Masters Degrees (Medical Virology)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/3155

Browse

Now showing 1 - 20 of 52

An anti-SARS-CoV-2 quantitative study to determine the effect of time on the antibody titre, post-vaccination
(Stellenbosch : Stellenbosch University, 2024-01) Meyer, Burnet Adriaan; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: In 2019, the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) outbreak emerged in Wuhan, China, rapidly evolving to a pandemic. Vaccines such as messenger ribonucleic acid (mRNA)- based and Adenovirus-based, have been developed to counter severe Coronavirus Disease 2019 (COVID-19). However, many factors, for example, immunosuppression and prior infection can affect the antibody titre. This study evaluated the effectiveness in Anti-SARS-CoV-2 immunoglobulin (Ig) G production and decline over time, indicating the potential requirement for additional boosters. Furthermore, this study focused on how Adenovirus-based vaccine boosters and breakthrough infections affected the antibody titre. A total of 184 samples were collected from two cohorts which consisted of 67 participants. Samples were collected at baseline, week 3, and week 6 post-booster for Cohort A (laboratory workers at the Western Cape Blood Service). Cohort B (staff and students at the Faculty of Medicine and Health Sciences, Stellenbosch University) had samples collected at baseline, week 2, week 4 and week 8 post-booster. The samples were tested for the presence of the SARS-CoV-2 Anti-nucleocapsid (N) as well as Anti-spike (S) antibodies. The Anti-N antibodies indicated that a participant had a natural infection to SARS-CoV-2 while the Anti-S antibodies were utilised to examine the influence of age, gender and time on vaccine-induced antibodies. This findings of this study demonstrated that vaccine-induced antibodies were still detectable six months following the initial vaccination. A significant increase was observed for both cohorts, when comparing the baseline titre with the different timepoints, post-booster. Multiple samples were found to be Anti-N positive, suggesting that these participants were potential asymptomatic cases. Variations were found in the Anti-S antibody titre based on different age groups, although these variations were not statistically significant. Moreover, the results have indicated that gender does not affect the Anti-S antibody titre. This research enhances the understanding of antibody titre dynamics, contributing to a better grasp of immune longevity. Further studies are recommended to determine the impact of combining different boosters on the antibody titre, and assess the difference in antibody titre between monovalent boosters with bivalent boosters.
The apoptotic potential of different HIV-1 subtype C Tat mutations in cell culture
(Stellenbosch : Stellenbosch University, 2013-03) Isaacs, Shahieda; Engelbrecht, Susan; Glashoff, Richard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
The efficiency in which HIV-1 can infect, spread and evade the attack of therapeutic agents can be attributed to a high mutation rate and frequent recombination events. These factors have collectively contributed to the diversity observed in HIV-1 and resulted in a multitude of subtypes, sub-subtypes, circulating recombinant forms (CRF’s) and unique recombinant forms (URF’s). The aim of this study was to investigate HIV-1 diversity in Cape Town using a small cohort of treatment naive patients being investigated for HIV Associated Neurocognitive Disorders (HAND). Four different genomic domains: gag, pol, accessory and gp41 genes were sequenced to subtype the virus. HIV-1 tat was further investigated because the dicysteine motif has been reported to play a role in HAND. Viral RNA and proviral DNA was extracted from 64 patients and used for the amplification and sequencing of the genes. Rega and jpHMM online tools were used to identify HIV-1 subtypes and recombinants while Neighbor-joining phylogenetic trees were constructed for phylogenetic analysis. The pol gene was further investigated using SCUEAL to detect possible intra-subtype recombination and was also screened for the presence of transmitted drug resistance. In addition tat sequence datasets retrieved from the Los Alamos sequence database were investigated and compared with the newly generated sequences for the detection of point mutations and amino acid signature patterns. Sequencing identified most of the samples as subtype C; however six inter-subtype recombinants (AE, A1G, A1CU and two BC) and 9 intra-subtype C recombinants were identified. In addition 13% of pol sequences were identified with resistance mutations. Signature pattern analysis identified a high level of variability in the tat sequences: 68% were identified with C30S31; 29% with the C30C31 mutation and a single sequence with a novel mutation C30A31. Functional analysis of these mutations indicated that all mutations investigated were capable of inducing apoptosis in cell culture. The C30C31 mutation generated the highest level of apoptosis, closely followed by the C30A31 mutation. However no statistical significance could be detected between tat mutations and the observed levels of apoptosis.
