neurocognitive disorders (HAND).(Stellenbosch : Stellenbosch University, 2022-04) Ruhanya, Vurayai; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.ENGLISH SUMMARY: HIV-associated neurocognitive disorders (HAND), which involve impairment or disruption of neurocognitive functioning have become one of the most frequent complications in adult HIV-1 infections with global estimates ranging from 42% to 45%. The screening and diagnosis for HAND relies on multiple clinical and neuro-psychometric methods. However, these methods have a low reliability because they are not precise as most of possess inadequate psychometric properties and diagnostic accuracy. Therefore, this study aimed to describe and characterise viral and immunological determinants of HAND and evaluated their relationship with specific clinical, neuromedical and neuropsychological data to identify putative easy-to-measure biological markers for diagnosis of the condition. This study demonstrated that higher peripheral blood lymphocyte-derived HIV-1 proviral DNA is a predictor of global and domain-specific neurocognitive impairment among individuals infected with HIV-1 subtype C. The study also determined proviral load cut-off /threshold value for neurocognitive impairment and associated diagnostic accuracy. It also identified IP-10 and RANTES as a plasma chemokine bio-signature for HIV-associated neurocognitive impairment with diagnostic accuracy comparable to standard psychometric tests used to screen and describe severity of HAND. In addition, the study identified 3 viral genetic signatures for cognitive impairment, namely Lysine at codon 24, (24K) and Arginine at codon 29 (29R) on Tat protein and Tyrosine (Y) at position 45 (45Y) of Vpr. These three signature amino acids were related to classical markers for monitoring HIV infection. Finally, we identified 4 conserved fragment sequences, PEDQGPQREPYNEWTLE (5 to 21), LGQYIY (42 to 47), TYGDTW (49 to 54), PEDQGPQREPYNEW (5 to 18) on viral protein R, that were associated with higher plasma viral load and proviral load. The study has identified novel cytokine/chemokine and viral biomarker signatures for HIV associated neurocognitive impairment with low to moderate diagnostic accuracy. The findings demonstrated a need for interdisciplinary approach to elucidate possible molecular interactions between the peripheral blood immune markers, viral signatures and the CNS that are linked to observed clinical outcomes of neurodegradation in HIV infection. The identified biomarkers can be further investigated for use as screening tools and treatment endpoints for HAND.
- ItemThe apoptotic potential of different HIV-1 subtype C Tat mutations in cell culture(Stellenbosch : Stellenbosch University, 2013-03) Isaacs, Shahieda; Engelbrecht, Susan; Glashoff, Richard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.The efficiency in which HIV-1 can infect, spread and evade the attack of therapeutic agents can be attributed to a high mutation rate and frequent recombination events. These factors have collectively contributed to the diversity observed in HIV-1 and resulted in a multitude of subtypes, sub-subtypes, circulating recombinant forms (CRF’s) and unique recombinant forms (URF’s). The aim of this study was to investigate HIV-1 diversity in Cape Town using a small cohort of treatment naive patients being investigated for HIV Associated Neurocognitive Disorders (HAND). Four different genomic domains: gag, pol, accessory and gp41 genes were sequenced to subtype the virus. HIV-1 tat was further investigated because the dicysteine motif has been reported to play a role in HAND. Viral RNA and proviral DNA was extracted from 64 patients and used for the amplification and sequencing of the genes. Rega and jpHMM online tools were used to identify HIV-1 subtypes and recombinants while Neighbor-joining phylogenetic trees were constructed for phylogenetic analysis. The pol gene was further investigated using SCUEAL to detect possible intra-subtype recombination and was also screened for the presence of transmitted drug resistance. In addition tat sequence datasets retrieved from the Los Alamos sequence database were investigated and compared with the newly generated sequences for the detection of point mutations and amino acid signature patterns. Sequencing identified most of the samples as subtype C; however six inter-subtype recombinants (AE, A1G, A1CU and two BC) and 9 intra-subtype C recombinants were identified. In addition 13% of pol sequences were identified with resistance mutations. Signature pattern analysis identified a high level of variability in the tat sequences: 68% were identified with C30S31; 29% with the C30C31 mutation and a single sequence with a novel mutation C30A31. Functional analysis of these mutations indicated that all mutations investigated were capable of inducing apoptosis in cell culture. The C30C31 mutation generated the highest level of apoptosis, closely followed by the C30A31 mutation. However no statistical significance could be detected between tat mutations and the observed levels of apoptosis.
