Virology

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Acquired and transmitted drug resistance in HIV-1 subtype C : implications of novel mutations on replication capacity, cleavage and drug susceptibility.
(2015) Singh, Urisha.; Gordon, Michelle Lucille.
Introduction Large scale roll-out of combination antiretroviral therapy (cART) has been successful in improving the quality of life of HIV-1 infected individuals in South Africa (SA). However the development and transmission of drug resistance threatens the future success and longevity of cART in the country. Studies have shown that resistance to Protease inhibitors (PI’s), in the absence of mutations in Protease (PR), is increasing in SA. Whilst some studies attribute this to poor treatment adherence, others have shown that mutations in Gag contribute to PI resistance. The majority of these studies however have been conducted on HIV-1 subtype B, despite HIV-1 subtype C being the most prevalent subtype globally. Given that Gag is highly polymorphic between subtypes, studies focusing on HIV-1 subtype C are required. Despite the high rate of virologic failure of patients on PI inclusive treatment regimens, no transmitted drug resistance (TDR) studies have identified PI associated TDR mutations. This could be due to the high fitness cost associated with PR mutations which would result in rapid reversion or low frequency of mutations within the viral quasispecies. Most TDR studies in SA, as in other resource limited settings, have used recently infected cohorts to measure TDR. It is however unlikely that rapidly reverting mutations would be detected in recent infection. Furthermore, these studies have all used Sanger sequencing which only detects mutations at frequencies >15-20%. With recent studies showing that low frequency mutations present at frequencies as low as 1% impact treatment outcomes, the elucidation of these mutations using deep-sequencing techniques is necessary. For a true measure of TDR, studies employing acute infection cohorts and deep-sequencing techniques are required. The current study aimed to identify mutations in Gag-Protease associated with PI resistance/exposure, and to determine their impact on replication capacity and drug susceptibility. The prevalence of low frequency TDR mutations in an HIV-1 subtype C acute infection cohort was also investigated. Methods A cohort of 80 HIV-1 subtype C infected participants failing a PI inclusive treatment regimen (i.e. PCS cohort) from 2009–2013 in Durban, South Africa was used to assess the role of Gag in PI resistance. Gag mutations were divided into three groups: PI exposure associated Gag mutations; resistance associated Gag mutations (rGag) and novel Gag mutations (nGag). Frequencies of each of these mutations were compared amongst: 80 PCS cohort sequences, 2,481 HIV-1 subtype B treatment naïve sequences, 954 HIV-1 subtype C treatment naïve sequences and 54 HIV-1 subtype C sequences from acutely infected individuals, in order to identify PI associated mutations and natural polymorphisms. Next, recombinant viruses for all 80 participants were generated by co-transfection of a CEM derived T-cell line (i.e. GXR cells) with an NL43-deleted-gag-protease (NL43Δgag-protease) backbone and patient derived Gag-Protease amplicons. Thereafter, the replication capacity of each virus was assessed using a replication assay that employed a green fluorescent protein reporter cell line and flow cytometry. Associations between replication capacity and Gag-Protease mutations were established. Eighteen viruses with mutations of interest were then selected for use in drug susceptibility assays, where the impact of mutations on susceptibility to lopinavir (LPV) and darunavir (DRV) was assessed in a luciferase based assay. Lastly, the impact of novel Gag mutations on replication capacity and drug susceptibility was validated by generating site-directed mutant viruses with mutations of interest and using these mutant viruses in replication capacity and drug susceptibility assays. Furthermore the cleavage profile of each site-directed mutant virus was established by western blotting. Samples available from 47 HIV-1 subtype C acutely infected individuals collected from 2007-2014 in Durban, South Africa, was used to assess low frequency TDR mutations in HIV-1 subtype C acute infection. Firstly the RT and PR region of each virus was genotyped using the Viroseq HIV-1 genotyping system in order to identify the prevalence of TDR in the cohort. Thereafter 14 participant samples were selected, based on the availability of plasma at one week after onset of plasma viremia (OPV), for sequencing by ultra-deep pyrosequencing (UDPS). This served to identify low frequency mutations. Comparisons in TDR prevalence was made between Sanger sequencing and UDPS. Thereafter, the impact of low frequency TDR mutations on treatment outcomes was assessed by comparing time to virologic suppression for two participants with low frequency mutations to that of four participants without low frequency mutations. Results Protease resistance associated mutations (RAMs) occurred in 34/80 (42.5%) participants, whilst Gag mutations associated with PI resistance in subtype B were detected in 67/80 (84%) participants. Overall, 12 Gag mutations associated with PI exposure (i.e. E12K, V35I, G62R, V370A/M, S373P/Q/T, A374P, T375N, I376V, G381S, I389T, I401T and H219Q), eight rGag mutations (i.e. R76K, Y79F, V128I, A431V, K436R, L449F, R452K and P453L) and four nGag mutations (i.e. Q69K, S111C/I, T239A/S and I256V) were identified in the PCS cohort. The E12K, V370A/M, T375N, G381S, R76K and Y79F mutations all occurred as natural polymorphism in HIV-1 subtype C. The A431V, K436R, L449F, R452K, P453L, Q69K, S111C/I, T239A/S and I256V mutations were all associated with PI resistance/exposure. Interestingly all viruses with PR RAMs harboured rGag and nGag mutations, however rGag and nGag mutations were also found to occur without PR RAMs. Protease RAMs were associated with significantly reduced replication capacity. The K335R and A431V mutations were the only Gag mutations associated with significantly reduced replication capacity. Viruses with PR RAMs were associated with significantly reduced susceptibility to LPV (>15 FC in IC50) and DRV (>6 FC in IC50). Furthermore, the following combinations of rGag and nGag mutations were found to confer reduced susceptibility to LPV and DRV in the absence of PR RAMs: R76K+Y79F+K436R+L449P+I256V (5.2 fold increase in IC50 for DRV), R76K+R453L (23.88 fold increase in IC50 for LPV and a 6.73 fold increase in IC50 for DRV) and R76K+K436R+Q69K+S111C (7.40 fold increase in IC50 for LPV). Analysis of recombinant viruses showed that the Q69K nGag mutation rescued replication capacity of all viruses harbouring A431V+PR RAMs. This was validated by SDM, where Q69K rescued the replication capacity of site-directed mutant viruses harbouring A431V+V82A. The Q69K mutation was also associated with increasing polyprotein cleavage when found in conjunction with A431V+V82A. With regards to TDR, we demonstrated a prevalence of 57% of TDR mutations with UDPS and 2.2% with Sanger sequencing. Sanger sequencing identified the K103N non-nucleoside reverse transcriptase inhibitor (NNRTI)-associated TDR mutation. In addition to K103N (frequency: >99%), the following low frequency mutations were detected by UDPS: the K65R (1-1.5%) and D67N (3.88%) nucleotide reverse transcriptase inhibitor (NRTI)-associated TDR mutations, the F53L (17.6%) and M46L (6.3%) Protease inhibitor (PI)-associated TDR mutations, and the T97A (2.90%) integrase strand transfer inhibitor (InSTI)-associated TDR mutations. Participants with low frequency TDR mutations took 40 days longer to achieve viral suppression than participants without low frequency TDR mutations, when placed on fixed dose combination antiretroviral therapy.
Allele-specific polymerase chain reaction (ASPCR) to detect resistance mutations in minor variants of HIV-1 subtype C in patients failing highly active antiretroviral therapy (HAART).
(2014) Maharaj, Shevani.; Gordon, Michelle Lucille.
The World Health Organization (WHO) has recommended Tenofovir disoproxil fumarate (TDF) as one of the preferred first-line antiretrovirals (ARVs). TDF and Abacavir (ABC) were introduced into the South African National Antiretroviral Treatment Guidelines in 2010. However, exposure to TDF and ABC can result in the development of the K65R and L74V resistance mutations, respectively. The K65R mutation occurs preferably in subtype C viruses, due to the unique polymorphisms found at codons 64 and 65 (which are not present in subtype B). This is a cause for concern in South Africa, where subtype C is the most common HIV-1 subtype. In addition, these mutations may be present in the minor viral population (i.e <20% of the viral population) and it has been shown that the presence of a resistance mutation in a frequency as low as <0.5% may be associated with an increase in the risk of virological failure. This study investigated the prevalence of K65R and L74V in the minor viral population, using Allele-specific PCR (ASPCR), in a cohort of subtype C infected patients that failed their first-line treatment regimen that did not include TDF or ABC. RNA was extracted from stored plasma samples from a subset of the South African Resistance Cohort Study (SARCS) and the pol region was reverse transcribed and amplified using a one-step RT-PCR kit (Invitrogen; California, USA). For both the K65R and L74V mutations, ASPCR was performed using specific and non-specific primers. A specific and non-specific standard curve was optimised for each mutation (using a mutant plasmid control) and these standard curves were used to perform an absolute quantification. Subsequently, the percentage of each mutation (in each sample) was calculated by dividing the quantity of mutant sequences in the sample by the quantity of total viral sequences in the sample and multiplying this ratio by 100. The Limit of Detection (LOD) of the K65R ASPCR was 0.72%. Of the 84 patients that were assayed, the K65R mutation was detected in 7 (8.33%) of the patients. Five of the 7 samples were detected above 1% (i.e 3 were approximately 2%, 1 was 9.48% and 1 was 