OpenUCT :: Browsing by Department "Division of Virology"

Browsing by Department "Division of Virology"

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Open Access
Acinetobacter baumannii : an evaluation of five susceptibility test methods to detect tobramycin resistance in an epidemiologically related cluster
(2011) Moodley, Vineshree Mischka; Oliver, Stephen; Elisha, B Gay
Acinetobacter baumannii is a major pathogen causing nosocomial infections, particularly in critically ill patients. This organism has acquired the propensity to rapidly develop resistance to most antibiotics. At several hospitals within Cape Town, tobramycin and colistin remain frequently the only therapeutic options. The Vitek2 automated susceptibility testing (AST) is used in the clinical laboratory to determine selected susceptibility profiles. The suspicion of a possible AST-related technical error when testing for susceptibility to tobramycin in A. baumannii precipitated this study.
Open Access
The allelic distribution of -308 Tumor Necrosis Factor-alpha gene polymorphism in South African women with cervical cancer and control women
(BioMed Central Ltd, 2006) Govan, Vandana; Constant, Debbie; Hoffman, Margaret; Williamson, Anna-Lise
BACKGROUND:Cervical cancer is due to infection with specific high-risk types of human papillomavirus (HPV). Although the incidence of genital HPV infection in various population groups is high, most of these regress without intervention. Investigating genetic host factors and cellular immune responses, particularly cytokines, could help to understand the association between genital HPV infection and carcinogenesis. The tumor necrosis factor alpha (TNF-alpha) cytokine plays an important role in all stages of cervical cancer and has the ability to induce the regression of human tumors. Therefore the aim of the study was to investigate the allelic distribution of -308 TNF-alpha gene polymorphism in South African women with cervical cancer compared to control women. METHODS: Included in our study were women with histologically proven cancer of the cervix (n = 244) and hospital-based controls (n = 228). All patients and controls were from mixed race and black population groups in South Africa. The detection of a bi-allelic -308 (A/G) polymorphism in the promoter region of TNF-alpha was investigated using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique. The distributions of the allelic frequencies were stratified in both patients and controls into two South African ethnic population groups. RESULTS: In this study we observed no association between the distribution of -308 TNF-alpha polymorphism and the risk of developing cervical cancer even after combining the data from the two ethnic populations (X2 = 2.26). In addition, using the chi-squared test we found no significant association between the known risk factors for cervical cancer and the allele distribution of -308 TNF-alpha. However, the frequency of the rare high-producing allele -308A of TNF-alpha was significantly lower in the South African population when compared to Caucasians and Chinese population groups. CONCLUSION: We demonstrated no association between -308 TNF-alpha polymorphism and the risk of cervical cancer among two South African ethnic population groups. However, as the distribution of the -308A TNF-alpha was notably different between the control groups of South Africa and other population groups this result suggests that ethnic disparity may influence the levels of TNF-alpha produced.
Open Access
Analysis of cytomegalovirus UL97 drug resistance mutations in patients receiving Ganciclovir
(2018) Nkosi, Nokwazi Pearl; Hsiao, Nei-Yuan; Korsman, Stephen; Smuts, Heidi
Introduction: Cytomegalovirus (CMV) drug resistance mutations, because of the widespread use of ganciclovir, have been widely reported in international literature, particularly in the post-transplant setting. However, a genotypic assay to detect CMV drug resistance is not available in South Africa and the prevalence of these mutations is therefore unknown. We aimed to document the prevalence and types of CMV UL97 mutations following exposure to ganciclovir in adult and paediatric oncology patients, transplant recipients and HIV-infected patients in the local tertiary level hospitals: Red Cross War Memorial Children's Hospital, Groote Schuur Hospital and Tygerberg Hospital. Methods: The study had two components, the first component being a retrospective cross-sectional study using stored extracted DNA from patients with serially elevated CMV viral load levels. Thirty-three samples were tested for this component. The second component was a prospective case series on patients who were referred by clinicians for genotypic testing in whom CMV drug resistance was suspected. Eight samples were tested for this component. The CMV UL97 gene was amplified by conventional nested polymerase chain reaction (PCR) and Sanger sequencing performed. Results: CMV UL97 mutations were identified in five of thirty-three (15%) retrospectively screened samples while the prospective testing of eight patient samples identified drug resistance mutations in three patients (38%). Overall 8/41 (20%) patients had CMV UL97 mutations. A trend of higher risk for development of drug resistance mutations among haematological oncology patients 7/23 (30%) compared to solid organ transplant recipients 1/10 (10%) was observed, however, this difference was not statistically significant (P=0.306). Conclusion: This study, the first of its nature in South Africa, identified the presence of CMV UL97 mutations conferring resistance to ganciclovir in the haematological oncology, primary immunodeficiency and solid organ transplant patients in the Western Cape. The assay successfully detected CMV UL97 drug resistance mutations in whole blood and cerebrospinal fluid clinical samples. Ongoing viral replication in the background of intensive immunosuppression and prolonged antiviral therapy selects for the emergence of CMV UL97 drug resistance mutations.
