Research Articles (Estrelita Janse van Rensburg)

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An automated genotyping system for analysis of HIV-1 and other microbial sequences
(Oxford University Press, 2005-10-01) De Oliveira, Tulio; Deforche, Koen; Cassol, Sharon; Salminen, M.; Paraskevis, D.; Seebreghts, C.; Snoeck, J.; Janse van Rensburg, Estrelita; Wensing, A.M.J.; Van der Vijver, D.A.; Boucher, C.A.; Camacho, R.; Vandamme, Anne-Mieke; tulio.deoliveira@zoology.oxford.ac.uk
Motivation: Genetic analysis of HIV-1 is important not only for vaccine development, but also to guide treatment strategies, track the emergence of new viral variants and ensure that diagnostic assays are contemporary and fully optimized. However, most genotyping methods are laborious and complex, and involve the use of multiple software applications. Here, we describe the development of an automated genotyping system that can be easily applied to HIV-1 and other rapidly evolving viral pathogens. Results: The new REGA subtyping tool, developed using Java programming and PERL scripts, combines phylogenetic analyses with bootscanning methods for the genetic subtyping of full-length and subgenomic fragments of HIV-1. When used to investigate the subtype of previously published reference datasets that were analysed using manual phylogenetic methods, the automated method correctly identified 97.5–100% of non-recombinant and circulating recombinant forms of HIV-1, including 108 full-length, 108 gag and 221 env sequences downloaded from the Los Alamos database. Availability: The tool, which can be easily downloaded and installed on either a UNIX or Linux-based computer system, is available at http://www.bioafrica.net/subtypetool/html/
Correlation between p53 gene mutation, p53 protein labeling and PCNA expression in oral squamous cell carcinomas
(International Institute of Anticancer Research, 1998-01) Van Heerden, Willem Francois Petrus; Janse van Rensburg, Estrelita; Hemmer, J.; Raubenheimer, E.J.; Engelbrecht, Susan
The prevalence of oral squamous cell carcinoma (OSCC) among the black community in South Africa is unacceptably high. The association between p53 protein, and PCNA overexpression and the presence of p53 gene mutations was evaluated. Materials and Methods: One hundred and ten formalin-fixed, paraffin-embedded blocks of OSCC were selected for immunohistochemical studies for p53 protein and PCNA expression using the DO-7 and PC10 monoclonal antibodies, respectively. DNA was extracted from fifty-five block and exons 5 to 9 of the p53 gene were amplified with nested primers, thereafter sequencing was performed to confirm the presence of mutations detected by single stranded conformational polymorphism. Results: Fifty-six cases (51%) showed p53 expression, while fourteen mutations (25%) were detected. A significant difference was found between the PCNA index in p53 positive and p53 negative rumors while the mean PCNA index for the tumors with p53 mutations was not significantly different from the tumors without mutations. Conclusions: No association between p53 protein overexpression and p53 gene mutations could be demonstrated.
Functional analysis of novel SLC11A1 (NRAMP1) promoter variants in susceptibility to HIV-1
(BMJ Publishing Group, 2004-04) Donninger, Howard; Cashmore, T.J.; Scriba, Thomas Jens; Petersen, Desiree C.; Janse van Rensburg, Estrelita; Hayes, Vanessa M.