100%) and 2 were detected below 1% (i.e 1 was 0.88% and the other was 0.93%). The limit of detection for the L74V ASPCR was 0.013%.We found the L74V mutation to be prevalent in 9 (10.7%) of 84 patients. In 4 of the 9 patients, the L74V mutation was found in ≥1% of the viral population (viz. 2.82%, 10.10%, 12.02% and 18.22%) and in the other 5 patients, the L74V mutation was detected in <1% of the viral population (2 were between 0.5% and1%, while 3 were detected between 0.013% and0.5%). In this study, ASPCR detected additional K65R and L74V mutations in the minor viral population of TDF and ABC-inexperienced patients that were missed by standard genotyping. These minorityK65R mutations could contribute to treatment failure in these patients when switched to TDF or ABC-containing ARV regimens. ASPCR is a useful tool for screening for minority mutations before starting or switching regimens.
The application of DNA hybridisation methods to a determination of the association of hepatitis B virus with cirrhosis and hepatoma.
(1987) Nair, Shamila.; Windsor, Isobel M.; Van den Ende, Jan.
Autopsy liver material from patients having died of chronic liver disease, cirrhosis, hepatocellular carcinoma (HCC) and causes unrelated to liver diseases was examined by dot blot hybridisation for the presence of HBV DNA. The results indicate that of the patients with chronic liver disease 6/9 were positive for HBV DNA in the liver tissue; of the patients with HCC 3/4 were positive for HBV DNA; of the patients with cirrhosis 4/4 showed the presence of HBV DNA in the liver. Thus by this technique 13/17 (76%) of these patients, all of whom were HBsAg positive, were shown to have HBV DNA present in liver tissue. However, autopsy liver samples were found to be unsuitable for Southern blot hybridisation. Biopsy liver/tumour tissue was examined for the presence of integrated or non-integrated HBV DNA by Southern blot analysis using the enzymes Eco R1 and Hind 111. 5/5 patients who were both HBsAg and HBeAg positive had extrachromosomal HBV DNA and 2/5 also showed the presence of integrated HBV DNA. 3/4 patients who were HBsAg positive and HBeAg negative had extrachromosomal HBV DNA and all three also had integrated HBV DNA. One control patient was negative for both markers and also for Southern blot hybridisation with the HBV DNA probe. These results support the hypothesis that HBV is a factor in the development of HCC, and indicate that the dot blot hybridisation method would be suitable for routine evaluation of patients with chronic liver disease or cirrhosis.
Biochemical characterization of highly mutated South African HIV-1 subtype C protease.
(2021) Eche, Simeon.; Gordon, Michelle Lucille.
Understanding the underlying molecular mechanism of HIV-1 protease (PR) inhibition by HIV-1 protease inhibitors (PIs) is essential to gain mechanistic insight into the evolution of resistance to HIV-1 PIs. HIV-1 PIs have improved patient care management, but the accumulation of drug resistance mutations in the HIV-1 PR gene diminishes their inhibitory capacity. The current study investigated the kinetic and structural characteristics of highly mutated South African HIV-1 subtype C PR from clinical isolates obtained from individuals failing a lopinavir (LPV) inclusive regimen at the point of switch to darunavir (DRV) based therapy. In this study, enzyme activity and inhibition assays were used to determine the biochemical fitness of HIV-1 PR variants and the inhibitory constants of HIV-1 PIs for drug-resistant HIV-1 subtype C proteases. The mechanistic insight into the impact of the accumulated drug resistance mutations on the HIV-1 PR structure and its interaction with LPV and DRV was obtained using fluorescence spectroscopy and molecular dynamic simulation. The study showed that the unfavorable binding landscape caused by the accumulation of drug-resistance mutations resulting from LPV associated drug pressure would shape the outcome of DRV-based therapy after a switch in the treatment regimen. This is related to the distortion of the HIV-1 PR structure associated with increased solvent exposure and instability of the HIV-1 PR dimer caused by these mutations leading to a shorter lifetime of the enzyme-inhibitor complex. Analysis of the binding kinetics of LPV and DRV with the HIV-1 PR variants showed that the drug resistance mutations caused an imbalance between the association and dissociation rate constants favoring a fast dissociation rate. The latter resulted in a reduced inhibitor residence time. Our findings showed that LPV had a longer residence time than DRV when bound to the HIV-1 PR variants; this shows LPV can be a suitable platform for developing newer HIV-1 PIs with a longer residence time. However, the enzyme inhibition mechanism shows both LPV and DRV act via a two-step tight-binding mixed inhibition mechanism, suggesting the existence of a second binding site on HIV-1 PR for these inhibitors. The information provided in this thesis adds to existing knowledge about HIV-1 PI drug resistance and for the design of novel HIV-1 PIs with the potential to evade drug resistance mutations.