- ItemCharacterization of HIV-1 subtype B near full-length genome sequences identified at the start of HIV epidemic in South Africa(Stellenbosch : Stellenbosch University, 2017-03) Obasa, Adetayo Emmanuel; Jacobs, Graeme Brendon; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH SUMMARY: South Africa is home to approximately 20.0% of the global Human Immunodeficiency Virus (HIV) infected population. The first reported cases of HIV-1 in the country were described in 1982 amongst the homosexual male population. This was attributed to HIV-1 subtypes B (HIV-1B) and D (HIV-1D). Since the late 1980s HIV-1 subtype C (HIV-1C), spread mainly through heterosexual contact, has been the driving force of the epidemic. To date, only six HIV-1B near full-length genome (NFLG) sequences from South Africa are available in the Los Alamos National Laboratory database (LANL). During this study we retrieved five HIV-1B positive samples from homosexual and bi-sexual males, stored for up to 30 years, from the early 1980s, for further characterization. The NFLG amplification reactions were performed using a modern Polymerase Chain Reaction (PCR) protocol designed to target two overlapping proviral DNA HIV genome fragments, 5.5 kb and 3.7 kb in size, respectively. All positive PCR products were sequenced to characterize the viruses. The sequences were checked and edited manually using Sequencher V5. Multiple sequence alignments were created using Clustal W and Maft V7. The sequences were subtyped using the REGA V3.0, RIP V3.0 and jumping profile Hidden Markov Model (jpHMM) online subtyping programmes. Maximum likelihood phylogenetic trees were drawn using MEGA V6. Four of the five HIV-1 patient sequences were subtyped as pure HIV-1B. One sequence, ZA|85|R605, was characterized as a novel HIV-1 BD recombinant. This is the first NFLG HIV-1 BD recombinant ever described and indicates that recombination events were most likely already happening at the early stage of the South African epidemic. Two patient sequences, ZA|87|R1296 and ZA|87|R459, clusters with HIV-1B sequences from the United States of America (USA). The sequence from patient ZA|87|R68 clusters with a HIV-1B sequence from France and the sequence of ZA|87|R526 clusters with another South African HIV-1B sequence. Homosexual flight stewards, international tourists and migrants from the European and North American countries were most likely responsible for the introduction of the HIV-1B epidemic into South Africa. The findings of this study provides valuable insights from the beginning of the HIV-1 epidemic in South Africa. We highlight the importance of characterizing complete viral genomes from early archival specimens to give a more detailed picture of landmarks of the HIV/AIDS pandemic. We show that NFLG sequencing is an important tool for the identification of recombinant viral strains. This study can form the basis for continued research in our attempt to reconstruct the epidemiology and evolutionary history of HIV in South Africa. The HIV-1 epidemic is dynamic in nature and is constantly changing.
- ItemDevelopment of selective real-time PCR (SPCR) asays for the detection of K103N resistance mutation in minor HIV-1 populations(Stellenbosch : Stellenbosch University, 2011-12) Seleka, Mpho Maria; Engelbrecht, Susan; Van Zyl, Gert; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: Background: The conventional sequence analysis is the most common method used for the detection of drug-resistant mutants. Due to its sensitivity limitations, it is unable to detect these mutants when comprising less than 20% (minor populations) of the total virus population in a sample. However, real-time PCR-based assays offer a rapid, sensitive, specific and easy detection and quantification of such mutants. The HIV-1 variants harbouring the K103N mutation are associated with resistance to nevirapine (NVP) and efavirenz (EFV). The persisting drug-resistant mutants decay slowly to low levels, and therefore they are called minor drug-resistant mutants. Consequently, they affect subsequent treatment with the drugs of the relevant class. Objectives: The objective of this study was to design two TaqMan real-time PCR-based assays called selective-polymerase chain reaction (SPCR), namely the total viral copy SPCR assay and the K103N-SPCR assay. The former detects HIV-1 of subtype C reverse transcriptase sequences, whereas the latter detects K103N drug-resistant variants in these sequences. Design and Methods: In developing the SPCR assays, sets of appropriate primers and probes for the HIV-1 subtype C reverse transcriptase (RT) were developed to use in the K103N-specific reaction and the total copy reaction. Twelve DNA plasmid standards with sequence diversity were constructed for the assay from two HIV-1subtype C samples known to harbour the K103N mutation (AAC or AAT) in our Department‟s Resistance Databank. Their RT regions were amplified, cloned and verified with sequencing. Site-directed mutagenesis was used to induce mutations at 103 amino acid position in some of these clones to generate more standards with either one of the three codons (AAA, AAC and AAT). The two assays were optimized and validated, and a standard curve was generated for each assay using 10-fold serial dilution (5x107-5x100 DNA copy/μL) of a K103N-mutant plasmid standard. The optimized and validated SPCR assays were used to screen 40 nested PCR products of previously genotyped patient samples for minor K103N variants. Results: Two sensitive and reproducible selective real-time PCR (SPCR) assays, with cut-offs of 8.23 and 10.33 and a detection limit of 0.01% for the K103N resistance variants, were successfully developed. The assays detected a prevalence of 25.64-46.15% for the K103N resistance mutation in 39 patient samples. The genotyping (population sequencing) missed 40-53.85% of these variants. Conclusion: In conclusion, sensitive and reliable selective real-time PCR assays to detect and quantify minor K103N variants of HIV-1 in nested PCR products were successfully developed. The assay had a lower detection limit of 0.01%.
