Masters Degrees (Medical Virology)
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Browsing Masters Degrees (Medical Virology) by browse.metadata.advisor "Jacobs, Graeme Brendon"
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- ItemCharacterization of HIV-1 subtype B near full-length genome sequences identified at the start of HIV epidemic in South Africa(Stellenbosch : Stellenbosch University, 2017-03) Obasa, Adetayo Emmanuel; Jacobs, Graeme Brendon; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH SUMMARY: South Africa is home to approximately 20.0% of the global Human Immunodeficiency Virus (HIV) infected population. The first reported cases of HIV-1 in the country were described in 1982 amongst the homosexual male population. This was attributed to HIV-1 subtypes B (HIV-1B) and D (HIV-1D). Since the late 1980s HIV-1 subtype C (HIV-1C), spread mainly through heterosexual contact, has been the driving force of the epidemic. To date, only six HIV-1B near full-length genome (NFLG) sequences from South Africa are available in the Los Alamos National Laboratory database (LANL). During this study we retrieved five HIV-1B positive samples from homosexual and bi-sexual males, stored for up to 30 years, from the early 1980s, for further characterization. The NFLG amplification reactions were performed using a modern Polymerase Chain Reaction (PCR) protocol designed to target two overlapping proviral DNA HIV genome fragments, 5.5 kb and 3.7 kb in size, respectively. All positive PCR products were sequenced to characterize the viruses. The sequences were checked and edited manually using Sequencher V5. Multiple sequence alignments were created using Clustal W and Maft V7. The sequences were subtyped using the REGA V3.0, RIP V3.0 and jumping profile Hidden Markov Model (jpHMM) online subtyping programmes. Maximum likelihood phylogenetic trees were drawn using MEGA V6. Four of the five HIV-1 patient sequences were subtyped as pure HIV-1B. One sequence, ZA|85|R605, was characterized as a novel HIV-1 BD recombinant. This is the first NFLG HIV-1 BD recombinant ever described and indicates that recombination events were most likely already happening at the early stage of the South African epidemic. Two patient sequences, ZA|87|R1296 and ZA|87|R459, clusters with HIV-1B sequences from the United States of America (USA). The sequence from patient ZA|87|R68 clusters with a HIV-1B sequence from France and the sequence of ZA|87|R526 clusters with another South African HIV-1B sequence. Homosexual flight stewards, international tourists and migrants from the European and North American countries were most likely responsible for the introduction of the HIV-1B epidemic into South Africa. The findings of this study provides valuable insights from the beginning of the HIV-1 epidemic in South Africa. We highlight the importance of characterizing complete viral genomes from early archival specimens to give a more detailed picture of landmarks of the HIV/AIDS pandemic. We show that NFLG sequencing is an important tool for the identification of recombinant viral strains. This study can form the basis for continued research in our attempt to reconstruct the epidemiology and evolutionary history of HIV in South Africa. The HIV-1 epidemic is dynamic in nature and is constantly changing.