Open Access
Analysis of HIV early infant diagnosis and linkage to care in the Western Cape: a laboratory perspective
(2012) Hsiao, Nei-Yuan
Prevention of mother-to-child transmission (PMTCT) of HIV is the cornerstones of HIV prevention programs. The principle of using antiretrovirals (ARV) to reduce the risk of transmission from mother to child is well established as a range of PMTCT regimens with varying efficacies have been widely studied and reviewed1. In South Africa and other Sub-Saharan countries, single dose Nevirapine, amongst other cost- effective regimens, have been adopted as part of the national HIV prevention program2 since 2003.
Open Access
Anti-vector immune responses to an MVA vaccine
(2011) Muller, Tracey; Burgers, Wendy
This study characterised the humoral and cellular immune responses to MVA from a candidate MVA-vectored HIV vaccine in non-human primates, and examined the effect of anti-vector immunity on the response to the HIV immunogens.
Open Access
CCR2-V64I polymorphism is associated with increased risk of cervical cancer but not with HPV infection or pre-cancerous lesions in African women
(BioMed Central Ltd, 2010) Chatterjee, Koushik; Dandara, Collet; Hoffman, Margaret; Williamson, Anna-Lise
BACKGROUND: Cervical cancer, caused by specific oncogenic types of human papillomavirus (HPV), is the second most common cancer in women worldwide. A large number of young sexually active women get infected by HPV but only a small fraction of them have persistent infection and develop cervical cancer pointing to co- factors including host genetics that might play a role in outcome of the HPV infection. This study investigated the role of CCR2-V64I polymorphism in cervical cancer, pre-cancers and HPV infection in South African women resident in Western Cape. CCR2-V64I polymorphism has been previously reported to influence the progression to cervical cancer in some populations and has also been associated with decreased progression from HIV infection to AIDS. METHODS: Genotyping for CCR2-V64I was done by PCR-SSP in a case-control study of 446 women (106 black African and 340 mixed-ancestry) with histologically confirmed invasive cervical cancer and 1432 controls (322 black African and 1110 mixed-ancestry) group-matched (1:3) by age, ethnicity and domicile status. In the control women HPV was detected using the Digene Hybrid Capture II test and cervical disease was detected by cervical cytology. RESULTS: The CCR2-64I variant was significantly associated with cervical cancer when cases were compared to the control group (P = 0.001). Further analysis comparing selected groups within the controls showed that individuals with abnormal cytology and high grade squamous intraepitleial neoplasia (HSIL) did not have this association when compared to women with normal cytology. HPV infection also showed no association with CCR2-64I variant. Comparing SIL positive controls with the cases showed a significant association of CCR2-64I variant (P = 0.001) with cervical cancer. CONCLUSIONS: This is the first study of the role of CCR2-V64I polymorphism in cervical cancer in an African population. Our results show that CCR2-64I variant is associated with the risk of cervical cancer but does not affect the susceptibility to HPV infection or HSIL in South African women of black and mixed-ancestry origin. This result implies that the role of CCR2 is important in invasive cancer of the cervix but not in HPV infection or in the development of pre-cancers.