- ItemFrom bedside to bench and back : future options for antiretroviral drugs in non-B HIV-1 subtypes(Stellenbosch : Stellenbosch University, 2020-12) Njenda, Duncan Tazvinzwa; Engelbrecht, Susan; Jacobs, Graeme Brendon; Neogi, Ujjwal; Sonnerborg, Anders; Spetz, Anna-Lena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.Introduction: HIV-1 drug resistance remains a burden in low- and middle-income countries (LMIC). Regardless of the advances in antiretroviral (ARV) therapy, there is an increase in the trend of acquired and pre-treatment drug resistance mutations (DRM) in LMIC affected by HIV-1 non-B subtypes. The overall aim of this thesis is to understand the potential subtype-specific differences in viral fitness and drug susceptibility against the drugs that target different stages of viral replication that includes, protease-mediated cleavage and maturation (Paper I), reverse transcription (Paper II) and integration (Paper III). Methods:The thesis presents cross-sectional drug resistance data from predominantly HIV-1 non-B subtypes. It also includes virological drug sensitivity and enzyme-based in vitro assay results of recombinant viruses derived from predominantly HIV-1 non-B and few HIV-1B infected patients. The following areas were investigated: a) the role that HIV-1 subtype C (HIV-1C) gag-p6 gene could play in response to proteaseInhibitors PIs in the absence of known PI primary mutations (Paper I). b) Ex vivo potency of the newer drug 4’-Ethynyl-2’-Fluoro-2’ deoxyadenosine (EFdA),first and second generation non-nucleoside reverse transcriptase inhibitors (NNRTIs)and Tenofovir alafenamide(TAF) against diverse HIV-1 subtypes (Paper III). c)Ex vivo potency of Integrase strand transfer inhibitors (INSTIs) against diverse HIV-1subtypes as well as genotypic resistance data comparing INSTI naïve and INSTI-experienced patients. Results: In paper I, the study showed an increase in viral fitness for HIV-1C viruses carrying the PYxE insertion in gag-p6 when compared to the wild-type (WT) HIV-1C viruses. Furthermore, some PYxE-carrying viruses had low sensitivity to LPV and (TAF) when tested in drug sensitivity assays. Clinical data analysis showed a higher pre-therapy viral load and a decrease in CD4+ T-cell counts for patients harboring PYxE-carrying viruses when compared to WT. Paper II. demonstrated that EFdA has a high inhibitory potential independent of HIV-1 subtype and high antiviral activity against resistant viruses. However, HIV-1C viruses had a significantly reduced susceptibility to NNRTIs, specifically Rilpivirine and Etravirine. In paper III, the drug susceptibility of INSTIs results indicated that INSTIs such as Dolutegravir (DRV), Bictegravir (BIC) and Cabotegravir (CAB) inhibited non-B subtypes significantly as compared to HIV-1B subtypes. Conclusion: Inferences can be made from the results to suggests that subtype-specific differences play an essential role in influencing the ARV susceptibility which could further impact the treatment efficacy in sub-optimal adherence. To reduce the trend of increasing DRMs in non-B HIV-1 subtypes which are mainly dominating in LMICs, adherence support and viral load monitoring should be prioritized. A rapid adaptation of INSTIs and newer drugs that have long-acting potential is encouraged. However, pre-clinical studies and clinical trials that are mainly restricted to the HIV-1B enrolled patients, should be inclusive of non-HIV-1B infected patients before the massive roll-out of INSTIs and newer drugs continues in non-HIV-1B dominated settings.