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.
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.
Clinical and epidemiological aspects of HIV and Hepatitis C virus co-infection in KwaZulu-Natal province of South Africa.
(2008) Parboosing, Raveen.; Lalloo, Umesh Gangaram.
HIV is known to affect the epidemiology, transmission, pathogenesis and natural history of HCV infection whilst studies on the effects of HCV on HIV have shown conflicting results and are confounded by the influence of intravenous drug use and anti-retroviral therapy. This study was conducted in KwaZulu-Natal Province in South Africa where HIV is predominantly a sexually transmitted infection. Intravenous drug use is rare in this region and the study population was naive to anti-retroviral therapy. For this study, specimens from selected sentinel sites submitted to a central laboratory for routine HIV testing were screened for anti-HCV IgG antibodies. HIV positive HCV-positive patients were compared to HIV-positive HCV-negative patients in a subgroup of patients within this cohort in order to determine if HCV sero-prevalence was associated with clinical outcomes in a linked anonymous retrospective chart survey. The prevalence of HCV was 6.4% and that of HIV, 40.2%. There was a significantly higher prevalence of HCV among HIV infected patients as compared to HIV negative patients (13.4% vs. 1.73% respectively). HCV-HIV co-infected patients had significantly increased mortality (8.3 vs. 21%). A significant association was found between HCV serostatus and abnormal urea and creatinine levels. Hepatitis B surface antigen seropo-sitivity was not found to be a confounding factor. This study has found that hepatitis C co-infection is more common in HIV positive individuals and is associated with an increased mortality and renal morbidity.
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.
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.
The development, optimisation and comparison of various virological assays and their uses in antiviral assessment of compounds wih potential anti-HIV activity.
(2009) Singh, Varish.; Parboosing, Raveen.
The development and optimization of anti-viral screening methods are essential to develop newer more effective, treatments against HIV. The XTT method is a widely described method for antiviral screening. Both continuous HIVinfected cells and experimentally infected T-cells have been used in the XTT assay. We compared these methods to screen several plant-derived extracts for cytotoxicity. Several considerations were taken into account when performing these tests (effect of media, solvents and plant enymes). Experiments were performed to investigate these effects. In addition, p24 and viral load quantification were compared as antiviral screening methods. The study showed that several modifications were necessary when performing the XTT assay on plant extracts, due to the effect of media, solvents and plant enymes. The XTT assays and p24 assays performed using experimentally infected cells are far more specific than those using chronically infected cells. The use of viral loads as an antiviral screening method consistently demonstrated the expected efficacy of AZT.
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.
The epidemiology of dual HIV infection in the KwaZulu-Natal Anti-Retroviral Roll-out Programme.
(2007) Naidoo, Anneta Frances.; Parboosing, Raveen.
KwaZulu-Natal has the highest prevalence of HIV in South Africa. The prevalence of dual infection in a normal-risk population in this region is unknown. Dual HIV infection has important implications for diagnosis, treatment response and vaccine development. This cross-sectional study aimed to establish and optimize methods for subtyping and detection of dual infection in KZN. Samples were from chronically-infected patients on ARV treatment within the ARV Rollout Programme, from sites throughout KZN. Subtyping of the samples was performed using HMA. Four samples had indeterminate results by HMA and were then cloned and sequenced. Phylogenetic analysis showed that one of the four samples was a dual infection. This study showed 1/46(2%) samples to be dually infected which suggests that the prevalence of dual infection is low in the sample population. The low prevalence of dual infection reported could be due to the low-risk profile of the sample population. It was concluded that the low prevalence of dual infection is unlikely to have a considerable impact on HIV management.
Functional and clinical consequences of immune-driven sequence variation of gag-protease in HIV-1 subtypes A, C, D and recombinants.