The application of wastewater-based epidemiology to Hepatitis E virus in South Africa
(Stellenbosch : Stellenbosch University, 2024-02) Roberts, Bronwyn; Maponga, Tongai; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Background Hepatitis E virus (HEV) has a global presence, but the highest burden of disease is found in Africa and Asia. Unfortunately, in Sub-Saharan Africa, there is a gap in knowledge on the molecular epidemiology of the disease, as often serological methods only are applied. In recent years a high seroprevalence of the virus has been detected in populations in the Western Cape province. Evidence of viraemia has been published on a few South African patients from the Western Cape. Viral ribonucleic acid (RNA) was also detected in swine samples from an abattoir and in porcine meat products in Cape Town. Based on the existing literature, HEV genotype 3 has been detected in patients and swine samples. In this study, the aim was to determine whether HEV was circulating in communities in the Western Cape by testing wastewater extracts for the presence of HEV RNA. The study also sought to determine the HEV genotype in wastewater sample extracts. Methods One hundred and forty-three wastewater extracts were tested for the presence of HEV RNA using real-time polymerase chain reaction (PCR). Samples were sourced from four locations: two wastewater treatment works and sewer manholes from two Stellenbosch University residences. Nested reverse-transcriptase PCR (RTPCR) targeting three regions of the HEV genome was applied to samples which tested positive for HEV RNA. The sequenced regions were a portion of open reading frame (ORF) 2 covering 347 base pairs (bp), a 286 bp region of ORF1 and a 126 bp region of the overlapping ORF2/3. The generated PCR products were then sequenced with Sanger sequencing. The generated consensus sequences were placed in a phylogenetic tree with reference sequences for HEV genotypes 1 to 8, to determine which genotype is likely circulating in the selected communities. Results Out of the 143 wastewater samples, 130 valid results were generated. Out of 130, 21 (16.2%%) were positive and 109 (83.9%) were negative. Of the 21 positive samples, 5 (23.8%) were collected from Athlone wastewater treatment works (WWTW), 9 (42.9%) from Zandvliet WWTW, 1 (4.8%) from Meerhoff residence in Tygerberg and 6 (28.6%) from Metanoia residence in Stellenbosch. The positive samples had a median cycle threshold value of 37.9 (interquartile range: 36.7-39.7). Out of the 21 positive samples, 4 (19.1%) sequences were obtained from the ORF2/3 region. The sequences clustered most closely with a sequence from a South African patient and HEV genotype 3 reference sequences. Conclusions Based on the real-time PCR results, it appears that HEV is circulating among communities in the Western Cape province. Based on the detection of HEV genotype 3 in the wastewater samples, it suggests that zoonotic transmission may be the mostly likely route of infections. Further investigation to identify porcine and other food products which may be contaminated with HEV are necessary to break chains of transmission.