- ItemHIV-1C dynamics and evolutionary trends in Botswana(Stellenbosch : Stellenbosch University, 2016-12-09) Moyo, Sikhulile; Engelbrecht, Susan; De Oliveira, Tulio; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical VirologyENGLISH ABSTRACT : Introduction: HIV incidence estimates are critical for monitoring HIV transmission dynamics, and for design and evaluation of the impact of interventions. Biomarkers and assays for cross-sectional surveillance of HIV incidence are greatly needed because of the high costs and time needed to maintain prospective cohorts to determine HIV incidence. New cross-sectional assays for estimation of HIV incidence are attractive due to their improved performance and cost-effectiveness. In this dissertation, methods for identification and characterization of recency of HIV infection are described. An in-depth review of HIV recency determination methods, including novel cross-sectional application of molecular methods, is given in “From serological assays to genomics.” Multi-assay approaches were evaluated in order to increase the sensitivity and specificity of the commercial incidence assays in the context of high treatment coverage and stable but high HIV prevalence in Botswana. A novel biomarker based on HIV viral diversity was investigated as a complementary or standalone tool to characterize HIV recency. In this thesis, an innovative use of pairwise diversity and the time to the most recent common ancestor (tMRCA) in a heterosexual HIV-1 subtype C (HIV-1C) epidemic were introduced as novel approaches for HIV incidence estimation. We evaluated the properties of the new potential tools for estimating time since infection, including their specificity and predictive performance in the context of the HIV-1C epidemic in Botswana. Methods: Characterization of HIV recency and novel biomarkers for estimation of HIV infection incidence is based on application of immunologic and molecular methods: a) Evaluation of the long-term specificity (false recent classification rates) of serological tests for recent infection, and algorithms for estimating HIV-1C incidence utilizing samples from patients with known long-standing HIV infection. b) Application of within-host viral diversity for estimation of HIV-1C recency in Botswana using samples collected from patients with known time since seroconversion in the primary HIV-1C infection cohort. c) Investigation of intra-host viral pairwise diversity and the time to the most common recent ancestor (tMRCA), as potential markers for HIV infection recency. Results: We estimated for the first time false recency rates (FRR) of the commercially available BED and Limiting Antigen (LAg) assays in Botswana. We demonstrated that combined algorithms reduce FRR to the recommended < 2%. Including viral load in the assay algorithm resulted in an FRR of 0.4% for LAg. Analysis of the within-host viral pairwise diversity provided more accurate estimation of HIV recency, as compared with the recommended LAg and BED using the receiver operator characteristic analysis (ROC). We demonstrated that intra-host viral pairwise distances reduce misclassification and increase the accuracy of serologic assays. tMRCA and intra-host viral pairwise distances correlated with time since HIV infection provide additional novel tools for reliable estimation of HIV recency. Conclusion: HIV infection recency can be determined cross-sectionally using a combination of serological and molecular biomarkers. Including viral load and an assessment of prior exposure to ARVs is critical for accurate estimation of HIV incidence. Intra-host pairwise diversity and tMRCA are able to predict time since HIV infection and can be used to improve accuracy in estimation of HIV infection recency.
- ItemIn-house genotypic antiretroviral resistance test : optimisation and validation for use in research and diagnostics(Stellenbosch : University of Stellenbosch, 2011-03) Claassen, Mathilda; Van Zyl, Gert Uves; Engelbrecht, Susan; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.It is estimated that 32.8 million people are living with Human Immunodeficiency Virus (HIV) globally with the number of people receiving antiretroviral therapy in low- and middle- income counties increasing to more than 5 million people in 2009. These successes are threatened by treatment failure and the development of resistance to treatment. With an estimated 3.7% patients failing first line treatment after 2 years and 17.9% after 4 years on treatment there is a need for a practical and cheap in-house drug resistance assay that can be used to provide drug resistance data to clinicians and to use as a research tool to investigate drug resistance. In this study we attempted to optimize and validate an in-house drug resistance assay, adapted from Jacobs et al, 2008, to be used as a diagnostic tool and to study the presence of antiretroviral resistance in patients on the Western Cape Mother-To-Child-Transmission (MTCT) regimen. Quality control samples were received from The National Institute of Communicable Diseases AIDS Virus Research Unit, The Round Robin HIV-1 genotyping assessment system from the University of Würzburg and the QCMD assessment system were used for the optimization and validation of an in-house drug resistance assay. The ViroSeq™ HIV-1 Genotyping System was used for comparison of sample and mutation detection. It was possible to optimise and validate a genotyping assay for diagnostic testing and research use by comparison with the ViroSeq™ HIV-1 Genotyping System and evaluation with external quality assessment systems. This assay could subsequently be used to determine the development of genotypic-antiretroviral resistance in patients treated according to the provincial prevention of mother-to-child-transmission (PMTCT) protocol in the Western Cape (single dose nevirapine (sd-NVP), combined with a short course Zidovudine (AZT)). Patient samples were collected from pregnant women who took part in the Western Cape PMTCT program and visited the Tygerberg Obstetrics Clinic and Delft Community Hospital. EDTA blood was obtained to measure CD4-cell count, viral load, and to do genotyping for viral subtype and the presence of resistance mutations. Information on prior exposure to antiretroviral therapy was also collected. A detected resistance rate of 17.1% in this predominantly HIV-1 subtype C population is lower than previously recorded when sd-NVP was administered to HIV-1 subtype C positive patients in PMTCT programs. This could indicate that a dual PMTCT regimen including AZT and NVP reduces the risk of resistance to NVP relative to a regimen that uses sd-NVP. The genotyping assay uses four primers to amplify the PR and the RT gene separately to obtain PCR products, of 487 and 804 base pairs respectively for sequencing. The two PCR products were sequenced with three and five primers respectively to sequence the complete PR and approximately 250 amino acids of the RT gene. The sequences generated, thus, are analysed and aligned with the Sequencer V4.7 software to obtain a consensus sequence of approximately 1200 base pairs for analysis of resistance mutations in the protease and reverse transcriptase genes. The developed assay was hence further simplified and improved, by combining the PR and RT assay into one, which was optimised and validated for use in the routine diagnostic setting. The final genotyping assay uses 8 primers for sequencing to obtain a 1200 bp sequence for genotyping that contains the protease and the 5’ of the reverse transcriptase genes in which antiretroviral resistance associated mutations are found. The assay was accredited by SANAS in 2008.