The divalent cation transporter is the natural resistance associated macrophage protein 1 (formerly NRAMP1 and now named SLC11A1) for solute carrier 11A1 (OMIM accession number 600266). The gene that codes for this transporter has been studied intensively for its role in conferring susceptibility to infectious diseases such as tuberculosis, leprosy, meningococcal meningitis, visceral leishmaniasis, and HIV infection, as well as to autoimmune diseases such as rheumatoid arthritis, diabetes, sarcoidosis, inflammatory bowel disease, and, more recently, Kawasaki disease. Most studies have investigated a functional GT repeat sequence in the promoter region of this gene and have identified two commonly occurring repeat alleles and four rare repeat alleles. The common alleles are T(GT)5-AC(GT)5AC(GT)10 (allele 2) and T(GT)5AC(GT)5AC(GT)9 (allele 3; GenBank accession number AF229163, 5768 to 5808). Allele 2, which decreases gene expression, has been associated with susceptibility to infectious diseases; the more common allele 3 enhances gene expression to protect against infectious diseases while enhancing susceptibility to autoimmune diseases. Although HIV is classified as an infectious disease, it affects the autoimmune system, which may explain why allele 3 is associated with susceptibility to HIV-1. This study aimed to screen the promoter region of SLC11A1 for novel sequence variations in people from sub- Saharan Africa infected with HIV-1 compared with uninfected people and to determine the effect of novel variants on normal promoter function.
Detection of EBV DNA in oral squamous cell carcinomas in a black African population sample
(International Institute of Anticancer Research, 1995-05) Janse van Rensburg, Estrelita; Engelbrecht, Susan; Van Heerden, Willem Francois Petrus; Raubenheimer, E.J.; Schoub, Barry D.
The purpose of this study was to determine the presence of Epstein-Barr virus (EBV) DNA in oral squamous cell carcinoma (OSCC) patients from a black African population. Formalin fixed paraffin embedded blocks of OSCC of two randomly selected groups were investigated. Group 1 consisting of 57 blocks containing OSCC with a fragment of normal appearing adjacent/overlying epithelium. Group 2 consisted of 48 blocks containing only OSCC tissue without any normal appearing epithelium. The control group consisted of 38 non-malignant, non-viral associated lesions. A standard polymerase chain reaction (PCR) was used to amplify the Bam HI W-fragment using a nested primer set. EBV DNA was demonstrated in 14/57 (25%) blocks from Group 1, in 13/48 (27%) blocks from Group 2 and in 16/38 (42%) blocks from the control group. No evidence for a direct role of EBV in the process of malignant transformation of intraoral epithelial cells was found in this study.
Variation in HIV type 1 V3 region env sequences from Mozambique
(Mary Ann Liebert, 1998-06-10) Engelbrecht, Susan; Koulinska, Irene; Smith, Tracey-Lee; Baretto, J.; Janse van Rensburg, Estrelita
No abstract available.
HIV type 1 V3 region subtyping in KwaZulu-Natal, a high-seroprevalence South African region
(Mary Ann Liebert, 1998-07-20) Moodley, D.; Smith, Tracey-Lee; Janse van Rensburg, Estrelita; Moodley, J.; Engelbrecht, Susan
No abstract available.
Human papillomavirus DNA in oral squamous cell carcinomas from an African population sample
(International Institute of Anticancer Research, 1996-03) Janse van Rensburg, Estrelita; Engelbrecht, Susan; Van Heerden, Willem Francois Petrus; Raubenheimer, E.J.; Schoub, Barry D.
BACKGROUND: The incidence of oral squamous cell carcinoma (OSCC) is on the increase in developing countries. MATERIALS AND METHODS: Formalin fixed paraffin embedded blocks of OSCCs from a black South African population sample of peri-urban and rural origin were selected as follows: Group 1 - 57 OSCCs with a mean age of 59 years; Group 2 - 43 OSCCs all cases younger than 40 years; Group 3 - 46 OSCCs with blocks containing only tumour tissue without any normal epithelium and Group 4, a control group of 38 non-neoplastic epithelial lesions. Type specific primers were used in a standard PCR to amplify a segment of the E6 region of HPV 6, 11, 16 and 18. RESULTS: HPV 11 and 16 DNA were found in one sample each from groups 1 and 2 respectively. CONCLUSION: HPV is not an etiologic factor in the development of OSCC in the population studied.
