Masters Degrees (Virology)

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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.
Molecular diagnosis and typing of HTLV-I in KwaZulu-Natal.
(1998) Tarin, Michelle Lucille.; York, Denis Francis.; Bhigjee, Ahmed Iqbal.
Two areas of the HTLV-I genome were targeted for an in-house molecular diagnostic test, namely the pol and env regions. The pol primers proved the most sensitive (100%)and specific (100%). Amplification using the env primer pair was not reproducible, and was not pursued further. The AmpliSensor assay (Acugen Systems, Lowell, MA) was also tested. The assay was very specific, but not as sensitive as our in-house PCR. To investigate the predominant HTLV-I subtype in the region, a 1535 by env gene was isolated from peripheral blood obtained from five local HTLV-I seropositive patients. Four of the patients presented with HAM/TSP, and the fifth presented with a skin disease. Nucleotide sequencing of the amplified products revealed the local strains to be very conserved, differing by 0.1% to 0.9% among themselves. No apparent difference was noted for the two clinical manifestations. Phylogenetic analysis was performed using repesentative strains from around the world. The local strains clearly fell within the cosmopolitan subtype. The local strains were most closely related to the North American strains suggesting an unexpected link between the two countries.
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.
An investigation into the serological and molecular diagnosis of Jaagsiekte Sheep Retrovirus (JSRV)
(2005) Padayachi, Nagavelli.; York, Denis Francis.
The Jaagsiekte Sheep Retrovirus (JSRV), an exogenous type B/D-retrovirus with about 10-15 endogenous counterparts in all normal sheep genomes, causes Jaagsiekte (JS) or ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer of sheep. This sheep lung cancer has been identified as the best natural out-bred model that can be used to study human epithelial tumours. The close similarity between the pathology of the sheep disease and Human Bronchiolo-alveolar carcinoma are highly suggestive that the human disease could have a similar aetiology and mechanism to the sheep disease. However, in the case of sheep at the time of the study there was a need for an assay that could be used to screen for infected sheep. The isolation, cloning and subsequent sequencing of the first full-length exogenous and endogenous forms of JSRV contributed greatly towards JSRV research. Until recently the diagnosis of OPA was based mostly on clinical presentation with confirmation by micro and macro examination of the affected lungs by expert pathologists. In the absence of a specific humoral response no serology-based tests were available to diagnose the disease early in live animals. Control and management of the disease was primarily by regular flock inspections and prompt culling of the suspected cases. The objective of this research project was therefore to assess and investigate the serological and molecular diagnosis of JSRV. In an attempt to develop a serology based assay three proteins were identified as candidate diagnostic antigens, the group specific antigen JSRV p26, the transmembrane and the orf-X proteins. Genes coding for all three proteins were isolated, cloned and expressed. The JSRV p26 was sufficiently purified and its potential as a diagnostic antigen was evaluated in both a Western blot and ELISA. Our studies confirmed that there were no circulating antibodies to the JSRV capsid protein. Evidence suggested that the immune response was localised to the lungs. Lung lavage samples were therefore collected from infected and normal sheep and analysed for the presence of JSRV p26 antibodies using an in-house JSp26 peroxidase conjugate in an antigen capture assay. This assay lacked sensitivity but the results indicated that there was a specific localised immune response to JSRV in the lungs of OPA affected sheep. This was confirmed with an in-house antigen capture assay that we developed. JS antigen was detected in the lung and nasal fluid of affected sheep, but not in equivalent samples from normal sheep. Three molecular assays were investigated for their sensitivity and specificity, the LTR-gag PCR, U3/LTR hemi-nested PCR and the PCR that covered the V1/V2 region. The U3/LTR hemi-nested assay was 2 logs more sensitive than the LTR-gag PCR. However, it detected the endogenous JSRV5.9A1 loci at higher concentrations. This was overcome by designing a more specific primer P3M for the first step of the U3/LTR hemi-nested PCR and the use of the AmpliTaq Gold DNA polymerase. This assay proved to be both sensitive and specific enough to screen for the infectious exogenous JSRV in peripheral blood samples from individual sheep. It is now possible to use this assay to selectively eradicate the disease from a flock through a selective culling programme. Furthermore, the assay could be made quantitative by the inclusion of concentration standards.
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.
Impact of p2/NC cleavage site polymorphisms on HIV-1 subtype C viral fitness.
(2012) Wilson, Serron.; Gordon, Michelle Lucille.