(2016) Kiguoya, Marion Wangui.; Ndung'u, Peter Thumbi.; Mann, Jaclyn Kelly.
Abstract available in PDF file.
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.
Gag-protease driven viral replication capacity among HIV-1 subtypes: Implications for disease progression, epidemic spread and vaccine design.
(2020) Farinre, Omotayo Oluwaremilekun.; Ndung'u, Peter Thumbi.; Mann, Jaclyn Kelly.
The HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. In this study, I tested the hypothesis that the subtypes prevalent in the East African epidemic, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower. Materials and methods: Gag-protease sequencing was performed on plasma samples from 213 and 160 antiretroviralnaïve participants from West and East Africa, respectively. Online bioinformatic tools were used to infer HIV-1 subtypes and recombination patterns. Replication capacities of patientderived gag-protease chimeric viruses from West (n=178) and East (n=114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in viral replication capacity and amino acid variants impacting replication capacity were identified using appropriate statistical methods. Results: Subtypes identified in West Africa were CRF02_AG (65%, n=139), G (7%, n=15), A3 (5%, n=10), other CRFs (12%, n=26), various pure subtypes (9%, n=19) and A1G recombinants (2%, n=4). Subtypes A1 (64%, n=103), D (22%, n=35), AD (11%, n=17) and AC (3%, n=5) were identified in East Africa. Chimeric viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact viral replication capacity. Furthermore, the Gag A83V polymorphism was associated with reduced viral replication capacity in CRF02_AG (median < 0.86). HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with significantly lower viral replication capacity in subtype A infected individuals from East Africa. Conclusion: Overall, the data showed that viruses from West Africa displayed higher replication capacity than those from East Africa, which is consistent with the hypothesis that lower viral replication capacity is associated with higher population prevalence.
GB Virus C / Hepatitis G Virus (GBV-C/HGV) infection in KwaZulu Natal, South Africa : its diagnosis, distribution and molecular epidemiology.
(2003) Sathar, Mahomed Aslam.; York, Denis Francis.
Recently a new Flavivirus, GB Virus C also referred to as Hepatitis G virus (GBV-C/HGV) was identified in humans with indeterminate hepatitis . Whilst in non-African countries this discovery led to an enormous enthusiasm to elucidate an association with liver disease, very little was known about the prevalence and pathogenicity of GBV-C/HGV infection in KwaZulu Natal, South Africa, where Hepatitis B Virus (HBV) infection is endemic and infection with the Human immunodeficiency virus (HIV) is a catastropic health problem. Sera from patients with liver disease (chronic liver disease [n = 98]; alcoholic liver disease [n = 50]); high risk groups (haemodialysis patients [n = 70]; HIV positive mothers and their babies [n = 75]) and control groups (alcoholics without liver disease [n = 35] and blood donors from the four racial groups [n = 232]) were screened for GBV-C/HGV RNA and Anti-E2 antibodies by reverse transcription polymerase chain reaction (RT-PCR) and an enzyme linked immunosorbent assay (ELISA), respectively. Overall 43.9% (43/98) of patients with chronic liver disease; 60 % (30/50) of patients with alcoholic liver disease; 47.1% (33/70) of haemodialysis patients; 60% (21/35) of alcoholics without liver disease and 31.9% (74/232) of blood donors (Africans] 44/76; 5.9%); Asians (5/52; 9.6%); Whites (15/49; 30.6%) and "Coloureds" [mixed origin] (9/54; 16.6%)]) were exposed to GBV-C/HGV infection as determined by the detection of Anti-E2 &/or RNA in serum. There was a significant difference in the prevalence of GBV-C/HGV infection (RNA &/or anti E2) between African blood donors and the other racial groups (p < 0.001), between blood donors and haemodialysis patients (p = 0.02) and or patients with chronic liver disease (p =0.04). There was no significant difference in the prevalence of GBV-C/HGV between African blood donors (45/76, 59.2%) and alcoholics with and without liver disease (30/50, 60% and 21/35, 60%, respectively). Anti-E2 antibodies and GBV-C/HGV RNA were almost mutually exclusive. GBV-C/HGV infected dialysis patients tended to have had more transfusions (p = 0.03) and had a longer duration of dialysis than non infected patients, indicating that the majority of patients on maintenance haemodialysis acquire their GBV-C/HGV infection through the transfusions they receive. There was no evidence for in utero and/or intrapartum transmission of GBV-C/HGY. However, there is some mother-to-infant transmission of GBV-C/HGV, though it is very