Prevalence of EBV in oral squamous cell carcinomas in young patients
(International Institute of Anticancer Research, 1995-09) Van Heerden, Willem Francois Petrus; Janse van Rensburg, Estrelita; Engelbrecht, Susan; Raubenheimer, E.J.
BACKGROUND: Recent studies reported a difference in the age distribution of oral squamous cell carcinoma (OSCC) between black and white South Africans with OSCC more prevalent in black patients under the age of 50 compared to whites. MATERIALS AND METHODS: Paraffin embedded blocks of OSCC were divided into two groups: one with a mean age of 56.2 years and the second group all younger than 40 years of age. A control group of 30 non-neoplastic intraoral lesions were selected. A standard PCR reaction was used to amplify the BAM H1 W-fragment of the EBV. RESULTS: EBV DNA was demonstrated in 11/45 (24%) cases from the first group and in 11/45 (24%) cases from the second group. EBV DNA was present in 11/30 (37%) cases from the control group. CONCLUSIONS: This study showed that the prevalence of EBV in OSCC was not influenced by the age of the patient.
Identification of env subtypes in fourteen HIV type 1 isolates from South Africa
(Mary Ann Liebert, 1995-10) Engelbrecht, Susan; Laten, Janetta D.; Smith, Tracey-Lee; Janse van Rensburg, Estrelita
All subtypes of HIV-1 have been identified in Africa. The envelope glycoprotein of the HIV-1 is the most variable region of the virus, with the third variable region, including the V3 loop, a major target of vaccine research. This paper reports the identification of the HIV-1 subtypes present in 14 viral strains, isolated between 1984 and 1992 in South Africa. HIV-1 strains were isolated routinely at Tygerberg hospital in the Western Cape region, with genomic DNA isolated from virus-infected cultures. After presenting the distance calculations and describing the tree constructions and bootstrap analysis, the authors emphasize the need for ongoing molecular epidemiological analysis of HIV-1 subtypes to track the current epidemic in South Africa. More rapid methods will facilitate subtyping to monitor the circulation and spread of HIV-1 subpopulations in the country.
Characterisation of the South African HIV-1 subtype C complete 5’ LTR, nef and regulatory genes
(Mary Ann Liebert, 2002-01) Scriba, Thomas Jens; De Villiers, Tania; Treurnicht, Florette K.; Zur Megede, Jan; Barnett, Susan W.; Engelbrecht, Susan; Janse van Rensburg, Estrelita
Human immunodeficiency virus type 1 (HIV-1) subtype C has become the major etiological agent in the global and especially African epidemic. To gain better understanding of the genetic diversity and rapid transmission of HIV-1 subtype C, we have characterized the complete 59 long terminal repeat (LTR) region along with the regulatory genes tat and rev as well as the accessory gene nef of 14 South African HIV-1 subtype C isolates. Phylogenetic analysis revealed a subtype C 59 LTR cluster, as well as subclustering of our nef sequences with various subtype C strains separate from the India and China subclusters. At least 3 NF-kB sites were present in the 59 LTR of most isolates and 13 isolates had the subtype C-specific Rev truncation. Some length variation in exon 2 and the absence of a critical cysteine were found in Tat. Residue variation in the myristoylation signal and motifs involved in CD4 and MHC-I down-regulation was recorded in our nef gene sequences.
Evaluation of envelope vaccines derived from the South African subtype C Human Immunodeficiency Virus Type 1 TV1 strain
(American Society for Microbiology, 2005-11) Lian, Ying; Srivastava, Indresh; Gomez-Roman, V.R.; Zur Megede, Jan; Sun, Yide; Kan, Elaine; Hilt, Susan; Engelbrecht, Susan; Himathongkham, Sunee; Luciw, Paul A.; Otten, Gillis; Ulmer, Jeffrey; Donnelly, John J.; Rabussay, Dietmar; Montefiori, David; Janse van Rensburg, Estrelita; Barnett, Susan W.
Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1V2), followed by boosting with oligomeric protein (o-gp140TV1V2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.
