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Masters Degrees (Virology)

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    Comparison of SARS-CoV-2 sequencing using the ONT GridION and the Illumina MiSeq.
    (2022) Tshiabuila, Derek Kalala.; De Oliveira, Tulio Paiva N Andrade.
    Corona Virus Disease 2019 (COVID-19) is an ongoing pandemic that has spread rapidly around the world and has seen over 431 000 000 identified cases and 5 930 000 deaths caused by this disease by the end of January 2022. Many viral lineages have arisen from Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) as public health measures from numerous countries have failed to contain the spread of the virus. Sequencing of SARS-CoV-2 has enabled the identification and classification of the viral lineages, while real-time tracking of the emergence and spread of these lineages has been facilitated by the open sharing of genomic surveillance data and collaborative online platforms. Several studies have suggested that various mutations may have a functional effect on the virus, such as a substitution in the spike protein (D614G) may result in increased transmissibility whilst an N439K substitution in the receptor-binding domain (RBD) may assist in neutralizing monoclonal antibodies. It is therefore necessary that a fast and reliable sequencing technology be used to rapidly and correctly produce SARS-CoV-2 genomes that can be used to identify viral lineages. Many sequencing laboratories have begun using Nanopore sequencing as it promises high throughput, realtime sequencing, at an affordable cost and many of their sequencing platforms allow for portability. The sequencing technology has, however, not been verified to produce consensus SARS-CoV-2 genomes that are comparable to Illumina Sequencing which is currently the gold standard Next Generation Sequencing (NGS) technology for SARS-CoV-2 sequencing. In this study, we compared the Illumina and Nanopore sequencing platforms by comparing the SARS-CoV-2 genomes produced by the Illumina MiSeq and Oxford Nanopore Technology (ONT) GridION X5. The results show that the GridION is currently unsuitable for SARS-CoV-2 genomic surveillance as consensus genomes produced by the platform have a lower quality than those produced by the MiSeq which reduces the reliability of the data obtained from the genomes. These results can be used to better understand the Nanopore sequencing technology and how it differs from the Illumina technology which will help in updating the Nanopore technology to produce consensus genomes at a faster rate than the Illumina technology whilst still having a similar quality.
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    Central nervous system (CNS) derived human immunodeficiency virus type 1 (HIV-1) subtype C long terminal repeat (LTR) genetic and functional variation mediates high viral load in this compartment of tuberculous meningitis (TBM) co-infected patients.
    (2023) Ntshangase, Wenzile Senorita.; Madlala, Paradise Zamokuhle.
    Background: Human immunodeficiency virus type 1 (HIV-1) ribonucleic acid (RNA) is characteristically lower in the central nervous system (CNS) than in plasma of antiretroviral treatment naïve patients. Paradoxically, there is higher HIV-1 viral load in the cerebral spinal fluid (CSF) than plasma of treatment naïve patients co-infected with tuberculous meningitis (TBM). The mechanisms that govern high viral replication in the CNS of TBM co-infected antiretroviral therapy naïve patients remain to be determined. Methodology: The study population comprised of 17 TBM and 3 non-TBM participants selected from an HIV-1 positive and TBM co-infected cohort. The HIV-1 viral RNA was reversed transcribed into complementary deoxyribonucleic acid (cDNA) thus the U3/R region of 3’ long terminal repeat (LTR) was amplified from CSF and plasma RNA by KAPA HiFi HotStart PCR Kits (ThermoFisher Scientific, Invitrogen™, USA). The patients CSF and plasma derived LTR were subsequently cloned into a pGL3 plasmid and further transfected in Jurkat and Astrocyte cell lines to assess the LTR transcriptional activity using Bright-Glo™ Luciferase Assay System (Promega, Madison, WI, USA). Results: CSF derived LTR had a significantly (p<0.0001) higher basal and Tat induced transcriptional activity compared to plasma derived LTR in Astrocyte (SVG) cell line. Similarly, CSF derived LTR had significantly higher (p=0.0024) Tat induced transcriptional activity compared to plasma derived LTR in Jurkat cell lines. LTR sequences containing an Adenine (A) at position 5 of the Sp1III binding site were associated with significantly high basal (p<0.0001) and Tat induced (p=0.0002) transcriptional activity compared to the LTR sequences containing a Thymine (T) at the same position when it was assessed in SVG cell. A similar case was observed in Jurkat cell lines. Consistently, CSF LTR sequences containing an A at position 5 of the Sp1III transcription binding site were associated with significantly higher HIV-1 viral load compared to LTR sequences containing a T at the same position (p=0.0093). Conclusion: Our data clearly show that CSF derived LTR from TBM co-infected individuals exhibit significantly higher transcriptional. Particularly, sequences containing the A5T mutation are significantly associated with higher LTR transcriptional activity and viral load.
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    Implementation of an efficient sample pooling strategy for high throughput diagnostic testing of Severe Acute Respiratory Syndrome Corona Virus-2.
    (2022) Anyaneji, Ugochukwu Jacob.; De Oliveira, Tulio Paiva N Andrade.; Petruccione, Francesco.; Giandhari, Jennifer.; Lessells, Richard John.; Singh, Lavanya.