Open Access
Cellular immune responses to human papillomavirus (HPV) type 16 at the cervix of women with HPV-associated squamous intraepithelial neoplasia
(2005) Milner, Michelle; Passmore, Jo-Ann; Williamson, Anna-Lise
Cervical cancer is the most common cause of cancer-related death in black South African women. Human papillomavirus (HPV) has been found to be a necessary causative agent of cervical cancer and has been reported to be associated with 84% of cervical intraepithelial neoplasia (CIN). HPV type 16 (HPV-16) is the most prevalent HPV type associated CIN and cervical cancer with ±56% of women with cervical disease being infected with HPV 16. Yet studies have shown that 47-85% of CIN regressed, suggesting that perhaps an effective immune response could result in HPV clearance and lesion regression. Since HPV infection does not disseminate and there is no systemic phase of infection, it is hypothesized that local cervical immune responses are important in lesion regression and clearance of HPV infection. There are, however, very few studies of mucosal immune responses to HPV infection. The aim of this study was to determine the type of mucosal immune response elicited by the CD4 and CD8 T cell subsets to HPV infection at the cervix of women diagnosed with varying grades of CIN and to compare these to systemic responses.
Open Access
Characterisation of HIV superinfection : genetic evolution and adaptive immune responses
(2011) Harvey, Hayley Janet; Williamson, Carolyn
In this thesis we aimed to determine the timing and frequency of intra-subtype C superinfection, and to determine if the reason for superinfection was a greater genetic distance within epitopes of the superinfecting virus compared to those of circulating strains from the same cohort.
Open Access
Characterisation of HIV-1 Envelope features of breakthrough infections from the CAPRISA 004 Microbicide Trial
(2016) Ismail, Sherazaan Dineo; Williamson, Carolyn; Selhorst, Philippe
The CAPRISA 004 trial demonstrated the safety and a 39% efficacy of a 1% tenofovir (TFV) gel for the prevention of HIV-1 acquisition in young African women. It was subsequently shown that women assigned to the TFV arm who became infected had higher viral loads, slower anti-HIV-1 antibody avidity maturation, and higher Gag-specific IFN-γ+ CD4+ T cell responses; although replication capacity, as measured by Gag-Pro recombinant viruses, did not differ between arms. We thus aimed to investigate if there were differences in Envelope function, or TFV susceptibility, which may be selected for during transmission in those who became infected despite being assigned to the TFV arm. Viruses from 39 out of 48 recently HIV-1 infected individuals from the trial (matched on time post-infection and the presence of protective HLAs) were isolated. Isolate env genes were sequenced using a single genome amplification approach and were compared to plasma sequences from the same time-point. To evaluate phenotypic characteristics of env, inhibition assays were performed using the following inhibitors: tenofovir, maraviroc, T20, PSC-RANTES and anti-CD4 antibody clone SK3. In addition, envs for 19 participants were cloned and used to generate pseudoviruses which were evaluated for entry efficiency. Viral isolates were identical or very similar to viruses in circulation in vivo; however had a lower diversity, indicating that they were representative of in vivo virus but did not reflect the entire quasispecies in plasma. The TFV arm viruses were not more resistant to TFV than those in the placebo arm. A comparison of variable loop characteristics, distance to a consensus representative of viruses circulating in the region, and sensitivity to inhibitors or entry efficiencies of the viruses, also found no difference in genotypic nor phenotypic properties between study arms. When assessing the impact of viral phenotype on markers of disease progression, it was found that sensitivity to inhibitors did not contribute to VL or CD4+ count in this cohort. To evaluate envelope in isolation of the rest of the genome, pseudoviruses were generated from 11 participants. We found that PSV entry efficiency did not correlate with VL at isolation, 3 months post-infection and set-point, or with CD4+ counts at set-point. However, pseudovirus inhibitor sensitivities were significantly different to those of isolates for the inhibitors T20, anti-CD4 antibody SK3 and PSC-RANTES. Overall, the isolate env genotypic and phenotypic characteristics investigated in this study did not differ between trial arms. Interestingly, pseudoviruses showed significant differences in their sensitivity to entry inhibitors when compared to their corresponding isolate, highlighting the importance of caution when interpreting data from in vitro studies, and motivates for further evaluation of in vitro models.