- ItemInvestigating the fitness benefit of reverse transcriptase (RT) mutation A62V when co-occurring with M184V and K65R in HIV-1 subtype C(Stellenbosch : Stellenbosch University, 2016-03) Njenda, Duncan Tazvinzwa; Van Zyl, Gert Uves; Engelbrecht, Susan; Jacobs, Graeme Brendon; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: Background and Aims Tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) or emtricitabine (FTC) combined with efavirenz is the predominant first-line antiretroviral regimen in the Southern African region. Resistance to TDF and 3TC/FTC is largely through the occurrence of the drug resistance mutations (DRMs) K65R and M184V, respectively. Preliminary data from a large laboratory-based dataset of HIV drug resistance that showed a high prevalence of these mutations in patients who received the TDF regimen also revealed a significant co-occurrence of A62V with M184V and K65R. The aim of this study was to investigate the functional interaction and effect on viral fitness that A62V has when it co-occurs with M184V and K65R reverse transcriptase mutations in HIV-1 Subtype C. Materials and Methods Using Infusion™ cloning and site-directed mutagenesis techniques, eight full-length genome infectious clones containing the HIV-1 subtype C polymerase gene were synthesised having all combinations of DRMs - A62V, M184V and K65R, either being present or absent. The mutations in these constructs were verified by sequencing. The constructs were transfected into 293T cells for virion production and harvested virus was infected in the TZM-bl cell line in head to head growth competition experiments and assayed for growth kinetics using an allele-specific quantitative real-time polymerase chain reaction (PCR) assay. Results The growth competition experiment between two viruses (A62V+K65R+M184V vs K65R+M184V) evaluated by taking the mean of 3 biological replicates in the assay in the absence of antiretroviral drugs, revealed that A62V mutation has no significant impact on fitness (Wilcoxon signed rank test p-value = 0.56). The overall coefficient of variation (CV) in the experiment was 12.8% indicating the high reproducibility of the growth competition assay using real-time PCR measurement of relative growth. Conclusion and recommendations A62V mutation has no effect on fitness when it co-occurs with M184V and K65R. The co-occurrence with M184V and K65R remains unexplained and might be due to an effect on TDF resistance in combination with K65R.This requires investigation in future studies as TDF regimens are part of 1st line therapy in many Sub-Saharan countries in the treatment of HIV-1. Finally, the cloning and mutagenesis techniques used coupled with a very sensitive and reproducible real-time quantitative PCR assay provide an efficient system for detection of mutation fitness interactions and can be used in any future work to study HIV mutation fitness interactions.
- ItemInvestigating 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.
- ItemInvestigation of the HIV diversity in the Cape Winelands, Overberg and West Coast districts of the Western Cape Province of South Africa(Stellenbosch : Stellenbosch University, 2017-03) Mikasi, Sello Given; Jacobs, Graeme Brendon; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH SUMMARY: The Western Cape Province of South Africa has a well-established program that monitors active combination antiretroviral therapy (cART) against HIV-1. The HIV-1 prevalence rate in the Province has increased from 5.0% in 2011 to 18.0% in 2015. South Africa has the highest rate of infections worldwide (19.2%). In this study, we analyzed the Protease (PR), Reverse Transcriptase (RT) and Intergrase (IN) regions of HIV-1 for diversity and resistanceassociated mutations (RAMs) from samples obtained from the Cape Winelands, West Coast and Overberg districts of the Province, where no such study has ever been conducted. Samples were received from our diagnostic laboratory for HIV-1 viral load testing, through the National Health Laboratory Services (NHLS). Two hundred and five (205) patient samples with a viral load of 2000 copies/ml and above were included, based on Gall et al., (2012) who showed that a sensitivity of at least 2000 copies/ml is a limit of amplification for the SuperScsript ® III one-step RT with Platinum Taq DNA Polymerase kit, used in this study. We screened for HIV-1 diversity and RAMs using the pol PR, RT and IN regions with a laboratory-based PCR and sequencing protocol. Sequence-specific subtype analyses were executed with the REGA HIV subtyping tool 3.0, Recombinant Identification Program (RIP) 3.0 and subtype classification using evolutionary algorithms (SCUEAL) software. Sequences were screened for RAMs using the Stanford University HIV Drug Resistance Database (HIVdb) 8.1. We successfully PCR amplified 170 (82.9%) PR and 166 (80.9%) RT fragments. For the IN region, only 176 samples had sufficient plasma and RNA left after genotyping of the PR and RT regions. For IN we successfully amplified 143 (81.3%) of the patient samples. A total of 197 (96.1%) samples could be amplified for at least one of the pol regions. Of these, 62 (53.4%) PR, 103 (62.0%) RT and 93 (86.1%) IN sequences were obtained, respectively. We could successfully sequence 173 (84.4%) of the samples included. HIV-1 subtype C was predominant (n = 144; 93.7%), with 5.3% of other subtypes detected. This includes A1 (n = 2; 1.3%), B (n = 4; 2.6%), D (n = 1; 0.7%) and H (n =1; 0.7%). No major RAMs were detected against PI and IN inhibitors. Minor RAMs were detected in 4 PR (3.7%) and 15 IN (16.1%) sequences analysed. RAMs against RT inhibitors were detected in 63 (61.7%) of the sequences analyzed. This includes 39 NRTI mutations (36.1%) and 71 NNRTI mutations (63.5%) identified. As the national cART program continues to expand, HIV-1 diversity, viral load monitoring and drug resistance screening remains critical for the success of cART outcomes and reducing transmission rates. Our results reflect that subtype C is still the driving force of the epidemic in South Africa. However, we cannot ignore the potential impact of non-C subtypes. Sequence analyses confirm that the majority of patients receiving viral load testing have major RAMs against RT inhibitors used in first line therapy. Better surveillance systems for HIV diversity and drug resistance testing are required to ensure success of cART.