    The rapid identification and isolation of infected individuals remains a key strategy for controlling the spread of SARS-CoV-2. Frequent testing of populations to detect infection early in asymptomatic or presymptomatic individuals can be a powerful tool for intercepting transmission, especially when the viral prevalence is low. However, RT-PCR testing – the gold standard of SARS-CoV-2 diagnosis – is expensive, making regular testing of every individual unfeasible. Sample pooling is one approach to lowering costs. By combining samples and testing them in groups the number of tests required is reduced, substantially lowering costs. Here we report on the implementation of pooling strategies using 3-d and 4-d hypercubes to test a professional sports team in South Africa. We have shown that infected samples can be reliably detected in groups of 27 and 81, with minimal loss of assay sensitivity for samples with individual Ct values of up to 32. We report on the automation of sample pooling, using a liquid-handling robot and an automated web interface to identify positive samples. We conclude that hypercube pooling allows for the reliable RT-PCR detection of SARS-CoV-2 infection, at significantly lower costs than lateral flow antigen tests.
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    Innovative and affordable HIV-1 drug resistance testing for resource limited settings.
    (2022) Manyana, Sontaga Cris.; Chimukangara, Benjamin.
    Background: HIV drug resistance (HIVDR) remains a major threat to achieving sustainable viral suppression on antiretroviral treatment (ART). Most countries including those in resource limited settings (RLS) have adopted use of dolutegravir (DTG), a more potent integrase strand transfer inhibitor, leading to an increase in the demand for integrase resistance testing. Current HIVDR testing methods in RLS focus on genotyping the HIV protease (PR) and reverse transcriptase (RT) genes, separate from the integrase (IN) gene. However, amplification of PR and RT separate from IN is expensive and increases the workload for HIVDR genotyping. Therefore, affordable and labour efficient methods that genotype all relevant HIV-1 genes (i.e., the PR, RT and IN genes) are required to guide clinical decisions, especially in RLS where cost is a major limiting factor. Thus, this study aimed to design an affordable in-house HIVDR genotyping method suitable for use in RLS. Methods: Remnant plasma samples were obtained from a CAPRISA 103 study and viral RNA was extracted from 500μl of plasma. We validated the assay using remnant plasma samples from an external quality assessment (EQA) programme. Complimentary DNA synthesis and first-round PCR were performed followed by second-round nested PCR which was designed to amplify an ~2.9kb HIV-1 pol region (PR, RT and IN genes) using 1% gel electrophoresis. Successful second-round nested PCR products were purified using ExoSAP-IT Express PCR Product Cleanup reagent. Sanger sequencing was performed and quality of the sequences were manually edited using Geneious Prime software. HIVDR mutations were assessed using the Stanford HIV drug resistance database. HIVDR mutations using the designed method were compared to previous results obtained on the same samples. Sequence quality was also evaluated using phylogenetic analysis in Geneious software with maximum likelihood tree reconstruction using a generalized time reversible model with proportion of invariable sites and gamma distribution (GTR + I + G), and with 100 bootstrap replicates. Method cost-estimates were done by comparing costs and turn-around time to current genotyping methods. Results: Of 115 plasma samples obtained, 19 samples were not processed due to inadequate plasma volume. Of the 96 processed, we obtained sequence data for 78 (81%). Of those, 75 (96%) had at least one HIVDR mutation in the PR and RT genes, with no major-IN mutations observed. Only one sample had an E157Q INSTI-accessory mutation. When compared to previous genotypes, only 2/79 (3%) had different phenotypic predictions that affected the choice of subsequent regimens. Of 7 EQA samples, 4 were HIV-1C, 2 were HIV-1D, and 1 was HIV-1A. Genotypic resistance data generated using the IDR method showed 100% concordance with EQA panel results. The overall cost per sample was estimated at ~US$43, with a turn-around time of ~15 hours. Conclusion: We successfully designed an in-house HIVDR method suitable for genotyping HIV-1C PR, RT and IN genes, at an affordable cost of US$64 and shorter turn-around time reduced from ~21 hours to ~15 hours, compared to currently available methods. This HIVDR genotyping method accommodates changes in ART regimens and will help to guide HIV-1 treatment decisions in RLS.
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    Investigating the impact of HIV infection on ILC3s in human lymph nodes.
    (2022) Herbert, Nicholas Graeme.; Kloverpris, Henrik.
    People living with HIV (PLWH) develop extensive fibrosis and collagen deposition throughout their lymphoid tissues not reversed by antiretroviral therapy (ART). Innate lymphoid cells (ILCs) play essential roles in tissue homeostasis and repair, however, no studies exist on ILCs in lymph nodes (LNs) during HIV infection. We hypothesized that ILCs are modulated by HIV infection and are involved in the subsequent immune responses. We obtained fresh celiac, cystic, bile, falciform, common hepatic and mesenteric LNs immediately after gastrointestinal surgery from patients recruited from areas in KwaZulu-Natal, South Africa – home to the highest HIV prevalence in the world. LNs from PLWH receiving ART exhibited extensive collagen deposition compared to uninfected controls characteristic of HIV-infected LN pathology. Single-cell transcriptional profiling revealed activation of the dominant ILC3 subset during HIV infection, suggesting ILC3s are directly involved in the HIV immune response. HIV-infected LNs expressed more heterogenous ILC3 subsets, including ‘ex-ILC3s’. We found signatures suggesting that HIV infection induces terminal differentiation of homeostatic ILC3 populations, whereby an ex-ILC3 population becomes distinct and may contribute to a type 1 immune response. Since HIV infection leads to sustained inflammation in LNs, this terminal differentiation and emergence of ex-ILC3s may be irreversible. Moreover, we found elevated levels of TGF production by ILC3s during HIV infection which may suggest that these cells play a role in fibrosis formation, directly or indirectly, through fibroblast-induced collagen deposition. Here, I performed the first singlecell analysis of ILCs in HIV-infected LNs and identified ILC3s as potential contributors to LN fibrosis, a major pathological consequence of HIV infection that warrants further investigation
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    Genetic characterization of viral blips in patients following suppressive HIV ART.