Open Access
Characterization of an Fc-receptor for human IgG in the tegument of human cytomegalovirus
(1991) Hardie, Diana Ruth
Open Access
Characterization of genotypic and phenotypic properties of transmitted Human Immunodeficiency virus type 1 variants circulating in Mbeya Tanzania
(2013) Nofemela, Andile; Williamson, Carolyn; Woodman, Zenda
Includes abstract. Includes bibliographical references.
Open Access
Classification of HIV virological failure using whole blood versus plasma viral load
(2016) Khan, Aabida; Hsiao, Marvin
Introduction: HIV viral load testing is the preferred monitoring approach for HIV infected patients on combination antiretroviral therapy (cART) as it is more sensitive than CD4 count and clinical monitoring. In resource limited settings, timely plasma separation and transportation to testing laboratories is a major barrier to the access of HIV viral load testing. The 2015 World Health Organisation guidelines recommend that cART should be initiated in all adults and children living with HIV regardless of disease stage or CD4 count, thereby escalating the demand for HIV viral load testing. Potential solutions to expand implementation and scale up of viral load testing in low and middle income countries are whole blood testing through point of care (POC) viral load assays or dried blood spots (DBS) collected at the health facility. Utilization of whole blood instead of plasma would simplify sample collection, storage and transportation requirements and be cost effective. However, the paucity of studies comparing whole blood HIV viral load across different test platforms, especially in the correct classification of virological failure, has resulted in the lack of a standardised programmatic approach to whole blood viral load testing. Methods: We evaluated four HIV whole blood viral load test methods namely Alere q HIV-1/2 POC, Abbott RealTime HIV-1 DBS original and updated protocols, and Roche CAP/CTM DBS free virus elution (FVE) protocol, against the standard of care, plasma viral load, on 299 samples across the viral load spectrum from South African patients on cART. Virological failure was defined at >1000 copies/ml. Proportions of correct classification of virological failure and overall correlation with plasma were used for evaluating each method's performance. Results: Alere q, Abbott original and updated, and Roche FVE correctly classified virological failure in 61%, 89%, 87% and 76% of all samples tested respectively. The performance varied across plasma viral load categories. Alere q showed good correlation above plasma viral load of 1000 copies/ml, with correct classification of virological failure in 100% of samples. However, below the plasma threshold of 1000 copies/ml, Alere q demonstrated significant over-quantification, resulting in reduced specificity and upward misclassification of virological failure in 39% of all samples tested. Abbott original and updated also had good sensitivity of 98% and 91% respectively and the best overall correlation with plasma (r² = 0.76 and 0.72 respectively), but there was upward misclassification in 10% and 8% of samples tested respectively. Roche FVE had the best specificity of 99% but with significantly reduced sensitivity of 53%, especially between 1000–10,000 copies/ml of plasma, resulting in downward misclassification in 24% of all samples tested. Greatest variability between the different testing methods was seen when plasma viral load was 40-1000 copies/ml. Correlation was best for all whole blood viral load assays at >10,000 copies/ml. Conclusion: The key finding highlighted by this study is the great variability between the different whole blood test methods. Various factors influence the ability to quantify whole blood HIV viral load such as input volume used in each assay vary, sample treatment/processing (DBS versus fresh blood samples versus FVE), extraction (RNA selective, total nucleic acid extraction), amplification target and detection methods are different for each of the platforms tested. Based on our study, Alere q and Abbott DBS need to raise their whole blood threshold for virological failure in order to reduce upward misclassification and Roche FVE needs to achieve better sensitivity around its limit of detection. Receiver operating characteristic curve analysis can be used to determine the optimum threshold of virological failure for each assay.