- ItemHIV-1 molecular diversity and drug resistance mutations amongst immuno-competent, therapy naïve infants/children and adults in Yaounde, Cameroon(Stellenbosch : Stellenbosch University, 2017-12) Gichana, Josiah Otwoma; Jacobs, Graeme Brendon; Ikomey, George Mondinde; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.Background: In Cameroon, HIV infections range between 550, 000 to 690, 000 for adults aged 15 to 49 years and a prevalence rate at 4.5%. In children of 0 – 14 years, HIV infections range between 34, 000 to 44, 000. The country harbors both HIV type 1 (HIV-1) and HIV type 2 (HIV-2). HIV groups found in Cameroon include M, N, O, P variants. Group M subtypes are the most prevalent, with CRF02_AG accounting for approximately 40% of all HIV infections. This is unlike other regions globally where other group M subtypes like C are the predominant ones. The high genomic diversity of HIV-1 and the emergence of drug resistant associated mutations (RAMs) continue to be a major challenge in designing standardized laboratory protocols for HIV testing, vaccine development and providing successful lifelong therapy to HIV infected patients. In Cameroon, drug resistance rates for therapy naïve individuals are currently at 3.8% in adults and 3.6% in children. This study aimed at identifying HIV-1 diversity and evaluate drug resistant mutations (DRMs) in two different cohorts of therapy-naïve infants/children and adults in Cameroon. Methods: A total of 180 plasma samples were collected from therapy naïve HIV positive patients that included: (1) 55 plasma samples from proxy-consented infants/children aged 9-72 months old with unknown prevention of mother-to-child transmission (PMTCT) exposure and (2) 125 plasma samples from adults of 15 to 50 years old. The CD4+ T-cell count was performed using standard methods following manufacturer’s instructions. To study the HIV-1 diversity and resistance in the two cohorts, partial pol Protease (PR), Reverse Transcriptase (RT) and Integrase (IN) regions of the HIV-1 genome were targeted for conventional PCR amplification and Sanger DNA sequencing. Phylogenetic inference using Neighbor-Joining (NJ) trees were used to cluster and infer subtypes. Results: In the infants/children cohort, the CD4+ T-cell count ranged between 500-2000 cells/m3 (a median of 33.0%) and the HIV-1 viral load between 3000-6000 copies/ml (a median of 4.96 RNA copies/ml). A total of 37/55 (67.3%) paediatric cohort samples were amplified for at least one of the HIV-1 pol fragments. These include 29/55 (52.0%) for the PR, 27/55 (49.0%) for the RT and 28/55 (51.0%) for IN. The most predominant HIV-1 strain was G/CRF02_AG at 62.5% (n = 15). Other subtypes detected include subtype A (20.8%; n = 5), C (8.3%; n = 2) and F2 (8.3%; n = 2). Three sequences (11.1%) could not be assigned to any subtype with confidence. Levels of DRMs to Protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTI were 27.6% (only minor DRMS were observed for PR), 3.7% and 40.7%, respectively. The NRTI mutations observed showed high-level resistance to Zidovudine (AZT), Tenofovir (TDF), Didanosine (DDI) and Stavudine (D4T), and low to intermediate-level resistance to Lamivudine (3TC), Abacavir (ABC), and Emtricitabine (FTC). The NNRTI mutations observed showed high level resistance to Nevirapine (NVP) and Efavirenz (EFV) with reduced susceptibility to Etravirine (ETR) and Rilpivirine (RPV). In the adult cohort, the RT fragment (n = 55) was used for phylogenetic analysis with majority of the sample sequences clustered with HIV-1 subtype G/CRF02_AG which accounted for 40% (n = 22), CRF22_01A1 (10.9%; n = 6), C (1.8%; n = 1), B (1.8%; n = 1), other complex forms – 37_cpx/11_cpx (3.6%; n = 2). Twenty three samples (41.8%) could not be assigned to any subtype with confidence. The levels of drug resistance for adults was 5.4% for both NRT and NNRT inhibitors - 4.0% had low level resistance to EFV, ETR, NVP and RPV while 1.4% had intermediate to high level resistance to ABC, FTC, TDF, EFV and NVP. Conclusion: Cameroon continues to harbor many HIV-1 subtypes and circulating recombinant forms (CRFs) as observed in both cohorts. Furthermore, rare group O and other group M subtypes like C were noticed within the study cohorts suggesting an improvement in sensitivity of detection methodologies currently used. Drug resistance is a major challenge to current antiretroviral drug regimens as illustrated by the detection of RAMS in both cohorts of this study. Continuous surveillance of the HIV diversity and drug resistance is therefore necessary to better manage the HIV-1 pandemic.