    (2021) Jhamba, Lindiwe Amanda.; Gounder, Kamini.; Ndung'u, Peter Thumbi.
    Antiretroviral therapy (ART) has resulted in the decline of HIV-related mortality worldwide. On initiating treatment, most patients can suppress plasma viral RNA to undetectable levels (<20 copies/mL). Patients on ART frequently experience intermittent viremia (viral blips), however the genetic nature and source of these rebounding viruses while on suppressive ART remains unclear. The study of these genetic characteristics would be essential in the development of targeted vaccines and adjunct treatments. We identified four HIV-1 subtype C infected women from the Females Rising through Education, Support and Health (FRESH) acute infection cohort who experienced viral blips following suppressive ART (median 584 days). Two participants initiated treatment during the chronic infection phase (~625 days post detection) and the other two initiated treatment immediately upon first HIV-1 RNA detection. RNA was extracted from stored plasma samples of participants’ transmitter/founder (T/F) virus (~3 days post detection), pre-treatment initiation and during viral blips (>2,000 copies/mL). Gag and env genes were amplified by single genome amplification followed by sequencing. The protease and reverse transcriptase region of the pol gene were also amplified and bulk sequenced. Phylogenetic relatedness and genetic differences were visualized using Maximum-likelihood trees and Highlighter plots respectively (Los Alamos HIV-1 database). The gag and env blip sequences of the acute-treated participants were similar to those of the T/F, while those of the chronic-treated participants were genetically distinct from the T/F but similar to the pre-treatment initiation virus (PreART). In the acute-treated participants, all transmitted HLA-associated gag cytotoxic CD8+ T lymphocyte (CTL) escape identified was retained in the blip-derived sequences, however the chronic-treated participants experienced an increase of ~0.8% CTL escape at the blip time point. This increase coupled with development of a reduced replication capacity mutation (HLA-B*57:01/58:01 T242N), may be indicative of immune pressure prior to ART initiation. Mutations associated with bnAb escape in the CD4 binding, gp120/gp41 and V1V2 sites were identified only in the PreART and blip sequences of the chronic-treated participants, whereas the acute-treated retained the same amino acid residues at T/F and blip. With the exception of one chronic-treated participant who developed mutations associated with resistance to efavirenz, the viral blips were not associated with mutations linked to drug resistance. This data suggests that those who initiate treatment late are less likely to benefit from an immune response-inducing vaccine or bnAb therapy.
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    The role of Nef-mediated SERINC5 down-regulation on HIV-1 disease progression.
    (2021) Naicker, Marshlin Delon.; Mann, Jaclyn Kelly.
    HIV-1 Nef is a small accessory protein that plays a vital role in enhancing HIV-1 pathogenesis, evidenced by a strongly attenuated disease course following infection with a virus with gross Nef defects. Nef has multiple cellular effects, which enhance HIV-1 replication and immune evasion. Major activities of Nef include CD4 down-regulation, HLA-I down-regulation, and CD4-independent enhancement of virion infectivity. Recent studies have uncovered Nefmediated down-regulation of the host restriction factor SERINC5 as an important mechanism by which Nef enhances virion infectivity. However, there is a lack of studies defining the role of this function in HIV-1 pathogenesis. Previous studies indicated that Nef-mediated CD4 down-regulation and enhancement of infectivity are likely the major contributors to Nef’s effect of enhancing pathogenicity; the relative significance of each Nef function for HIV-1 disease progression remains incompletely understood. Given the key role of Nef-mediated SERINC5 down-regulation in enhancing virion infectivity, the primary aim of the present study was to determine if this Nef activity contributes significantly to disease progression in individuals infected with HIV-1 subtype C, which is the dominant HIV-1 subtype worldwide. To investigate this, SERINC5 down-regulation activity of 106 Nef clones derived from patients with early HIV-1 subtype C infection were evaluated in a CD4+ T cell line using a flow cytometry-based assay and subsequently related to viral load set point and to the rate of CD4+ T cell decline using linear regression analysis. The second aim of this study was to assess the overall contribution of SERINC5 down-regulation to Nef function, using linear regression analysis with E values as a proxy for overall Nef function in vivo. The third aim of the study was to identify amino acid variants that significantly alter Nefmediated SERINC5 down-regulation using a codon-by-codon sequence-function analysis tool available online. No significant relationship was found between each Nef function and viral set point (SERINC5 down-regulation, p=0.28) or rate of CD4+ T cell decline (SERINC5 down-regulation, p=0.48). CD4 down-regulation (p=0.02) and SERINC5 down-regulation (p=0.003) were significant determinants of the E value in univariate analyses, and SERINC5 down-regulation remained significant in the multivariate analysis (p=0.003). We found several amino acids that were significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity. In conclusion, none of the Nef functions in our study, including SERINC5 down-regulation, were found to be significant individual contributors to disease progression. However, interestingly we found CD4 down-regulation and SERINC5 down-regulation to be the largest contributors, of the Nef functions considered here, to overall Nef function and that the contribution of SERINC5 down-regulation was the most significant. Taken together, this could be explained by multiple Nef functions acting together to facilitate the enhancement of viral spread and immune evasion in vivo that ultimately enhance disease progression. We found several amino acid variants that either increased or decreased Nef’s ability to down-regulate SERINC5; however, further studies in the form of site-directed mutagenesis are warranted to further understand their effect on SERINC5 down-regulation activity. In summary, the results suggest that SERINC5 down-regulation is a strong contributor to overall Nef function and identifies potential genetic determinants of this Nef function that may have relevance for vaccines or therapeutics.