Subtype C accounts for the majority of HIV infections and in South Africa, is the dominant subtype. The Gag cleavage sites of subtype C viruses show a high degree of natural variation compared to subtype B and group M sequences, with the p2/NC site having the highest degree of variation among all cleavage sites and between all subtypes. This study therefore aimed to determine the functional effect of this variation on viral fitness. A library of drug naïve subtype C sequences were screened using computational analysis to predict binding affinity between HIV protease and the Gag substrate at the p2/NC site. Ligands with high predicted affinity had hydrophobic cleavage sites with substantial diversity at positions P5-P3. Lower ranking ligands were mostly similar to the consensus subtype C. Three ligands were selected for fitness assays from each the high ranking and low ranking groups. Chimeric viruses expressing selected cleavage sites were generated by site directed mutagenesis. Replication capacity assays of these viruses showed moderate differences in fitness but failed to demonstrate a correlation with computational estimates of binding affinity. Enzymes assays were performed to further investigate substrate preferences and the binding mechanism of protease. To this end, recombinantly expressed HIV-1 protease was tested against a range of substrates the matching the p2/NC cleavage sites used in the replication capacity assay. Results of the enzyme assay did not correlate with either the computation studies or the replication capacity assay results, suggesting a sequence independent binding and recognition mechanism of HIV-1 protease. Taken together the results suggest that processing of Gag is determined by tertiary folding of the polyprotein and not amino acid sequence at the cleavage site.
The role of the protease cleavage sites in viral fitness and drug resistance in HIV-1 subtype C.
(2010) Giandhari, Jennifer.; Gordon, Michelle Lucille.
There is an increasing number of patients failing second line highly active antiretroviral therapy (AZT, DDI and LPV/r) in South Africa, where HIV-1 subtype C predominates. Mutations at gag cleavage sites (CS) have been found to correlate with resistance mutations in protease (PR). Therefore, it is important to collect data on subtype C protease and gag sequences from patients as these mutations may affect the efficacy of protease inhibitor (PI) containing drug regimens. In this study, 30 subtype-C infected second-line failures were genotyped using the ViroSeqTM resistance genotyping kit and the gag region from these isolates were then characterised. These sequences were then compared to 30 HIV-1 subtype C infected first-line failures (PI-naïve) and subtype B, C and group M naïve sequences that were downloaded from the Los Alamos Sequence Database. Amino acid diversity at the CS was measured using Mega version 4.0. To investigate the effect of CS mutations on replication capacity, a mutation was introduced by site-directed mutagenesis (Stratagene’s QuikChange Site-Directed Mutagenesis kit). Of the 30 second-line failures that we genotyped, only 16 had resistance mutations in PR and 23 in gag. The most frequent major PI mutations were: I54V/L, M46I, V82A, and I84V and in gag CS were V390L/I and A431V. Interestingly the A431V mutation significantly correlated with protease mutations M46I/L, I54V and V82A. The virus carrying the A431V mutation in vitro was found to have a lower replication capacity compared to the wild type. These findings emphasize the need for further investigation of gag mutations and their contribution to the evolution of HIV resistance to PIs.
Molecular epidemiology of HIV-2 infection in KwaZulu-Natal Provnce, South Africa.
(2013) Singh, Lavanya.; De Oliveira, Tulio De Paiva Nazareth Andrade.
Infection with HIV-2 has important implications for the diagnosis, treatment and management of the infection. The objective of this study was to describe the seroprevalence and molecular epidemiology of HIV-2 in KwaZulu-Natal – the province with the highest HIV prevalence in South Africa, which in turn is the country with the highest HIV prevalence in the world. HIV-1 positive samples were screened using a rapid test for HIV-2. Samples showing antibody positivity were subject to molecular confirmation by PCR and / or serological confirmation by Western blot. There was a large difference in results (10.6% by Western blotting versus 0% by PCR). This discrepancy between molecular and serological confirmation by Western blot. There was a large difference in results (10.6% by Western blotting versus 0% by PCR). This discrepancy between molecular and serological confirmation was attributed to cross-reacting antibodies. The use of rapid tests and Western blots for HIV-2 diagnosis in South Africa, should, therefore, be interpreted with caution. Based on the results of this study, HIV-2 is most probably not present in KwaZulu-Natal.
An investigation of virologic failure and the spectrum of drug resistance mutations in a paediatric ART programme in rural KZN, SA.
(2014) Pillay, Sureshnee.; Danaviah, Sivapragashini.; Bland, Ruth Margaret.