- ItemInvestigating the fitness benefit of reverse transcriptase (RT) mutation A62V when co-occurring with M184V and K65R in HIV-1 subtype C(Stellenbosch : Stellenbosch University, 2016-03) Njenda, Duncan Tazvinzwa; Van Zyl, Gert Uves; Engelbrecht, Susan; Jacobs, Graeme Brendon; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: Background and Aims Tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) or emtricitabine (FTC) combined with efavirenz is the predominant first-line antiretroviral regimen in the Southern African region. Resistance to TDF and 3TC/FTC is largely through the occurrence of the drug resistance mutations (DRMs) K65R and M184V, respectively. Preliminary data from a large laboratory-based dataset of HIV drug resistance that showed a high prevalence of these mutations in patients who received the TDF regimen also revealed a significant co-occurrence of A62V with M184V and K65R. The aim of this study was to investigate the functional interaction and effect on viral fitness that A62V has when it co-occurs with M184V and K65R reverse transcriptase mutations in HIV-1 Subtype C. Materials and Methods Using Infusion™ cloning and site-directed mutagenesis techniques, eight full-length genome infectious clones containing the HIV-1 subtype C polymerase gene were synthesised having all combinations of DRMs - A62V, M184V and K65R, either being present or absent. The mutations in these constructs were verified by sequencing. The constructs were transfected into 293T cells for virion production and harvested virus was infected in the TZM-bl cell line in head to head growth competition experiments and assayed for growth kinetics using an allele-specific quantitative real-time polymerase chain reaction (PCR) assay. Results The growth competition experiment between two viruses (A62V+K65R+M184V vs K65R+M184V) evaluated by taking the mean of 3 biological replicates in the assay in the absence of antiretroviral drugs, revealed that A62V mutation has no significant impact on fitness (Wilcoxon signed rank test p-value = 0.56). The overall coefficient of variation (CV) in the experiment was 12.8% indicating the high reproducibility of the growth competition assay using real-time PCR measurement of relative growth. Conclusion and recommendations A62V mutation has no effect on fitness when it co-occurs with M184V and K65R. The co-occurrence with M184V and K65R remains unexplained and might be due to an effect on TDF resistance in combination with K65R.This requires investigation in future studies as TDF regimens are part of 1st line therapy in many Sub-Saharan countries in the treatment of HIV-1. Finally, the cloning and mutagenesis techniques used coupled with a very sensitive and reproducible real-time quantitative PCR assay provide an efficient system for detection of mutation fitness interactions and can be used in any future work to study HIV mutation fitness interactions.
- ItemInvestigation of the HIV diversity in the Cape Winelands, Overberg and West Coast districts of the Western Cape Province of South Africa(Stellenbosch : Stellenbosch University, 2017-03) Mikasi, Sello Given; Jacobs, Graeme Brendon; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH SUMMARY: The Western Cape Province of South Africa has a well-established program that monitors active combination antiretroviral therapy (cART) against HIV-1. The HIV-1 prevalence rate in the Province has increased from 5.0% in 2011 to 18.0% in 2015. South Africa has the highest rate of infections worldwide (19.2%). In this study, we analyzed the Protease (PR), Reverse Transcriptase (RT) and Intergrase (IN) regions of HIV-1 for diversity and resistanceassociated mutations (RAMs) from samples obtained from the Cape Winelands, West Coast and Overberg districts of the Province, where no such study has ever been conducted. Samples were received from our diagnostic laboratory for HIV-1 viral load testing, through the National Health Laboratory Services (NHLS). Two hundred and five (205) patient samples with a viral load of 2000 copies/ml and above were included, based on Gall et al., (2012) who showed that a sensitivity of at least 2000 copies/ml is a limit of amplification for the SuperScsript ® III one-step RT with Platinum Taq DNA Polymerase kit, used in this study. We screened for HIV-1 diversity and RAMs using the pol PR, RT and IN regions with a laboratory-based PCR and sequencing protocol. Sequence-specific subtype analyses were executed with the REGA HIV subtyping tool 3.0, Recombinant Identification Program (RIP) 3.0 and subtype classification using evolutionary algorithms (SCUEAL) software. Sequences were screened for RAMs using the Stanford University HIV Drug Resistance Database (HIVdb) 8.1. We successfully PCR amplified 170 (82.9%) PR and 166 (80.9%) RT fragments. For the IN region, only 176 samples had sufficient plasma and RNA left after genotyping of the PR and RT regions. For IN we successfully amplified 143 (81.3%) of the patient samples. A total of 197 (96.1%) samples could be amplified for at least one of the pol regions. Of these, 62 (53.4%) PR, 103 (62.0%) RT and 93 (86.1%) IN sequences were obtained, respectively. We could successfully sequence 173 (84.4%) of the samples included. HIV-1 subtype C was predominant (n = 144; 93.7%), with 5.3% of other subtypes detected. This includes A1 (n = 2; 1.3%), B (n = 4; 2.6%), D (n = 1; 0.7%) and H (n =1; 0.7%). No major RAMs were detected against PI and IN inhibitors. Minor RAMs were detected in 4 PR (3.7%) and 15 IN (16.1%) sequences analysed. RAMs against RT inhibitors were detected in 63 (61.7%) of the sequences analyzed. This includes 39 NRTI mutations (36.1%) and 71 NNRTI mutations (63.5%) identified. As the national cART program continues to expand, HIV-1 diversity, viral load monitoring and drug resistance screening remains critical for the success of cART outcomes and reducing transmission rates. Our results reflect that subtype C is still the driving force of the epidemic in South Africa. However, we cannot ignore the potential impact of non-C subtypes. Sequence analyses confirm that the majority of patients receiving viral load testing have major RAMs against RT inhibitors used in first line therapy. Better surveillance systems for HIV diversity and drug resistance testing are required to ensure success of cART.