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    Cytotoxic and anti-proliferative effects of Moringa Oleifera Lam.on hela cells.
    (2021) Govender, Krishnambal.; Parboosing, Raveen.; Moodley, Indres.
    Moringa oleifera Lam., known to most as the ‘drumstick tree’, is a non-fastidious botanical that is native to India, and is cultivated on a global scale as a sustainable crop, for sustenance, medicinal and beauty applications, amongst others. The antitumour, antibacterial and antifungal effects of M. oleifera are well-documented, however, its specific effects on human papillomavirus (HPV)-induced malignancy have not been established. High-risk HPV subtypes 16 and 18 are implicated in the carcinogenesis of more than 90% of cervical cancers. Despite well-established national cervical screening programmes, cervical cancer still remains the most common cancer affecting females in South Africa. This may partly be attributed to the high incidence of HIV infection in South Africa. Some of the hallmarks of cancer are, the up-regulation of telomerase, over-expression of E2F1 transcription factor, and over-expression of cyclin E and cyclin B1. The aim of this current study was to establish whether 24-hour treatment with hexane and ethanol leaf extracts of M. oleifera modulate telomerase, E2F1, cyclin E, and cyclin B1. The apoptotic pathway and phase of cell cycle arrest were also investigated. The HeLa cell line, an aggressive cervical cancer cell line in which high-risk HPV-18 viral strands have been identified, was used in this study A novel effect of M. oleifera leaf extract was evident in the inactivation of telomerase. The inactivation of telomerase implies that p53 function was restored by the repression of E6 gene expression. Another novel outcome of the study is that M. oleifera down-regulates E2F1, accounting for the dose-dependent antiproliferative effects seen. The inactivation of telomerase was demonstrated by caspase-3 and caspase-7 activation, which confirmed intrinsic apoptosis. The down-regulation of E2F1 possibly occurs through the repression of the E6 oncoprotein and the activation of p53. The quantitative assessment of cyclin E and cyclin B1, showed an overall down-regulation, and G2-M cell cycle arrest. Taken together, this study provides convincing evidence that M. oleifera hexane and ethanol leaf fractions have potential antitumour effects, by targeting multiple abnormally elevated markers for down-regulation. Other M. oleifera fractions investigated in a parallel study, and were excluded due to p values being greater than 0.05 and inconclusive findings, in dichloromethane and aqueous fractions.
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    Measuring HLA-B allele expression across differential cell types.
    (2018) Ramphal, Upasana.; Ramsuran, Veron.
    Background: The human leukocyte antigen (HLA) region has shown to have the strongest disease associations and recent studies have shown that expression levels of these HLA molecules play a major role in the clinical course of diseases. Differences in the expression levels of these molecules have been found to have a major effect on their ability to present specific peptide antigens. HLA molecules are critical to the interaction between diseases and components of the immune system. Expression of such molecules, namely HLA-C and HLA-A, have been shown to associate with HIV disease outcomes. An increase in expression of HLA-C leads to protection against HIV whereas an increase in HLA-A expression leads to rapid HIV progression. Furthermore, studies have shown the region with the strongest genetic effect falls within the HLA-B gene, as determined by genome wide association studies. However, limited information is available for HLA-B allelic expression levels and the variation across differential cell types. Materials and Methods: Allelic expression levels of HLA-B were measured using cryopreserved PBMC samples from HIV negative and positive cohorts with HLA typing. Antibodies specific to the HLA-B protein were identified. The affinity of the antibodies relative to class-I alleles were determined. Based on these affinities, donors with specific alleles were selected for HLA-B cell surface measurement using the flow cytometer. mRNA levels were measured across HLA-A, -B, -C and -E genes within the following cell types T-cells, B-cells, Monocytes and NK cells. These levels and a comparison of HIV infected and uninfected mRNA levels from the same donor were measured using droplet digital PCR (ddPCR). Conclusions: Contrary to HLA-B mRNA expression levels, we find cell surface expression levels vary in an allele-specific manner. We further observed differential mRNA expression patterns for HLA-A, HLA-B, HLA-C and HLA-E across cell types. We also observed no mRNA expression variation across pre- and post- HIV samples. When comparing HLA-B mRNA and surface expression across alleles and donors no significant correlation was found. However, at an donor level, some alleles may be differentially regulated at the cell surface. This study built existing knowledge and fills in some of the gaps in knowledge surrounding HLA-B expression. We also report, for the first-time, variation in allele specific expression, variation in expression across differential cell types and lack of expression variation across pre- and post- HIV infection at the mRNA level. ”
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    Identifying novel transcriptional regulatory elements of HLA-A alleles through the evaluation of the 5’ un-translated region sequences.
    (2020) Singh, Saiyuri.; Ramsuran, Veron.