B lymphocyte activation and exhaustion in chronic HIV : novel surrogate markers of generalised immune activation and selective modulation of aberrant B cell responses using vasoactive intestinal peptide (VIP)
(Stellenbosch : Stellenbosch University, 2015-04) Reid, Timothy Dawson; Glashoff, Richard H.; Ipp, Hayley; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Introduction: Chronic HIV-1 infection is characterized by immune activation and dysregulation of immune homeostasis, which impacts on multiple immune cell types. The B-cell compartment, which plays an important role in the producing neutralizing antibodies, is also dysregulated in HIV- 1 infection. In this study we investigated peripheral blood B-cell subset distribution, and changes in expression of cellular activation, inhibition, and apoptosis signaling markers in both untreated chronic HIV-1 infected individuals and healthy uninfected controls. The neuropeptide immune modulator, vasoactive intestinal peptide (VIP) is known to selectively down-regulate activation of CD4+ T-cells in various disease settings including HIV-1, however to our knowledge, no studies have investigated the effect of VIP inhibition on B-cell activation. Materials & Methods: A total of 21 HIV+ve (CD4 count >250 cells/µl), and 19 HIV-ve individuals were recruited from the Emavundleni voluntary testing and counseling clinic in Crossroads, Western Province, South Africa. Whole blood was stained to distinguish B-cell subsets (activated memory (AM: CD21-CD27+), resting memory (RM: CD21+CD27+), mature naïve (MN: CD21+CD27-), or tissue-like memory (TLM: CD21loCD27lo). In addition expression of markers of B-cell activation (CD126, CD86, CD38, CD284, CD287), inhibition (CD72, CD85j, CD300a, CD305, CD307d), and apoptosis signaling (CD95), was assessed ex vivo by flow cytometry (BD FACSCanto II). For determination of functional responsiveness isolated B-cells (RosetteSep, Stemcell Technologies) were cultured for 18h (37°C, 5%CO2) without stimulation or stimulated with TLR ligands (LPS or R848). Stimulation experiments were also performed in the presence or absence of VIP. Results: Chronic HIV-1 infection affected B-cell subset distribution. The percentage (%) of TLM was increased by 59.24%, and %RM was decreased by 22.73% (both p<0.01). Total expression of the VIP receptor VPAC2 was decreased by 47.35% (p=0.0296). Subsets had a mixed phenotype ex vivo; HIV infection upregulated CD38 (by 59.56%, p=0.0004), CD72 (by 60.70%, p=0.0396), CD307d (by 68.63%, p=0.0015) on AM, while RM B-cells had increased expression of TLR4 (by 107.04%, p=0.0057) and TLR7 (by 208.14%, p=0.0199). TLM B cells (i.e. exhausted phenotype) displayed upregulated TLR7 (by 550%, p=0.0128) and CD307d (by 72.40% p=0.045) expression. MN B-cells had increased CD72 expression (by 70.98%, p=0.0026). R848 upregulated CD86 expression by 42.20% on AM (p<0.01), and by 56.06% on RM B-cells (p<0.01), which was significantly downregulated with VIP inhibition (both p<0.05). Similarly, CD95 expression on RM, TLM, and MN B-cells increased by 31.10% (p<0.001), 21.46% (p<0.01), and 39.92% (p<0.01) with R848 stimulation respectively, which was also significantly downregulated with VIP inhibition. Conclusion: These data indicate that B-cells in untreated HIV infection display increased levels of activation, and also the potential for increased susceptibility to apoptosis as evidenced by increased FAS (CD95) expression. VIP significantly down-regulated markers of activation, inhibition, and apoptosis signaling. Dysregulation of B-cells is thus apparent in asymptomatic stable chronic HIV-1 infection, which may impact on both inefficient neutralizing antibody production and hypergammaglobulinemia. The ability of VIP to prevent stimulationassociated marker upregulation may indicate that VIP is a potential therapeutic agent. Its immuno-modulatory properties were demonstrated to limit B-cell hyperactivation, and selectively down-regulate apoptosis and mark it out for further investigation.
The characterisation and expression of HIV-1 subtype C gag
(Stellenbosch : Stellenbosch University, 2002) Sampson, Candice Corene; Van Rensburg, E.J.; Engelbrecht, S.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.