- ItemMolecular characterisation of HIV-1 recombinants and non-subtype C viruses in South Africa(Stellenbosch : Stellenbosch University, 2019-03) Varathan, Olivette; Jacobs, Graeme Brendon; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: HIV/AIDS is a severe health burden, affecting 36.9 million people worldwide by the end of 2017. South Africa has the largest HIV-1 epidemic in the world, estimated at 7.2 million infected individuals by the end of 2017. The HIV-1 epidemic in South Africa is dominated by HIV-1 subtype C, accounting for an estimated 98.2% of the infections in the country, based on the viral sequences in the Los Alamos National Library (LANL) database. To date, to the best of our knowledge, 22 papers have been published on non-subtype C viruses in South Africa. This study aimed to characterise two near full-length genome sequences of non-subtype C viruses in South Africa. The study samples were obtained through the diagnostic services of the National Health Laboratory Services (NHLS), performing routine drug resistance testing within the Division of Medical Virology, Stellenbosch University. All samples received were sequenced in the partial pol region (~1.4kb) of the viral genome by the NHLS. The possible subtype of the virus was identified from the sequences using online subtyping programmes. All sequences and samples that identified as possible non-subtype C viruses were recorded in a separate non-subtype C cohort. In 2011, a possible C, D recombinant virus was first identified in this cohort. By the end of 2015, 30 similar recombinant viruses were observed in the cohort, indicating a possible emergence of this recombinant strain. Two of the samples that identified as possible C, D recombinants were selected for further near full-length genome (NFLG) characterisation. Proviral DNA, from sample EC148, was extracted from PBMCs and viral RNA, from sample WC416, was extracted from plasma. The RNA was reverse transcribed to DNA via cDNA synthesis. Both sample viruses were amplified by PCR in two rounds. The first round targeted the amplification of the HIV-1 NFLG (8978bp) and the second targeted the amplification of two overlapping fragments (5455bp and 4909bp). Positive PCR amplicons were purified and sequenced. The generated sequences were read and analysed before being used in online subtyping programmes. The jumping profile hidden markov model (jpHMM), REGA and recombination identification programmes (RIP) were used to preliminary assign subtypes to both samples. Phylogenetic analyses was inferred to confirm / reject the online subtyping programme results. Online subtyping programmes identified the virus sequences of samples EC148 and WC416 as complex A, C, D recombinants. Phylogenetic analysis confirmed the online subtyping programme results for the sequence of sample WC416 in identifying it as a complex A, C, D recombinant. Phylogenetic analysis indicated that the sequence of sample EC148 is consistent with the results observed from the online subtyping programmes. Each HIV-1 sequence identified as a unique complex recombinant form as the breakpoints between the different subtypes differed. The emergence of new and unique non-subtype C recombinants in South Africa indicates that the epidemic is complex and evolving. It is therefore important to monitor the spread of different HIV subtypes circulating in South Africa.