- ItemInvestigation of the molecular epidemiology of HIV-1 in Khayelitsha, Cape Town, using serotyping and genotyping techniques(Stellenbosch : University of Stellenbosch, 2005-12) Jacobs, Graeme Brendon; Engelbrecht, Susan; De Beer, Corena; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.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.
- ItemMolecular characterisation of HIV-1 recombinants and non-subtype C viruses in South Africa(Stellenbosch : Stellenbosch University, 2019-03) Varathan, Olivette; Jacobs, Graeme Brendon; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: HIV/AIDS is a severe health burden, affecting 36.9 million people worldwide by the end of 2017. South Africa has the largest HIV-1 epidemic in the world, estimated at 7.2 million infected individuals by the end of 2017. The HIV-1 epidemic in South Africa is dominated by HIV-1 subtype C, accounting for an estimated 98.2% of the infections in the country, based on the viral sequences in the Los Alamos National Library (LANL) database. To date, to the best of our knowledge, 22 papers have been published on non-subtype C viruses in South Africa. This study aimed to characterise two near full-length genome sequences of non-subtype C viruses in South Africa. The study samples were obtained through the diagnostic services of the National Health Laboratory Services (NHLS), performing routine drug resistance testing within the Division of Medical Virology, Stellenbosch University. All samples received were sequenced in the partial pol region (~1.4kb) of the viral genome by the NHLS. The possible subtype of the virus was identified from the sequences using online subtyping programmes. All sequences and samples that identified as possible non-subtype C viruses were recorded in a separate non-subtype C cohort. In 2011, a possible C, D recombinant virus was first identified in this cohort. By the end of 2015, 30 similar recombinant viruses were observed in the cohort, indicating a possible emergence of this recombinant strain. Two of the samples that identified as possible C, D recombinants were selected for further near full-length genome (NFLG) characterisation. Proviral DNA, from sample EC148, was extracted from PBMCs and viral RNA, from sample WC416, was extracted from plasma. The RNA was reverse transcribed to DNA via cDNA synthesis. Both sample viruses were amplified by PCR in two rounds. The first round targeted the amplification of the HIV-1 NFLG (8978bp) and the second targeted the amplification of two overlapping fragments (5455bp and 4909bp). Positive PCR amplicons were purified and sequenced. The generated sequences were read and analysed before being used in online subtyping programmes. The jumping profile hidden markov model (jpHMM), REGA and recombination identification programmes (RIP) were used to preliminary assign subtypes to both samples. Phylogenetic analyses was inferred to confirm / reject the online subtyping programme results. Online subtyping programmes identified the virus sequences of samples EC148 and WC416 as complex A, C, D recombinants. Phylogenetic analysis confirmed the online subtyping programme results for the sequence of sample WC416 in identifying it as a complex A, C, D recombinant. Phylogenetic analysis indicated that the sequence of sample EC148 is consistent with the results observed from the online subtyping programmes. Each HIV-1 sequence identified as a unique complex recombinant form as the breakpoints between the different subtypes differed. The emergence of new and unique non-subtype C recombinants in South Africa indicates that the epidemic is complex and evolving. It is therefore important to monitor the spread of different HIV subtypes circulating in South Africa.