ENGLISH ABSTRACT: The gag gene of HIV-1 encodes for one of the major structural proteins, which contains several conserved cytotoxic T cell (CTL) epitopes. Gag specific CTL responses are important in controlling viral load during acute infection and asymptomatic stages of the infection. Currently, only one complete South African HIV-1 subtype C gag sequence has been published. The first aim of this study was to characterise the complete gag gene of 15 HIV-1 subtype C isolates, to be used as a set of reference sequences in the design of a South African HIV-1 subtype C vaccine. Fifteen HIV-1 subtype C isolates selected for this study, were isolated during 1998 and 1999 from the HIV-1 positive patients attending the Infectious Disease Clinic at Tygerberg Hospital. The gag gene of these isolates was amplified by PCR, cloned into mammalian expression vectors and sequenced. Restriction digest analyses as well as phylogenetic analyses were performed on the sequencing data. Previously published mutational analyses and CTL epitopes were compared to the predicted amino acid sequences of the gag clones. Sequences of 23 complete gag genes representing the 15 HIV-1 subtype C isolates as well as one complete sequence of an HIV-1 subtype B isolate were compiled. Subtyping by restriction fragment length polymorphism (RFLP) would have correctly identified 14 of the 15 subtype C isolates as subtype C and one as unidentifiable. The subtype B isolate would have also been correctly identified. Phylogenetic analyses showed that our subtype C isolates clustered with reference subtype C strains from various countries, including Botswana, India, Israel, Tanzania and Zambia. Strains from Ethiopia and Brazil formed a separate subtype C cluster. The diversity between our isolates was comparable to the diversity seen between all the HIV-1 subtype C strains. Comparisons of previously published mutational analyses and CTL epitopes to the predicted amino acid sequences of the gag clones, showed conservation in most of the clones throughout the sequence. A second aim was to establish transfection and Western Blot techniques in our laboratory for use in future studies. An in vitro transcription! translation assay was performed on the gag clones and the protein producing clones were used to transfect mammalian cells using electroporation. A Western blot was then used to screen for Gag protein expression in the transfected cell Iysates. The in vitro transcription! translation assay showed that seven of the 23 clones could produce a protein of -55 kDa in size. Four out of the seven of these clones gave a weak expression of a-55 kDa protein after transfection in a mammalian cell line. Since the completion of the experimental work of this study, other cloned HIV-1 genes have successfully been transfected into mammalian cells using the electroporation technique and the proteins produced were screened for by Western blot. To conclude with; the native form of the gag gene does not elicit strong expression of the protein, but studies have shown that expression can be improved by sequence-modification of the gag nucleotide sequence. Due to the conservation of gag, the sequence of any subtype C strain can be used for the development of a Southern African vaccine.
Characterisation of new full-length HIV-1 subtype D viruses from South Africa
(2004-12) Loxton, Andre Gareth; Janse van Rensburg, E.; Engelbrecht, S.
ENGLISH ABSTRACT: The first episode of HIV-1 in South Africa was documented in 1982. Homosexual transmission of the virus was the predominate mode of transmission in an epidemic of mainly HIV-1 subtype Band D infections. To date, no full-length sequences of Subtype D strains from South Africa has been reported. Here we describe the characterization and some of the unique features of the Tygerberg HIV-1 subtype D strains. A near full-length 9 kb fragment was obtained through a one step PCR using high molecular weight DNA. Cloning was done successfully with the pCR-XLTapa cloning kit. Large quantities of plasmid DNA was grown and sequenced on both strands of the DNA. ORF determination and subtyping was followed by standard phylogenetic methods to construct evolutionary phylogenetic trees. Subtyping and similarity plots revealed that the sequences from Tygerberg are pure subtype D. All the Tygerberg strains had intact genes with no premature stop codons. At the tip of the V3 loop, the Tygerberg strains have the GOGO motif. R214 has a more variable vpu gene than the rest of the Tygerberg strains, but is still subtype D in this region. No premature stop codons have been observed in the tat gene and the glycosilation of the strains are less than the subtype D consensus. We are the first to report full-length sequences of HIV-1 subtype D strains from South Africa. The sequences represent non-mosaic genomes of subtype D. Our results confirm that the subtype D sequences from the beginning of the HIV-1 epidemic differ from the Subtype D sequences from recent isolates.