probable that in KZN GBV-C/HGV is transmitted by as yet undefined non-parenteral routes. Sequence and phylogenetic analysis of the 5' non-coding region (5' NCR) and E2 gene segments of the GBV-C/HGV genome identified an additional "genotype" (Group 5) of GBV-C/HGV that is distinct from all other known GBV-C/HGV sequences (Groups 1-4). Although there is a high prevalence of Group 5 GBV-C/HGV isolates in KZN, there was no significant difference in liver biochemistry between GBV-C/HGV infected and noninfected patients with liver disease or between blood donors in each of the four racial groups. There was no significant differences in CD4 (461.12 ± 163.28 vs 478.42 ± 181.22) and CD8 (680.83 ± 320.36 vs 862.52 ± 354.48) absolute cell counts between HIV positive patients co-infected with GBV-C/HGV and those not infected with GBV-C/HGV, respectively. However, significantly higher relative CD3 [80.0 ± 4.17% vs 70.99 ± 19.79%] (p = 0.015), gamma delta T cells (yLT) [3.22± 1.30% vs 2.15 ± 29.12%] (p = 0.052) and lower CD 30 [35.45 ± 17.86% vs 50.59 ± 9.20%] (p = 0.041) status were observed in GBV-C/HGV positive compared to GBV-C/HGV negative HIV infected patients, respectively. Although there is a high prevalence of novel Group isolates of GBV-C/HGV in KZN, the lack of elevated liver enzymes and clinical hepatitis in blood donors and haemodialysis patients suggests that GBV-C/HGV is not associated with liver disease. HBV and not GBV-C/HGV modifies the course of alcoholic liver disease. The relatively higher number of CD3 cells and increased yLT expression, together with a decrease in CD 30 cells tends to suggest an association with protection and or delayed progression of HIV disease in GBV-C/HGV infected patients. Whilst GBV-C/HGV is not associated with liver disease, it may be an important commensal in HIV infected patients.
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.
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.
Genotypic and phenotypic characterization of HIV-1 from cerebrospinal fluid and blood compartments in patients with cryptococcal meningitis.
(2017) Sojane, Katlego.; Ndung'u, Peter Thumbi.
The combination of HIV and cryptococcal meningitis (CM) is a major cause of morbidity and mortality in sub-Saharan Africa. The phylogenetic relatedness of HIV-1 subtype C (HIV-1C) variants in peripheral blood and the central nervous system (CNS) compartments of individuals with CM is unknown. Additionally, the major and alternative coreceptor usage of HIV-1C in those compartments are not clear and these have implications for the pathogenesis of the virus, and the use of coreceptor blocking therapies. For genotypic studies, 16 antiretroviral therapy naïve individuals with CM were randomly selected. We conducted single-genome, or bulk PCR amplification and sequencing of full-length HIV-1 env genes from plasma and/or cerebrospinal fluid (CSF) of the participants. Additionally, we estimated the prevalence of CXCR4-using variants in our cohort using coreceptor usage prediction algorithms (CPAs). Next, we evaluated the usage of CCR3, CCR5 and CXCR4 expressed on NP2/U87-CD4 cells by HIV-1C Envs derived from the plasma and/or CSF of 14 of the 16 participants. CCR3 alone, or in combination with the other receptors. Overall, our results have improved the understanding of HIV-1C pathogenesis in the peripheral blood and CNS compartments of individuals with end-stage infection and CM, and provides clinically relevant information for therapies including coreceptor antagonists in this setting.
HIV cell-to-cell spread leads to a differential transcriptional response and slows evolution of drug resistance relative to cell-free infection.
(2020) Hunter, Jessica Rose.; Sigal, Alexander.
HIV transmits between hosts but also transmits between cells in the same host. How this latter, cellular transmission occurs has been the subject of extensive study. Yet why HIV transmits between cells using two different infection modes: cell-to-cell spread and cell-free infection, is not clearly understood. This is because cell-to-cell spread is a more efficient mode of infection, where the virus is able to be successfully transmitted between cells despite natural inhibitors such as antibodies. Here, using in vitro experimentation, I have determined some of the implications of cell-to-cell spread of HIV for cell death, evolution, and inflammation. I have discovered potential costs to this infection mode, such as increased cell death, slowed evolution of resistance, and an increased interferon response which may interfere with viral replication. Hence, costs associated with cell-to-cell spread may prevent it being the dominant infection mode in cellular transmission.

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