Molecular characteristics of HIV-1 Subtype C viruses from KwaZulu-Natal, South Africa: implications for vaccine and antiretroviral control strategies
(American Society for Microbiology, 2003-02) Gordon, Michelle; De Oliveira, Tulio; Bishop, Karen; Coovadia, H.M.; Madurai, L.; Engelbrecht, Susan; Janse van Rensburg, Estrelita; Mosam, A.; Smith, A.; Cassol, Sharon
The KwaZulu-Natal region of South Africa is experiencing an explosive outbreak of Human Immunodeficiency Virus Type 1 (HIV-1) Subtype C infections. Understanding the genetic diversity of C viruses and the biological consequences of this diversity is important for the design of effective control strategies. We analyzed the protease gene, the first 935 nucleotides of reverse transcriptase, and the C2V5 envelope region of a representative set of 72 treatment-naïve patients from KwaZulu-Natal and correlated the results with amino acid signature and resistance patterns. Phylogenetic analysis revealed multiple clusters or "lineages" of HIV-1 subtype C that segregated with other C viruses from Southern Africa. The same pattern was observed for both black and Indian subgroups and for retrospective specimens collected prior to 1990, indicating that multiple sublineages of HIV-1 C have been present in KwaZulu-Natal since the early stages of the epidemic. With the exception of three nonnucleoside reverse transcriptase inhibitor mutations, no primary resistance mutations were identified. Numerous accessory polymorphisms were present in the protease, but none were located at drug-binding or active sites of the enzyme. One frequent polymorphism, I93L, was located near the protease/reverse transcriptase cleavage site. In the envelope, disruption of the glycosylation motif at the beginning of V3 was associated with the presence of an extra protein kinase C phosphorylation site at codon 11. Many polymorphisms were embedded within cytotoxic T lymphocyte or overlapping cytotoxic T-lymphocyte/T-helper epitopes, as defined for subtype B. This work forms a baseline for future studies aimed at understanding the impact of genetic diversity on vaccine efficacy and on natural susceptibility to antiretroviral drugs.
Variability at Human Immunodeficiency Virus Type 1 Subtype C Protease Cleavage Sites : an indication of viral fitness
(American Society for Microbiology, 2003-09) De Oliveira, Tulio; Engelbrecht, Susan; Janse van Rensburg, Estrelita; Gordon, Michelle; Bishop, Karen; Zur Megede, Jan; Barnett, Susan W.; Cassol, Sharon
Naturally occurring polymorphisms in the protease of human immunodeficiency virus type 1 (HIV-1)subtype C would be expected to lead to adaptive (compensatory) changes in protease cleavage sites. To test this hypothesis, we examined the prevalences and patterns of cleavage site polymorphisms in the Gag, Gag-Pol, and Nef cleavage sites of C compared to those in non-C subtypes. Codon-based maximum-likelihood methods were used to assess the natural selection and evolutionary history of individual cleavage sites. Seven cleavage sites (p17/p24, p24/p2, NC/p1, NC/TFP, PR/RT, RT/p66, and p66/IN) were well conserved over time and in all HIV-1 subtypes. One site (p1/p6gag) exhibited moderate variation, and four sites (p2/NC, TFP/p6pol, p6pol/PR, and Nef) were highly variable, both within and between subtypes. Three of the variable sites are known to be major determinants of polyprotein processing and virion production. P2/NC controls the rate and order of cleavage, p6gag is an important phosphoprotein required for virion release, and TFP/p6pol, a novel cleavage site in the transframe domain, influences the specificity of Gag-Pol processing and the activation of protease. Overall, 58.3% of the 12 HIV-1 cleavage sites were significantly more diverse in C than in B viruses. When analyzed as a single concatenated fragment of 360 bp, 96.0% of group M cleavage site sequences fell into subtype-specific phylogenetic clusters, suggesting that they coevolved with the virus. Natural variation at C cleavage sites may play an important role, not only in regulation of the viral cycle but also in disease progression and response to therapy.
