Masters Degrees (Medical Virology)

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Browsing Masters Degrees (Medical Virology) by Author "Delaney, Kayla Eileen"

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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.