Characterisation of rotavirus strains responsible for breakthrough diarrhoeal disease among Zambian children using whole genome sequencing
(Stellenbosch : Stellenbosch University, 2024-01) Mwape, Innocent; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Background Genetically altered viruses or variants have the potential to increase their virulence, pathogenicity, transmission, and ability to evade both natural and vaccine-induced immune responses leading to diarrhoeal disease in under five-year-old children who have received all recommended doses of Rotavirus (RV) vaccines, also known as breakthrough infections. Studies characterising RV strains responsible for breakthrough infections are rare in resource-limited countries like Zambia where RV-associated diarrhoeal disease is endemic. We aimed to characterise RV strains detected in fully vaccinated under five-year-old children residing in Zambia using next generation sequencing. Methods This was a case study nested under an open label randomised controlled RV vaccine clinical trial that evaluated safety and immune boosting effects of a third dose of Rotarix compared to a two-dose schedule. The Rotaclone kit was used to screen for RV in stool. We performed VP7 and VP4 genotyping on RV positive stool using Sanger sequencing. Whole genome sequencing was done on the Illumina Miseq platform. Genome assembly was done using Geneious software and multiple sequence alignment using Muscle in MEGA version 6. Results A total of 76 diarrhoeal stool specimens were collected and screened for RV of which 4/76 (5.2%) were positive. Genotypes of three of the four cases were identified as G1P[4], G12P[4] and G12P[8] using Sanger sequencing. Whole genome analysis revealed that the RVA/Human-wt/ZMB/CIDRZRV2088/2020/G1P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and RVA/Human-wt/ZMB/CIDRZ-RV2106/ 2020/G12P[4]-I1-R2-C2-M2-A2-N1-T2-E1-H2 strains were mostly DS-1-like with mono and multiple reassortant respectively, whilst the RVA/Human-wt/ZMB/CIDRZ-RV2150/2020/G12P[8]-I1-R1-C1- M1-A1-N1-T1-E1-H1 was a typical Wa-like strain. Comparison of VP7 antigenic epitope of strains causing breakthrough infections and Rotarix vaccine strains revealed several amino acid differences like G96P and M217E. Comparison of P[4] strains with VP4 of the Rotarix vaccine strain demonstrated two amino acids differences (P114Q and V115T) were P114Q is an immune escape mutation. Discussion and Conclusion Differences observed in amino acids in antigenic epitope suggested its role in the immune evasion of neutralising antibodies elicited by the vaccine. Findings from this study have potential to inform national RV vaccination strategies and the design of highly efficacious universal RV vaccines. Furthermore, there might be need to monitor strains that have escaped vaccine-induced immunity to prevent diarrheal diseases in children under five years of age.
Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells
(Stellenbosch : Stellenbosch University, 2003-03) De Villiers, Tania; Janse van Rensburg, E.; Engelbrecht, S.; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: At the end of 2002, human immunodeficiency virus (HIV) had infected 42 million people worldwide. The morbidity and mortality rate, as well as the epidemic proportions of the disease have led to concentrated scientific efforts to reveal the disease's pathogenesis and develop effective preventative and treatment measures. Advances have been made to inhibit viral replication by suppressing the virus' ability to replicate by developing antiretroviral treatments, although development of a save and effective vaccine is the only way to stem the pandemic. Advances in vaccine design, animal models and clinical research have led to the creation of promising candidate vaccines to counter this rampage, but most of these vaccines entering phase I-III clinical trials are based mainly only subtype B genomes. HIV-1 subtype C is the most commonly transmitted subtype worldwide, and is the predominant subtype in India, China, East and Southern Africa. A subtype C vaccine is critical for the developing nations such as South Africa, where antiretroviral therapies are largely unaffordable. The envelope gene (env) is an attractive target as immunogen to be included in a HIV vaccine. The envelope protein (Env) elicits neutralising antibodies and cytotoxic T-Iymphocyte (CTl) responses. This protein will therefore be useful in creating a humoral and cellular immune response in the host. A shortage in characterised subtype C env gene sequences from South Africa was recognised, and this study focussed on the characterisation of generated sequences, as well as the expression of selected env genes. These immunogens were created for possible use in a prime-boost vaccine modality. The env genes from recent circulating strains in South Africa were amplified by polymerase chain reaction (PCR). The genes were then cloned for sequencing and expression purposes. Phylogenetic relationships were determined by comparing the sequences to reference subtype strains and subtype C strains. Expression of the genes was assessed by Western Blot in 293 cells with HIV- 1 positive patient sera. Sequence analysis showed a more conserved third variable (V3) loop in South African subtype C sequences, with a more variable region downstream from the loop. The crown sequence (GPGQ) and positions of uncharged or negatively charged residues in the V3 loop indicated a non-syncytium-inducing (NSI) phenotype for the isolates. Phylogenetic analysis showed the sequences to all belong to the C subtype, and further that the sequences were not recombinant, which was confirmed by recombination analysis. The intersample diversity observed for strains from South Africa was significantly higher than distances observed to the subtype C consensus sequence. The South African sequences were distributed across several subclusters in a subtype C phylogenetic tree, highlighting the concept that these infections represent a more longstanding epidemic with multiple introductions from different geographic areas. Western Blot with HIV-1 positive patient sera showed the expression of uncleaved gp160 Env proteins, which were Rev dependent. This study has generated much needed subtype C South African env gene sequences that can be used as basis for modification for use as immunogens in a South African vaccine.
