Masters Degrees (Medical Virology)

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Browsing Masters Degrees (Medical Virology) by Subject "Antiretroviral agents"

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    Improved methods to recover HIV-1 integrated proviruses and integration sites
    (Stellenbosch : Stellenbosch University, 2020-12) Delaney, Kayla Eileen; Van Zyl, Gert Uves; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
    ENGLISH ABSTRACT: Background Long-lived HIV-1 infected cells that form part of the latent HIV-1 reservoir represent a major barrier to HIV-1 cure despite antiretroviral therapy (ART). The majority of these cells contribute to a persistent HIV-1 infection through clonal expansion. Many methods exist to study clonal expansion by identifying identical integration sites in different cells. However, the majority of proviruses are defective and until recently, methods did not exist to enable the simultaneous characterisation of integration sites and integrated proviruses, to identify which HIV-1 infected cell clones harbour intact proviruses. As recent methods are laborious and expensive, more efficient methods of linking intact proviral sequences and their cognate integration site are required. In addition, third-generation sequencing platforms allow for real-time long read-length sequence data which is ideal to characterise proviruses. These methods provide faster and simpler genome assembly than short read length second-generation sequencing platforms. Therefore, the overall aim of this study was to contribute to the HIV cure agenda, by improving our understanding of true HIV reservoirs by developing methods that would improve the characterisation of HIV-1 infected cell clones that harbour intact HIV genomes. Methods Intact proviral genomes are rare and attempts to enrich and link these proviral sequences to their integration site were investigated. The Integration Site Loop Amplification (ISLA) assay published by Wagner et al. (2014) was adapted for HIV-1 subtype C. Verification by Sanger sequencing showed that no integration sites were recovered and the “Premium whole genome amplification” method from Oxford Nanopore Technologies’ (ONT) third-generation sequencing technology was attempted as an alternative method. To investigate the utility of ONT sequencing, the “Amplicons by Ligation” method was utilised to sequence 9 near-full-length provisionally intact HIV-1 proviral sequences, previously sequenced by Illumina MiSeq. The consensus sequences for ONT sequencing were generated through a custom bioinformatic pipeline and compared to the Illumina MiSeq consensus sequences. Results The modified ISLA approach for HIV-1 subtype C or Premium whole genome amplification method did not succeed in recovering the HIV-1 proviral integration sites, of rare intact HIV-1 genomes. For near-full-length HIV genome sequencing, the “Amplicons by Ligation” protocol ONT sequencing achieved high coverage across the HIV genome and apart from hypervariable HIV-1 envelope there was a near perfect concordance between the consensus sequences generated with ONT and the previous Illumina MiSeq sequences, with an overall concordance of >99% for 8 out of 9 samples. Conclusion HIV-1 integration sites were not recovered in this study and efficient methods for simultaneous and efficient identification of intact proviral genomes and their integration sites remain unavailable. ONT sequencing allows for efficient and accurate sequencing of long fragments in real-time which may overcome technical barriers and eliminate laborious, time-consuming and expensive methods currently used for integration site identification and near-full-length HIV-1 genome sequencing. As part of the research towards future possible HIV cures, it remains a priority to investigate the persistence of cells harbouring intact HIV-1 genomes, and the role of clonal cellular proliferation in their survival.
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    Investigation 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.
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    A Simple Virus Recovery Assay to detect the Replication-Competent HIV-1 Reservoir in Children
    (Stellenbosch : Stellenbosch University, 2021-03) Van Zyl, Carli; Van Zyl, Gert Uves; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
    Background and Introduction: The early initiation of combined antiretroviral therapy (cART) in children has proved to significantly reduce the morbidity and mortality rate associated with human immunodeficiency virus type 1 (HIV-1) infection. However, even though cART can reduce plasma viral loads to being clinically undetectable, it does not cure HIV-1 infection due to the presence of a latent reservoir where HIV-1 persists as integrated, replication-competent proviruses. For this reason, the search to finding a cure for HIV-1 continues, either by targeting the HIV-1 reservoir directly (eradication approach) or by controlling rebound of viraemia (functional cure approach). To accurately assess the success of such cure strategies, assays to accurately measure the latent reservoir are essential,such as the quantitative viral outgrowth assay (qVOA). In this study, a simple viral outgrowth assay (VOA) was developed that utilises MOLT-4CCR5+ cells for propagation of HIV-1 as well as a sensitive reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assay for detectionof viral outgrowth. Methods Two children (patient 337756 and patient 341622) were selected that were part of the post-Children with HIV Early Antiretroviral Therapy randomised trial (CHER) to be assayed for replication-competent virus,with the VOA,from stored peripheral blood mononuclear cells (PBMCs) formas singleblood collection, which consisted of stimulation to induce viral release followed by co-cultureover28 days. Both children started therapy at two months of age, were ART suppressed for eight years, although both had a recent episode of viraemia (six months before the time point sampled), and both had total HIV-1 deoxyribonucleic acid (DNA) levels of >50copies per million cell equivalents at the time point sampled. Patient 337756 was 11years old and patient 341622 was 10years old at the time point sampled. A total of 29million and 21million PBMCs were assayed for patients 337756 and 341622, respectively. A VOA was implemented and optimised that utilisedMOLT-4CCR5+ cells for HIV-1 expansion and a sensitive, affordable RT-qPCR assay was developed that targets the integrase region ofpolin the HIV-1 genome to detect exponential viral outgrowth. T cells were stimulated by addition of phytohemagglutinin (PHA) and irradiated feeder cells. To determine the sensitivity ofthis RT-qPCR assay, it was compared to the traditional, but more expensive p24 antigen enzyme-linked immunosorbent assay (ELISA). Results Infectious virus was recovered from two out of three VOA wells in one of the two children(patient 337756). The in-house RT-qPCR assay was able to detect virus on day five for this child while the p24 ELISA only had a positive result on day 14. Interestingly, the second VOA well for the same child had low-level exponential outgrowth with the RT-qPCR assay, but the p24 had no positive signals. This proved that the RT-qPCR assay was more sensitive in detecting viral outgrowth compared to the p24 ELISA. The third VOA well for this patient had weak ribonucleic acid (RNA)signal on day 14 as well as DNA signal on day seven. The child who had no infectious virus recovery from stored PBMCs (patient 341622) also had a single instance of weak RNA signal on day 14 in one of the two VOA wells assayed. The single instances of RNA release might have been non-infectious, inducible virus that got released after stimulation. HIV-1 DNA detected in the supernatant were cell derived and may therefore indicate apoptosisor lysis of infected cells. Conclusion: The VOA implemented in this project proved to be more practicable by using MOLT-4CCR5+ cells compared to the gold standard qVOA that makes use of cluster of Differentiation8 (CD8+)-depleted lymphoblasts from different donors.The RT-qPCR assay also proved to be more sensitive than the p24 ELISA. Although, by streamlining and optimising this assay even more, it could be more valuable for future studies in evaluating HIV-1eradication strategies. Nevertheless, this VOA could be a valuable tool in studying the success of latency reversing agents (LRAs) in children on suppressive cART.