- ItemMolecular characterization of non-subtype C and recombinant HIV-1 viruses from Cape Town, South Africa(Stellenbosch : University of Stellenbosch, 2009-03) Wilkinson, Eduan; Engelbrecht, Susan; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology.ENGLISH ABSTRACT: HIV-1 was first diagnosed within South Africa in 1982. In the 1980’s homosexual transmission dominated the HIV-1 epidemic within the country. In the late 1980’s the second HIV-1 epidemic was recognized amongst heterosexual individuals. Today heterosexual transmission of HIV-1 dominates the epidemic in South Africa. Subtype C HIV-1 is responsible for the overwhelming majority of heterosexual infections. An estimated 95% of all infections in the country are thought to be subtype C related. To date only a few papers have been published on non-subtype C HIV within the country. This study characterized subgenomic and near full-length sequences of non-subtype C HIV-1 viruses from the Cape Town area. The gag p24, pol-integrase, and env gp41 regions of 11 of the 12 samples were characterized by amplification and direct sequencing. Phylogenetic analysis of the sequenced data, with online subtyping tools (REGA and jpHMM) and the drawing of NJ-trees revealed the presence of subtype A1, B, F1 and recombinant viral forms such as AD, AG and AC. One of the isolates was classified as a subtype C and was included for control purposes. Near full-length characterization of four of the samples were attempted, through full genome PCR amplification and sequencing. Analysis of sequenced data with the use of subtyping-, recombination identification, and tree drawing tools revealed a subtype B, and A1 isolate. The other two isolates were identified as possible AC and AD recombinants. The data that was generated will greatly improve our knowledge of non-subtype C isolates circulating within South Africa. Due to the possible impact that the high degree of genetic variation that HIV may have on vaccine design and development and ARV treatment and HIV diagnosis, ongoing research of the epidemiology and spread of HIV within South Africa are needed.
- ItemMutagenesis and functional studies of the HIV-1 vpr gene and Vpr protein obtained from South African virus strains(Stellenbosch : University of Stellenbosch, 2011-03) Romani, Bizhan; Engelbrecht, Susan; Glashoff, Richard H.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical VirologyENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of host cellular and other viral proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell cycle G2 arrest, modulation of gene expression, and suppression of immune activation. The functionality of subtype C Vpr, especially South African strains, has not been studied. The aim of this study was to describe the diversity of South African HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr proteins. Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped using phylogenetic analysis. Fragments containing natural mutations were cloned in mammalian expression vectors. A consensus subtype C vpr gene was constructed and site-directed mutagenesis was used to induce mutations in postions in which no natural mutations have been described. The functionality of all constructs was compared with the wild-type subtype B Vpr, by transfecting human 293T cell line to investigate subcellular localization, induction of apoptosis and cell cycle G2 arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan Low density arrays (TLDA) was also investigated. Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The following mutations were constructed using site-directed mutagenesis: P14I, W18C, Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr localized to the nucleus but the W18C mutation disturbed the nuclear localization of Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated gene expression was similar for all constructs, indicated that apoptosis was efficiently induced through the intrinsic pathway by the mutants. Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a similar ability for nuclear localization and apoptosis induction. The induction of cell cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C Vpr proteins. The natural mutations studied in the isolates did not disturb the functions of subtype C Vpr and in some cases even potentiated the protein to induce apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as defective, since enhanced functionality would be more indicative of an adaptive role. The increased potency of the mutated Vpr proteins suggests that Vpr may increase the pathogenicity of HIV-1 by adapting apoptotic enhancing mutations.
- ItemOrigin and phylodynamics of HIV-1 subtype C in South Africa(Stellenbosch : Stellenbosch University, 2013-12) Wilkinson, Eduan; Engelbrecht, Susan; De Oliveira, Tulio; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Division of Medical Virology.ENGLISH ABSTRACT: The HIV epidemic in the past couple of decades has spread at an alarming rate throughout Southern Africa. Today the region accounts for roughly one third of all HIV infections, while prevalence rates in other areas of sub-Saharan Africa remain low. In the following study, sampled sequences from Cape Town, spanning over a 21-year period were used to investigate the epidemic history of HIV, which was compared to epidemic trends across Southern Africa. Longitudinal sequence data sets were generated from stored patient samples from Cape Town through standard molecular techniques. Firstly, these sequences were used to estimate the date of origin of the HIV epidemic in Cape Town and to reconstruct a demographic history of the epidemic with advanced Bayesian inference methods. These analyses placed the estimated date of origin of the Cape Town epidemic around the mid 1960‟s with periods of strong epidemic growth observed during the mid 1980‟s and 1990‟s. Secondly, reference strains of HIV from Southern African countries were used to estimate the date of origin of the epidemic in the Southern African region. These analyses placed the date of origin of the epidemic in the Southern African region around the mid 1950‟s roughly ten years before the start of the epidemic in Cape Town/South Africa. These sequences were also used for the reconstruction of the demographic history of the epidemic in the region. A two phased growth in the HIV epidemic in the Southern African region was observed with exponential growth occurring in the mid 1980‟s and 1990‟s. Such findings are also supported by HIV prevalence estimates made by some of the leading HIV research centres and government health departments. Thirdly, a large number of homologous reference strains were used to establish the evolutionary relationship of HIV isolates from Cape Town with those from around the world. A close genetic relationship between Cape Town isolates with other South African and other Southern African isolates was observed in these analyses. Finally, large monophyletic clusters of Cape Town isolates, which was observed during the evolutionary inference, were further investigated. After detailed analyses it appears that these transmission clusters of HIV-1 have been in circulation amongst the infected population of Cape Town for several years or decades.