Open Access
Cloning and expression of a functionally active truncated N-glycosylated KSHV complement regulatory protein and immunohistochemical studies with the anti-KCP peptide antibody
(2005) Gomes Pereira, Neuza Alexandra; Kotwal, Girish J
Kaposi sarcoma herpes virus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement-binding protein, KCP) that binds complement proteins and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Kaposi's sarcoma is an angiogenic skin lesion that has been recognized as one of the most abundant tumours found in many parts of Southern Africa and which can occasionally become highly invasive, aggressive and capable of causing death, particularly amongst AIDS patients. It is of major significance to understand how complement control proteins (CCPs) such as KCP perform their biological functions at the molecular and structural levels, because of their potentials as therapeutic agents, their implications in the pathology and importance in the etiology of many disease conditions. This study was therefore undertaken to characterise the structure-function relationship of KCP. Based on primary sequence analysis and comparison to other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by PCR, three regions of the predicted ORF 4 from human herpes virus-8 (llliV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris and to produce separately, the 4 N-terminal Sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane binding domain (KCP-M, medium) and the full-length protein (KCPF, full). Expression of the viral proteins was confirmed by SDS-PAGE and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. All the KCP proteins migrated electrophoretically as higher bands compared to their expected sizes. The lower mobilities of the proteins may be due to g1ycosy1ation since there are potential N-and O-glycosylation sites in the protein's primary sequence. Also, diffused bands were obtained in all the electrophoretic gels and Western blots carried out, which is characteristic of glycoproteins. Furthermore, the antibody recognized several larger and smaller bands that may represent aggregates and/or degradation products respectively. Both partially purified KCP-S and KCP-S directly from expression media were able to inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice more efficient in inhibiting the classical pathway-mediated lysis of erythrocytes than the vaccinia virus complement control protein (VCP), which also contains 4 Sushi domains. The KCP-F and KCP-M proteins did not show any significant complement inhibitory activities. Preliminary immunohistochemical studies using the same antibody were carried out to determine the expression and distribution of KCP proteins in Kaposi's sarcoma.
Open Access
Comparative analysis of avian poxvirus genomes, including a novel poxvirus from lesser flamingos (Phoenicopterus minor), highlights the lack of conservation of the central region
(BioMed Central, 2017-12-06) Carulei, Olivia; Douglass, Nicola; Williamson, Anna-Lise
Background: Avian poxviruses are important pathogens of both wild and domestic birds. To date, seven isolates from subclades A and B and one from proposed subclade E, have had their genomes completely sequenced. The genomes of these isolates have been shown to exhibit typical poxvirus genome characteristics with conserved central regions and more variable terminal regions. Infection with avian poxviruses (APVs) has been reported in three species of captive flamingo, as well as a free-living, lesser flamingo at Kamfers dam, near Kimberley, South Africa. This study was undertaken to further characterise this virus which may have long term effects on this important and vulnerable, breeding population. Results: Gene content and synteny as well as percentage identities between conserved orthologues was compared between Flamingopox virus (FGPV) and the other sequenced APV genomes. Dotplot comparisons revealed major differences in central regions that have been thought to be conserved. Further analysis revealed five regions of difference, of differing lengths, spread across the central, conserved regions of the various genomes. Although individual gene identities at the nucleotide level did not vary greatly, gene content and synteny between isolates/species at these identified regions were more divergent than expected. Conclusion: Basic comparative genomics revealed the expected similarities in genome architecture but an in depth, comparative, analysis showed all avian poxvirus genomes to differ from other poxvirus genomes in fundamental and unexpected ways. The reasons for these large genomic rearrangements in regions of the genome that were thought to be relatively conserved are yet to be elucidated. Sequencing and analysis of further avian poxvirus genomes will help characterise this complex genus of poxviruses.