    Sub-Saharan Africa holds approximately half the population living with human immunodeficiency virus (HIV) in the world (~19.6 million), of which around 7.2 million cases are found in South Africa. Although antiretroviral therapy can suppress viral loads to below detectable levels in most cases, drug resistance is a growing problem. Therefore, identifying novel treatment strategies are warranted against HIV. The strongest human genetic associations with HIV disease have been found within the human leukocyte antigen (HLA) region. The expression levels of various HLA genes have been associated with HIV disease outcomes. Increased HLA-A mRNA expression results in poor HIV outcomes due to the inhibition of natural killer (NK) cells since high mRNA expression of HLA-A results in high protein expression of HLA-E which serves as an inhibitory receptor for NK cells. Identifying factors that regulate the expression of HLA-A has the potential to serve as an avenue for HIV drug target sites. DNA methylation has previously been identified as one of the factors responsible for HLA-A expression regulation. In this study, we aimed to identify additional regulatory mechanisms for the HLA-A gene. The identification of a putative CCCTC-binding factor (CTCF) binding site upstream of HLA-A suggested that CTCF may play a role in regulation of HLA-A. Sequence alignments about 2 kilobases (2KB) upstream of the transcriptional start site (TSS) were analysed for polymorphisms that associate with HLA-A expression. Six HLA-A promoter variants (rs9260084, rs9260086, rs9260092, rs9260101, rs9260116 and rs41560714) were observed to significantly associate with HLA-A mRNA expression. However, only one single nucleotide polymorphism (SNP), rs9260084 (-993G>A), was predicted to disrupt a CTCF binding site. Despite the predicted disrupted binding site, using a chromatin immunoprecipitation (ChIP) assay, we did not detect any difference in CTCF binding across the -993 G>A variants. Additional transcriptional regulators, Nuclear Factor 1 (NF1), Ras related protein (RAP1) and glucocorticoid receptor (GR), were predicted to have differential binding to -993G>A, -226G>A and -885C>G, respectively. The results provided here serve as a basis for further studies exploring the role HLA-A promoter variants have in regulating HLA-A expression. These variants may serve as potential target sites for future therapeutic intervention against HIV.
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    Identifying the cellular HIV-1 reservoir in lymph nodes of antiretroviral therapy suppressed individuals.
    (2019) Ferreira, Isabella Anna Theresa Markham.; Sigal, Alexander.
    HIV-1 infection is suppressed but not cured in the face of antiretroviral therapy (ART). Pinpointing the cellular HIV-1 reservoir, which allows HIV-1 to persist, is key to the eradication of the virus. Lymph nodes are known to be a reservoir site for HIV-1 persistence, and we have assembled lymph nodes from a cross-sectional cohort of participants on suppressive ART to better understand the cellular HIV-1 reservoir. We developed a novel single-cell RNA-Seq methodology to identify the cellular HIV-1 reservoir in the lymph node compartment in ART suppressed individuals. HIV-1 positive cells from these lymph nodes were stained with anti-HIV- 1 antibodies and selected using flow cytometric sorting. Seq-Well, a high throughput single-cell RNA-Seq approach, was then performed to detect gag and env HIV-1 transcripts in individual cells, as well as the infected subtype using the cellular transcriptome. In parallel, the consensus near full length viral clone from the lymph node was sequenced and used for alignment. Using our methods for identifying HIV-1 infected cells from lymph nodes from chronically infected individuals, we have identified both known and novel putative host markers that are associated with persistent infection. These included co-expression of APOBEC3G, NFAT5, and NFKB2 in cells that contained HIV-1 mRNA. Our results show that cells with transcriptomes consistent with a T cell origin are the main infected population, and we are in the process of deeply characterizing the cell subtypes involved that also express markers of HIV-1 infection.
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    The differential influence of HIV-1 subtype C,nucleoside analog resistance mutations: K65R, A62V, S68N and Y115F susceptibility to tenofovir.
    (2019) Didamson, Onyisi Christiana.; Gordon, Michelle Lucille.
    The use of Tenofovir Disoproxil Fumerate (TDF) for the treatment of HIV-1 infection has been recommended for the first-line as well as a second-line antiretroviral regimen in South Africa, due to its high antiretroviral activity and low toxicity level. However, the efficacy of the drug could be threatened by the emergence of drug resistance mutations. The development of TDF resistance poses a public health threat. TDF resistance can be acquired through a selection of the K65R mutation or the K70E mutation (though less frequently) under TDF selection pressure. Besides, K65R and K70E mutations, recent studies have identified other mutations associated with TDF resistance such as A62V, K65N, S68G/N/D, K70E/Q/T, L74I, V75L, and Y115F. These mutations were particularly observed to be in association with the K65R mutation and were reported to be more common in HIV-1 subtype C viruses. Also, these mutations could cause high-level resistance to TDF, especially when in combination with K65R. However, in-vitro studies are required to demonstrate their influence on viral fitness and TDF susceptibility. In this study, we investigated the impact of K65R, A62V, S68D, Y115F, and K65R+S68N on replication capacity and TDF susceptibility. The reverse transcriptase (RT) region was amplified from a drug-naive HIV-1 subtype C isolate obtained from a patient enrolled in the Tropism study (BREC: BF088/07) and cloned into a TOPO vector using a TOPO TA cloning kit. The HIV-1 RT mutations (K65R, A62V, S68D, Y115F, K65R+A62V, K65R+S68D, K65R+S68G, K65R+S68N, and K65R+Y115F) were introduced into the TOPO+RTsubC recombinant using the Quikchange lightning Multi site-directed mutagenesis kit. Next, recombinant viruses were created by co-transfection of the mutant RT amplicons and a pNL4-3-deleted-reverse transcriptase (RT) (pNL43ΔRT) backbone into GXR cells by electroporation. The replication capacity of the mutant viruses was assessed using a replication method that utilized a green fluorescent protein (GFP) reporter cell line and flow cytometry. We evaluated the replication capacity using the exponential growth curve function in Excel to determine the percentage GFP-expressing cells between days 2 and 6. The impact of the mutant viruses on susceptibility to TDF was performed in a luciferase-based assay. The 50% inhibitory concentration (IC50) was calculated using Graph Pad Prism. Drug susceptibility was expressed as the fold change in IC50 of mutant virus compared with the wild type virus. Of the 5 TDF- selected mutants analysed: A62V, K65R, and Y115F mutants display a reduction in replicative fitness whereas, S68D and K65R+S68N showed high viral fitness. Interestingly, the TDF- selected resistance mutations we analysed, showed high susceptibility (A62V, S68D, and Y115F) and reduced susceptibility (K65R and K65R+S68N) to TDF. Our findings support the hypothesis that TDF- selected mutations only confer reduced susceptibility to TDF. Hence, further study is needed on various combinations of TDF-selected resistance mutations to further solidify this claim.