Rhinovirus induction of the CXC chemokine epithelial-neutrophil activating peptide-78 in bronchial epithelium
(University of Chicago Press, 2003-06-01) Donninger, Howard; Glashoff, Richard H.; Haitchi, Hans-Michael; Syce, James A.; Ghildyal, Reena; Janse van Rensburg, Estrelita; Bardin, Philip G.
Epithelial-neutrophil activating peptide-78 (ENA-78) induces neutrophil migration, an early response to viral infection. Rhinovirus serotype 16 (RV16) was used to infect primary bronchial epithelial cells and a cell line (BEAS-2B). Release of ENA-78 protein was measured by enzyme-linked immunosorbent assay, ENA-78 mRNA production was quantified by reverse-transcription polymerase chain reaction, and ENA-78 promoter activity was assessed by use of a promoter construct. After infection with RV16, ENA-78 protein and mRNA increased significantly, and RV16 induced 3-fold increases in ENA-78 gene transcription. Nasal ENA-78 measured in patients with asthma with and without RV infection was more elevated in patients with RV infection present. Our study demonstrates that ENA-78 is produced in bronchial epithelial cells in response to RV16 infection. With other chemokines, it may be an important initiator of neutrophil airway inflammation during RV common colds and thus may play a role in the development of virus-associated airway pathologies.
HPV detection in primary intra-oral squamous cell carcinomas – commensal, aetiological agent or contamination?
(Blackwell, 2006-02) Boy, Sonja Catharina; Janse van Rensburg, Estrelita; Engelbrecht, Susan; Dreyer, Leonora; Van Heerden, Marlene B.; Van Heerden, Willem Francois Petrus; sonja.boy@up.ac.za
Background: High-risk human papilloma viruses (HPV) are reported to be significant independent risk factors for oral squamous cell carcinoma (OSCC). The prevalence of HPV in OSCC in a South African population sample was evaluated comparing three different HPV detection methods. Methods: Tumour and adjacent morphologically normal oral mucosa of 59 resections of primary OSCC were evaluated for the presence of HPV using real-time polymerase chain reaction (PCR), conventional in situ hybridization (ISH), and a signal amplification ISH technique (Dako GenPointTM). Results: HPV18 DNA was detected in seven cases using real-time PCR. No positivity was found with the other two ISH techniques. Conclusions: We support the view that HPV is probably unimportant in the pathogenesis of OSCC and hypothesize HPV detection techniques as the main reason for the positive results in many studies. Real-time PCR was confirmed as the most sensitive technique, but researchers are urged to incorporate strict internal controls when using this detection method.
Functionally-inactive and immunogenic Tat, Rev and Nef DNA vaccines derived from sub-Saharan subtype C human immunodeficiency virus type 1 consensus sequences
(Elsevier, 2005-01-19) Scriba, Thomas Jens; Zur Megede, Jan; Glashoff, Richard H.; Treurnicht, Florette K.; Barnett, Susan W.; Janse van Rensburg, Estrelita; estrelita@up.ac.za
The efficacy of cellular immune responses elicited by HIV vaccines is dependent on their strength, durability and antigenic breadth. The regulatory proteins are abundantly expressed early in the viral life cycle and CTL recognition may bring about early killing of infected cells. We synthesised DNA vaccine constructs that encode consensus HIV-1 subtype C Tat, Rev and Nef proteins. Proteins carrying inactivating mutations were tested for functional activity and highly expressing, inactive Tat, Rev and Nef mutants were identified and their reading frames fused into a TatRevNef cassette. Single- and polygene Tat, Rev and/or Nef constructs were immunogenic in BALB/c mice. These constructs may serve to increase the antigenic breadth for an HIV-1 vaccine that is relevant for sub-Saharan Africa.

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