Open Access
A comparative analysis of cowpox virus (CPV WT) and a deletion mutant lacking the gene encoding the inflammation modulatory protein (CPV IMP)
(2007) Paulsen, Janis; Douglass, Nicola; Williamson, Anna-Lise
Cowpox virus has been found to encode the inflammation modulatory protein (IMP) (Miller, C.G., 1997), a homologue vaccinia virus complement control protein (VCP). VCP belongs to regulation of complement activation (RCA) protein superfamily. It has been shown to inhibit both the alternative and classical pathways of complement activation by binding to the proteins C3 and C4, thereby preventing complementmediated opsonisation of virus, antibody-mediated lysis of infected cells and migration of inflammatory cells into the site of infection. VCP also possesses heparin binding sites.
Open Access
Comparing high-throughput methods to measure antibody dependent cellular cytotoxicity during HIV infection
(2014) Mbodo, Iyaloo; Passmore, Jo-Ann; Gamieldien, Hoyam
The prevalence of HIV-1 is highest in Sub-Saharan Africa. Protective immune responses directed against HIV are complex and involve both cellular and humoral immunity. Based on the recent finding that the best correlate of protection against the first protective prophylactic RV144 vaccine were HIV-specific antibody responses, including those mediating natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC), there has been considerable interest in measuring alternative roles for HIV-specific binding antibodies. The aim of this MSc dissertation was to optimise and compare two high-throughput flow cytometry based approaches - the GranToxilux and PanToxilux assays - to measure HIV-specific ADCC responses. To do this, NK cells from a panel of healthy HIV-negative individuals were screened for their ability to directly kill the tumour cell line K562, as a measure of direct NK cell cytotoxicity. The individual with the highest granzyme B and caspase activity against K562 cells was chosen as the universal NK cell donor for this study.
Open Access
Comparison of HIV-1 specific T cell immunity in the female genital tract and blood of HIV-infected women : impact of in vitro T cell expansion on HIV-specific T cell specificity, maturational status and functional complexity
(2010) Bere, Alfred; Passmore, Jo-Ann
This study shows that HIV-specific cervical T cells can be isolated by cytobrushing and in vitro polyclonal expansion is a useful approach to increase the number of T cells available from mucosal sites. Dynal beads (1:1) in the presence of IL-2, IL-7 and IL-15 resulted in the best yields of cervical T cells while anti-CD3 in the presence of IL-2 best conserved the ex vivo T cell profile. Expanded T cell lines, irrespective of expansion method used, generally maintain their cytokine response profile to HIV anti- gens. This study shows that HIV Gag-specific blood and cervical T cells were largely mono-functional with polyfunctional T cells being detected in women with high blood CD4 count and low plasma viral load. This study confirms that HIV-specific Gag T cell responses detected in the polyclonal expanded female genital tract T cells are associated with those measured in blood during HIV infection.
Open Access
Construction, stability and immunogenicity of recombinant BCG expressing HIV-1 subtype C gag under the control of MtrA promoter, with or without the leader sequences
(2011) Lebeko, Maribanyana R; Chapman, Ros; Williamson, Anna-Lise
This study aimed to compare recombinant mycobacteria expressing HIV-1 gag under the control of different promoters and leader sequences. This was done to determine whether the genetic stability of the recombinant mycobacteria could be improved by modification of these vector features and to gain insight into what types of immune responses may be elicited in mice.
Open Access
Correction to: Microbial function and genital inflammation in young South African women at high risk of HIV infection
(2022-03-09) Alisoltani, Arghavan; Manhanzva, Monalisa T; Potgieter, Matthys; Balle, Christina; Bell, Liam; Ross, Elizabeth; Iranzadeh, Arash; du Plessis, Michelle; Radzey, Nina; McDonald, Zac; Calder, Bridget; Allali, Imane; Mulder, Nicola; Dabee, Smritee; Barnabas, Shaun; Gamieldien, Hoyam; Godzik, Adam; Blackburn, Jonathan M; Tabb, David L; Bekker, Linda-Gail; Jaspan, Heather B; Passmore, Jo-Ann S; Masson, Lindi
Correction to: Microbiome 8, 165 (2020) https://doi.org/10.1186/s40168-020-00932-8
Open Access
Cytomegalovirus viraemia in immunocompromised children in Cape Town
(2009) Hsiao, Nei-Yuan; Hardie, Diana
Includes bibliographical references (leaves 54-62).