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    Genetic and functional diversity of central nervous system (CNS) derived Human Immunodeficiency Virus type 1 (HIV-1) tat from Tuberculous Meningitis (TBM) patients.
    (2018) Ramruthan, Jenine.; Madlala, Paradise Zamokuhle.
    INTRODUCTION Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (tat) is a regulatory gene that encodes the transactivator of transcription Tat protein. The Tat effectively increases the activity of the HIV-1 5’ long terminal repeat (5’ LTR) viral promoter to transcribe viral genes. The tat gene has two exons; the first 72 amino acids of Tat are encoded by the first exon, whilst amino acids 73 – 101 are encoded by the second exon. Exon 1 of Tat is sufficient for the transactivation of the 5’ LTR and therefore was the focus of this study. The Tat encoded by exon 1 consists of 5 functional domains these include: the acidic domain (domain I) comprising amino acids 1 – 21, this is a proline rich domain with high sequence variation; the cysteine-rich domain (domain II) comprising amino acids 22–37, is composed of 6 well conserved cysteine residues in subtype C Tat proteins, a mutation at any of the 6 cysteine residue results in loss of Tat activity; the core domain (domain III) comprising amino acids 38–48, is made of a hydrophobic motif and is relatively well conserved. Together, the first 48 amino acids of Tat comprising domains I – III, allow for the transactivation activity of Tat responsible for enhancing viral gene transcription. The basic domain (domain IV) is an RNA-binding domain made up of amino acids 49 – 57 which allows for the binding ability of Tat to the TAR loop structure of the 5’ LTR. Lastly the glutamine-rich domain (domain V) comprised of amino acids 58 – 72, also concentrated with basic amino acids, has the highest sequence variation in Tat. During the early stages of infection, HIV-1 enters the central nervous system (CNS) and replicates at marginal levels compared to high viral replication in the periphery. Yet, there is higher HIV-1 RNA levels in the in the cerebrospinal fluid (CSF) compared to plasma of tuberculosis meningitis (TBM) co-infected patients. However, the mechanisms driving the higher viral replication in the CNS of TBM patients are not well understood. Therefore, the major aim of this study is to characterise genetic and functional diversity of CNS and plasma derived Tat from TBM coinfected patients. We hypothesized that TBM coinfected patients will display genetically distinct HIV-1 tat variants in the CSF as a driver or consequence of higher viral replication in this compartment compared to plasma. METHODS Viral RNA was extracted from matched CSF and plasma samples obtained from 20 HIV- 1 chronically infected patients (17 TBM and 3 non-TBM) using the QIAmp viral RNA Mini kit (Qiagen Inc., Valencia, CA, USA). Extracted viral RNA was reverse transcribed into viral DNA using SuperScript IV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and amplified using two rounds of (nested) PCR with the Platinum ® Taq DNA Polymerase High Fidelity PCR kit (Thermo Fisher Scientific, Boston, MA, USA). Genetic diversity of plasma and CSF derived isolates was assessed in 19 patients (16 TBM and 3 non-TBM) by sequencing, neighbour-joining phylogenetic analysis and both interpatien and intrapatient diversity analysis. The Tat sequences with previously reported mutations that affect Tat function were selected for downstream functional assays. Twelve tat PCR amplicons were cloned into a pTargeT™ expression plasmid (Promega Corporation, Madison, WI). Recombinant pTargeT clones containing patient derived HIV-1 tat was propagated using the QIAfilter Plasmid Maxi Kit (Qiagen Inc., Valencia, CA, USA) to transfect the TZM-bl mammalian cells, which contains the luciferase gene luc under the control of the LTR promoter. A luciferase assay was done to measure the relative luminescence for each Tat mutant and this was correlated to markers of disease progression such as viral load. RESULTS The phylogenetic data from our study show that sequences from plasma and CSF derived HIV-1 tat clustered closely per patient. Genetic variation was seen as varying branch lengths between patient clusters. However, our data do not show significant nucleotide differences between the plasma and CSF tat sequences with a p-distance of 0.059 and 0.062 respectively (p = ns). Additionally, our data revealed that the amino acid sequences were the same between the CSF and plasma compartments, except in 5% of patients that showed differences in positions that were not previously reported to affect Tat activity. However, Tat diversity was observed to occur in all 5 domains of the first 72 amino acids of Tat namely: V4I, P21A, K24S, H29R, S31C, S46Y, R52W, S57R, P59S and D64G. The functional data from our study revealed that most patient derived Tat mutations occurred in combination with other previously reported mutations. Interestingly, Tat mutations that occurred together with P21A in five different patients showed a showed strong positive correlation with CSF viral load in the CNS (p = 0.003; r = 0.98). CONCLUSION We reject our hypothesis that CNS specific Tat mutations were responsible for the high viral load in the CNS of patients who have TBM, as the allele frequencies of reported amino acid substitutions were represented in equal proportions within plasma and CSF derived Tat variants. Furthermore, our functional data shows that majority of all Tat variants from the TBM group had a reduced capacity to transactivate the 5’ LTR. Whilst we cannot confirm that Tat is responsible for the higher viral replication seen in the CNS of TBM coinfected patients, our data demonstrate that all Tat variants with a P21Anmutation significantly correlates to viral replication in the CNS. Future studies should perform site directed mutagenesis to determine the exact mutations that mediate LTR activity.