Characterization 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.
Coreceptor expression and T lymphocyte subset distribution in HIV-infected and TB co-infected South African patients on anti-retroviral therapy
(Stellenbosch : University of Stellenbosch, 2009-12) Ngandu, Jean Pierre Kabue; De Beer, Corena; Glashoff, R. H.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: In 2007, AIDS caused an estimated 2.1 millions deaths worldwide; about 70% in sub-Saharan Africa. HIV preferentially targets activated CD4 T cells, expressing the major HIV receptor CD4, as well as the major chemokine coreceptors CCR5 and CXCR4. These coreceptors play a prominent role during HIV cell entrance phase, HIV transmission and also disease progression. They have been found to be differentially expressed by CD4 T cell subsets. Tuberculosis coinfection may enhance immune activation in vivo thus accelerating HIV disease progression and has become a major challenge in the control of TB in Africa. Introduction of HAART has reduced disease progression to AIDS, as well as risk of further morbidity and mortality. HAART results in a rapid decline of viral load and an initial increase of peripheral CD4 count, however little is known on the effect of HAART in regulation of coreceptor expression, immune activation status and CD4 T cell subset distribution in HIV infection and HIV/TB coinfection. This study is a cross-sectional analysis of coreceptor expression, immune activation status and CD4 T cell subpopulation distribution in South African HIV and HIV/TB coinfected patients before and after ARV. A total of 137 South African individuals were investigated, comprising 15 healthy normal donors (healthy subgroup), 10 patients with active pulmonary tuberculosis (PTB subgroup), 33 HIV-1 positive patients without active PTB (HIV subgroup), 23 positive patients with active PTB (HIV/PTB subgroup), 36 HIV-1 positive patients on ARV (HIV on ARV subgroup) and 20 HIV-1 positive patients with active PTB on ARV (HIV/PTB on ARV subgroup). CD4 absolute count and plasma viral load were determined for all donors. Freshly isolated PBMC were classified by flow cytometry into the following CD4+ T lymphocyte subsets: naïve (CD45+, CD27+), effector memory (CD45-, CD27-), central memory (CD45-, CD27+), and effector (CD45+, CD27-). Coreceptor expression and activation status was assessed by CCR5, CXCR4 and CD38 expression on CD4 T cell subsets. HIV, TB and HIV/TB coinfection was associated with a decrease in percentage CCR5+ T cells as compared to healthy controls, with the HIV/TB group showing the most extensive decrease. In treatment naive patients, CD4 T cells showed elevated surface expression of CCR5 and CD38 as determined by mean fluorescence intensity in HIV/TB co-infection compared to HIV infection alone. The percentage of antigen-experienced cells was higher in the HIV/TB co-infected group compared to the HIV group. The percentage of naïve T cells was decreased in both the HIV infected and the HIV/TB co-infected groups compared to healthy controls. HIV patients with more than 6 months of ARV showed decreased CCR5 and CD38 surface level expression in the HIV and the HIV/ TB co-infected subgroups. An increased percentage of naïve T cells was observed in the HIV infected subgroup, but not in the HIV/TB subgroup, similarly, a decreased percentage of antigen-experienced cells was observed in the HIV subgroup, but not in the HIV/TB co-infected subgroup. A positive correlation was found between CCR5 and CD38 expression, and CXCR4 and CD38 expression (Spearman coefficient of correlation respectively: r=0.59, p<0.001 and r=0.55, p<0.001). Furthermore we found plasma viral load positively associated with CD38 expression (r=0.31, p<0.001) and percentage activated CCR5+ expressing CD4 T cells positively related to viral load (r=0.31, p<0.001). Percentage naïve CD4 T cells was positively associated with CD4 count (r=0.60, p<0.001) and negatively correlated to viral load (r=-0.42, p<0.001). These results indicate that TB coinfection exacerbates certain aspects of dysregulation of CD4 T cell homeostasis and activation caused by HIV infection. In addition, ARV-associated decrease in coreceptor expression, immune activation status and a normalisation of CD4 T cell subset distribution was observed in HIV infected individuals, but not in HIV/TB coinfection. Despite viral suppression after ARV treatment, the decline in the immune activation marker CD38 and coreceptor CCR5 expression, increase in percentage naïve CD4 T cells and decrease of antigen-experienced cells did not reach the levels displayed in the healthy control group. This may indicate that ongoing (albeit reduced) T cell immune activation may occur in the presence of ARV. Further longitudinal studies are needed to closely monitor immune activation during ARV treatment. This study highlighted an association of TB disease with immune activation in HIV infection, the importance of T-cell activation in HIV pathogenesis and its impact on ARV treatment. Further studies are needed to identify causative factors that may lead to a persistent immune activation status during ARV treatment, and how TB coinfection confounds normal responses to ARV.