- ItemPhylogenetic analysis of HIV-1 in Mpumalanga(Stellenbosch : Stellenbosch University, 2013-03) Msimanga, Wela Patrick; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.The diversity of HIV-1 sequences derived from patients in Bushbuckridge, Mpumalanga, was investigated. The gag p24, pol p10 and p66/p51, pol p31 and env gp41 gene fragments from 51 patients were amplified and sequenced. Quality control on the sequences was carried out using the LANL QC online tool. HIV-1 subtype was assigned using the LANL QC (RIP), REGA and jpHMM online tools. Subtype for the pol gene fragment was further designated using the SCUEAL online tool. Most of the sequences, that is 89%, belonged to HIV-1 subtype C. LANL QC (RIP), REGA, jpHMM also detected recombinants in 11% of the sequences. One of the isolates could only have the env gp41 gene fragment amplified and sequenced, which was determined to be HIV-1 subtype B. Phylogenetic analysis using the Neighbor-Joining and Maximum Likelihood methods from MEGA v 5 showed that, except for the env gp41 designated as a subtype B, all sequences in the study clustered with HIV-1 subtype C. Significantly, phylogenetic analysis showed that not only are the Bushbuckridge, Mpumalanga sequences related to HIV-1 subtype C sequences from southern Africa, India, Ethiopia and Brazil, but it is possible there has been multiple introductions of HIV-1 in the province. SDRMs were observed in two samples.
- ItemThe role of clonally expanded HIV-1 infected cells in maintaining HIV reservoirs in adults and children on antiretroviral treatment(2020-12) Botha, Johannes Christiaan; van Zyl, Gert U; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: The HIV-1 pandemic remains a major public health dilemma. Treatment with combination antiretroviral therapy (cART) can, in the majority of patients, sufficiently suppress HIV viral replication. However, cART is not a cure as it does not affect long-lived reservoirs. HIV proviruses are harboured in the genome of long-lived CD4 helper cells that are maintained by having long half-lives and by being replenished through cellular proliferation. HIV infected cells that proliferate can be identified as clonal populations by all having the same integration site. A small proportion of proviruses are intact from where HIV rebounds after therapy interruption and is the barrier to achieving cure or remission. Therefore, many molecular assays have been designed to characterise HIV proviruses, with a focus on intact proviruses. From this the proviral landscape is estimated to consist of ~2% intact proviruses and up to 98% defective proviruses comprising deletions and hypermutations. However, highly deleted proviruses such as solo-long terminal repeats (LTRs) remain largely uncharacterised. Recently, it has become apparent that some proviral clones are able to express infectious HIV and cause clonal viraemia in patients on cART, which are not indicative of ongoing cycles of viral replication. This has further confirmed the importance of proviral clones in maintaining the HIV reservoir. Subsequently, aspects of proviral clones were investigated in this study. A novel assay capable of characterising severely deleted proviruses by targeting the unique integration sites was developed. The characterisation of proviral clones in paediatric patients with this novel assay revealed that severely deleted solo-LTR proviruses may be more prevalent than previously expected. Further investigation of these solo-LTR proviral clones was performed with another novel assay capable of quantifying proviruses based on unique integration sites. The longitudinal waxing and waning of solo-LTR proviral clones could be observed. Although solo-LTR proviruses do not contribute to the true reservoir, it suggests that current proviral landscape proportions require adjustment to account for all HIV integration events in cells. Clonal viraemia was studied in HIV positive adult patients on long term cART presenting with non-suppressible HIV viraemia. Three clusters of monotypic plasma viraemia and cell associated DNA HIV sequences were identified in one patient. These monotypic proviruses were shown to be replication competent by viral outgrowth. This provides evidence that clonally proliferated cells harbour at least a proportion of intact proviruses and therefore constitute an important component of the true HIV reservoir. Two additional patients were identified with uncommon cases of sustained viraemia. Firstly, a probable large cell clone harbouring non-infectious virus was found to leak proviral nucleic acid into plasma, resulting in apparent treatment failure. Secondly, possible clonal viremia was observed in a patient with very slow decaying viral load, despite objective evidence of adherence and initial undetectability of treatment-relevant drug resistance over a period of 2 years. We conclude that clonal viraemia may be underreported, and that cases like these show that clonal viraemia should be considered as an explanation of non-suppressed viraemia or apparent therapy failure in the management of HIV infected patients on cART.