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    Functional characterization of human immunodeficiency virus type 1 (HIV-1) subtype C transmitted/founder (T/F) viruses long terminal repeat (LTR) variants and association with disease outcome.
    (2020) Naicker, Shamara.; Madlala, Paradise Zamokuhle.
    Background: The persistence of latent viral reservoirs is a major roadblock to human immunodeficiency virus type 1 (HIV-1) cure development. Latent reservoirs harbour transcriptionally silent yet replication competent proviruses. However, the molecular mechanisms that govern HIV-1 latency at the transcriptional level is unknown. Therefore, we hypothesize that HIV-1 subtype C (HIV-1C) transmitted/founder (T/F) 5’ long terminal repeat (LTR) genetic variation may affect disease outcome. Methods: To address this, viral RNA was extracted from plasma samples obtained from 25 HIV-1 infected patients from the HPP and FRESH acute infection cohorts (QIAamp® Viral RNA Mini Kit, Qiagen, Hilden, Germany). Viral RNA was reverse transcribed to DNA using SuperScript™ III One Step RT-PCR System with Platinum™ Taq DNA Polymerase (Invitrogen, Massachusetts, United States). Nested PCR was performed (Platinum® Taq DNA Polymerase High Fidelity PCR Kit (Invitrogen, Massachusetts, United States) and PCR products cloned into the pGL3 Basic plasmid. LTR/pGL3 recombinant plasmids were sequenced using BigDye Terminator v3.1 Sequencing Kit (Invitrogen, Massachusetts, United States) to confirm correct sequences. The LTR-pGl3 recombinant plasmids were transfected into Jurkat cells alone or co-transfected with either consensus (wild type) subtype C Tat (conTat) or autologous tat (autoTat) to determine the effect of LTR genetic variation on expression of a luciferase reporter gene. Results: Interestingly, our data demonstrate that basal transcription activity significantly differs between LTR variants. Specifically, patients harbouring the Sp1 III: G2A mutation demonstrated significantly lower transcription compared to the wild type LTR. Although conTat co-transfection increased the LTR activity for most of the LTR variants, the T/F virus LTR containing the TATA box mutation (TATAA TAAAA) in combination with other LTR mutations was not induced. Interestingly, the transactivation activity of the autologous Tat was variable among patients. Specifically, the TATA box variant was marginally induced. Lastly, we observed that the majority of LTR variants were more responsive to stimulation by PMA as compared to TNF-α, SAHA and prostratin. Interestingly, our data demonstrate that autologous tat induced transcription positively correlated with viral load at transmission (p=0.0134, r=0.66) and at one-year post infection but was not significant (p=0.3905, r=0.26). Conclusion: These data suggest that the TATAA TAAAA mutation in combination with other LTR mutations may reduce transcription activity. Taken together our data suggest that HIV-1 subtype C T/F viruses LTR genetic variation may modulate viral gene transcription and impact disease outcome.
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    Sequence analysis of an HIV-1 subtype C acutely infected cohort from Durban, South Africa.
    (2018) Carries, Stanley.; Gordon, Michelle Lucille.
    The Human Immunodeficiency Virus is a global public health concern. The Joint United Nations Programme on HIV/AIDS estimated that 36.9 million people were infected with HIV globally at the end of 2017. Almost 20% of these resided in South Africa, making this the highest global HIV burden held by any one country. It is thus important that HIV infection be detected early as this may have important implications in the control of the pandemic. The early recognition of acute HIV infection could present early treatment options that could alter the natural history of the disease, or even eliminate infection. Detecting acute infection early could also provide a unique opportunity to understand HIV transmission and pathogenesis, including early host-virus interactions. In the present study, blood samples were collected from 18-23 year old HIV-1 subtype C acutely infected women from Umlazi Township in KwaZulu-Natal, South Africa, that had participated in a study called Females Rising through Education, Support and Health (FRESH). Eleven blood samples from this cohort, collected within 24 hours of onset of plasma viremia, were used for this study. The aim of the present research was to identify sites within pol that were experiencing positive selective pressure and the likely implications of these mutations on viral functional domains and host cytotoxic T-lymphocyte (CTL) epitopes. The study also sort to observe the loss of drug resistant mutations (DRM) in the viral sequences of participants who had multiple timepoints and to correlate mutation loss to structural changes. Datamonkey and Phylogenetic Analysis by Maximum Likelihood (PAML) were used to detect positively selected sites. Putative functional domains were detected using Prosite and CTL epitopes were identified using the Los Alamos Molecular Immunology Database. Ancestral reconstruction was performed using PAML and Bayesian Evolutionary Analysis by Sampling Trees (BEAST) was used to calculate the time to the most recent common ancestor. Altogether 16 unique positively selected sites were identified in this cohort. Putative functional domains were highly conserved in protease, while positive mutations in reverse transcriptase resulted in either a loss of functional domains in conserved regions or in the gain of functional sites in non-conserved regions. Owing to the important role that protease plays in viral maturation and infectivity, mutations within these conserved regions could possibly lead to defective viral particles with reduced viral infectivity. The K103N in reverse transcriptase, observed in one participant, was the only DRM inherited from its common ancestor. The major limitation of this study was the small sample size.