Background Better understanding of drug resistance patterns in HIV-infected children on antiretroviral therapy (ART) is required to inform public health policies in high prevalence settings. The aim of this study was to characterise the acquired drug resistance in HIV-infected children failing first-line ART in a decentralised rural HIV programme. Methods Plasma samples were collected from 101 paediatric patients (<15 years of age) identified as failing ART. RNA was extracted from the plasma, reverse transcribed and a 1.3kb region of the protease gene was amplified and sequenced using Sanger sequencing protocols. Sequences were edited in Geneoius and drug resistance mutations were identified using the RegaDB and the Stanford, Rega and ANRS resistance algorithms. The prevalence and frequency of mutations were analysed together with selected clinical and demographic data in STATA v11. Results A total of 101 children were enrolled and 89 (88%) were successfully genotyped; 73 on a non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based regimen and 16 on a protease inhibitor (PI)-based regimen at the time of genotyping. The majority of patients on an NNRTI regimen (80%) had both nucleoside reverse-transcriptase inhibitor (NRTI) and NNRTI resistance mutations. M184V and K103N were the most common mutations amongst children on NNRTI-based and PI-based regimens. 23% had one or more thymidine analogue mutation (TAM) and 6% had ≥3 TAMs. Only one child on a PI-based regimen harboured a major PI resistance mutation. Conclusions Whilst the patterns of resistance were largely predictable, the few complex resistance patterns seen with NNRTI-based regimens and the absence of major PI mutations in children failing PI-based regimens suggest the need for wider access to genotypic resistance testing in this setting.
Nucleoside reverse transcriptase inhibitors-associated mutations in the RNase H region of HIV-1 isolates in South African adults and children failing highly active antiretroviral therapy.
(2012) Ngcapu, Sinaye.; Gordon, Michelle Lucille.
Background: The South African national treatment program includes NRTIs in both first and second line highly active antiretroviral therapy regimens. Recently, mutations in the RNase H domain have been associated with resistance to NRTIs. Here we investigated the prevalence and association of RNase H mutations with NRTI resistance in isolates of HIV-1 subtype C infected individuals. Methods: RNase H sequences from 134 NRTI treated (104 adults and 30 children) and 134 drug-naïve sequences (30 KZN isolates and 104 downloaded from the Los Alamos Database) were analyzed. Spearman’s rank correlation and a Bayesian network were used to explore the relationship between mutations occurring within the RNase H domain and NRTI treatment. Results: 130 of 134 samples clustered phylogenetically with HIV-1 subtype C, with one subtype A, two subtype B and two subtype D. All 30 sequences from HAART-naїve patients were classified as subtype C. Five mutations in the RNase H region had significantly higher frequency when comparing ART-naïve and NRTI-experienced patients. These were: (E438GKR, L517ISV, K527GENQR, E529DK and Q547HKR) (Table 1). Three mutations (E432D, A446SVY and Q507HK) showed decreased proportions in treatment-experienced isolates when compared to ART- naїve isolates. E438GKR was seen in 6.72% of treated versus only 0% of naїve isolates (p= 0.0034), L517IV was found in 17.16% of treated isolates versus 7.46% of naїve isolates (p= 0.0245). Similarly, K527GENQRS was found in 41.04% of treated isolates versus 26.12% of naїve isolates (p= 0.0138), and E529DK was more prevalent in treated (17.91%) when compared to 2.99% of naїve subtype isolates (p <0.001). Finally, Q547HKR was seen in 5.22% of treated versus 0% of naïve subtype C patients (p= 0.0144). Interestingly, samples of twenty treatment experienced individuals that did not show of the classical NRTI mutations in the RT domain harbored E438GKR, L517ISV, K527GENQR, E529DK and Q547HKR. Conclusion: Results obtained from this study suggested that drug resistance could be caused by mutations in the RNase H domain either alone (T470S), or in combination with mutations in the pol region (D67N and L491P). Phenotypic studies are required to understand the prevalence and impact of RNase H mutations, particularly E438GKR, T470S, L517ISV, K527GENQR, E529DK and Q547HKR on NRTI resistance in HIV-1 subtype C as suggested by our data. Further studies using site-directed mutagenesis may also reveal the impact of these mutations on viral fitness.
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.
Minority HIV-1 drug resistance mutations in patients failing highly active antiretroviral therapy (HAART).
(2014) Khumalo, Phumzile.; Gordon, Michelle Lucille.
Abstract not available.
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.
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.
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.
Identification of mutational pathways to tenofovir resistance in subtype C isolates using a Bayesian Network.
(2016) Maphumulo, Ntombikhona F.; Gordon, Michelle Lucille.
No abstract.
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.
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.
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.
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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