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    The influence of HIV-1 genomic target region selection and sequence length on the accuracy of inferred phylogenies and clustering outcomes.
    (2017) Sibisi, Zandile.; De Oliveira, Tulio De Paiva Nazareth Andrade.
    To improve the methodology of HIV-1 cluster analysis, we addressed how analysis of HIV-1 clustering is associated with parameters that can affect the outcome of viral clustering. The extent of HIV clustering, tree certainty, subtype diversity ratio (SDR), subtype diversity variance (SDV) and Shimodaira-Hasegawa (SH)-like support values were compared between 2881 HIV-1 full genome sequences and sub-genomic regions of which 2567 were retrieved from the LANL HIV Database and 314 were sequenced from blood samples from a cohort in KwaZulu-Natal. Sliding window analysis was based on 99 windows of 1000 bp, 45 windows of 2000 bp and 27 windows of 3000 bp. Clusters were enumerated for each window sequence length, and the optimal sequence length for cluster identification was probed. Potential associations between the extent of HIV clustering and sequence length were also evaluated. The phylogeny based on the full-genome sequences showed the best tree accuracy; it ranked highest with regards to both tree certainty and SH-like support. Product 4, a region associated with env, had the best tree accuracy among the sub-genomic regions. Among the HIV-1 structural genes, env had the best tree certainty, SH-like support, SDR score and the best SDV score overall. The hierarchy of cluster phylotype enumeration mirrored the tree accuracy analysis, with the full genome phylogeny showing the highest extent of clustering, and the product 4 region being second best. Among the structural genes, the highest number of phylotypes was enumerated from the pol phylogeny, followed by env. The extent of HIV-1 clustering was slightly higher for sliding windows of 3 000 bp than 2000 bp and 1000 bp, thus 3000 bp was found to be the optimal length for phylogenetic cluster analysis. We found a moderate association between the length of sequences used and proportion of HIV sequences in clusters; the influence of viral sequence length may have been diminished by the substantial number of taxa. Full-genome sequences could provide the most informative HIV cluster analysis. Selected sub-genomic regions with the best combination of high extent of HIV clustering and high tree accuracy, such as env, could also be considered as a second choice.
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    Identification of mutational pathways to tenofovir resistance in subtype C isolates using a Bayesian Network.
    (2016) Maphumulo, Ntombikhona F.; Gordon, Michelle Lucille.
    No abstract.
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    Prevalence of minority HIV-1 drug resistant quasi-species in children patients at virologic failure in a rural KwaZulu-Natal cohort.
    (2016) Mthiyane, Hloniphile Ruth.; Danaviah, Sivapragashini.; De Oliveira, Tulio De Paiva Nazareth Andrade.
    Abstract available in PDF file.
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    In vitro testing of the predicted viral fitness landscape for the HIV-1 Nef protein.
    (2015) Rajkoomar, Erasha.; Mann, Jaclyn Kelly.; Ndung'u, Peter Thumbi.
    Abstract available in PDF file.
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    Characterizing protease inhibitor failure in HIV-1 subtype C, using ultra deep pyro-sequencing and homology modelling.
    (2015) Singh, Avashna.; Gordon, Michelle Lucille.
    The extensive roll-out of combination antiretroviral therapy (cART) has significantly improved the life expectancy for HIV-1 infected individuals in South Africa. Despite the inclusion of potent Protease Inhibitors (PIs) in second-line cART, many patients still fail treatment. The extent to which PI resistance contributes to treatment failure is not completely clear. In this study we report the prevalence of PI mutations amongst individuals failing a second-line Lopinavir (LPV/r) inclusive regimen. We also investigated if low frequency minority variants at LPV/r failure influence Darunavir (DRV/r) failure in a subset of patients using Ultra Deep Pyro-sequencing. Structural changes at DRV/r failure were investigated using Homology modeling. Models were constructed using the SWISS-MODEL webserver and visualized in Chimera v1.8.1. Darunavir was docked into each of the structures using the CLC Drug Discovery workbench ™ and Molecular Dynamics simulations was performed using the AMBER12 package. Our study reports a 24% prevalence of PI resistance mutations, slightly higher than other studies. A distinct pattern of PI resistance mutations was found: M46I+I54V+L76V+V82A, present in 13/37 (35%) of those with PI mutations. Darunavir resistance mutations detected following DRV/r failure included V11I, V32I, L33F and I54L. There were no minority variants detected at LPV/r failure that could have influenced DRV/r failure. Distinct conformational changes were evident in both the LPV/r-resistant and DRV/r-resistant model. Molecular docking showed that the inhibitory potency of DRV was lowered in the mutated DRV/r-resistant model and to a lesser extent in the LPV/r-resistant model. These results show that resistance mutations greatly contribute to DRV drug susceptibility. This work will contribute to the clinical management of patients failing treatment and will also assist in the design of new and improved ARVs.