Delays in HIV-1 infant PCR testing may leave children without confirmed diagnoses
(Stellenbosch : Stellenbosch University, 2021-12) Mahlakwane, Kamela L.; Van Zyl, Gert; Preiser, Wolfgang; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
Background The early diagnosis and confirmation of HIV infection in newborns is crucial for expedited antiretroviral therapy initiation. Confirmatory testing must be done for all children with a reactive HIV PCR result. There is no comprehensive data on confirmatory testing and rejection of HIV PCR test requests at National Health Laboratory Service laboratories. Aim and objectives To assess relevant measures for routine infant HIV PCR testing: rate of rejected test requests, turnaround time, and rate of confirmatory testing. Method A retrospective review was performed on the laboratory-based data of all HIV PCR tests that were performed on children ≤24 months old (n=43,346), and data of rejected HIV PCR requests (n=1,479) over a two-year period (2017-2019). These data were extracted from the laboratory information system. Data were analyzed from sample collection to release of results, assessing the TAT and follow-up patterns. Results The proportion of HIV PCR requests that were rejected was 3.3%, of which 83.9% were rejected for various pre-analytical reasons. The majority of test results (89.2%) met the required 96-hour TAT. Of the reactive initial test results, 53.5% had a follow-up sample sent, of which 93.1% were positive on follow-up. Of the initial indeterminate results, 74.7% were negative on follow-up. Conclusion A significant proportion of HIV PCR requests were rejected for various pre-analytical reasons. The high number of initial reactive tests, without evidence of follow-up, may suggest that a shorter TAT would be required to allow confirmatory testing, before children are discharged.
Development of selective real-time PCR (SPCR) asays for the detection of K103N resistance mutation in minor HIV-1 populations
(Stellenbosch : Stellenbosch University, 2011-12) Seleka, Mpho Maria; Engelbrecht, Susan; Van Zyl, Gert; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Background: The conventional sequence analysis is the most common method used for the detection of drug-resistant mutants. Due to its sensitivity limitations, it is unable to detect these mutants when comprising less than 20% (minor populations) of the total virus population in a sample. However, real-time PCR-based assays offer a rapid, sensitive, specific and easy detection and quantification of such mutants. The HIV-1 variants harbouring the K103N mutation are associated with resistance to nevirapine (NVP) and efavirenz (EFV). The persisting drug-resistant mutants decay slowly to low levels, and therefore they are called minor drug-resistant mutants. Consequently, they affect subsequent treatment with the drugs of the relevant class. Objectives: The objective of this study was to design two TaqMan real-time PCR-based assays called selective-polymerase chain reaction (SPCR), namely the total viral copy SPCR assay and the K103N-SPCR assay. The former detects HIV-1 of subtype C reverse transcriptase sequences, whereas the latter detects K103N drug-resistant variants in these sequences. Design and Methods: In developing the SPCR assays, sets of appropriate primers and probes for the HIV-1 subtype C reverse transcriptase (RT) were developed to use in the K103N-specific reaction and the total copy reaction. Twelve DNA plasmid standards with sequence diversity were constructed for the assay from two HIV-1subtype C samples known to harbour the K103N mutation (AAC or AAT) in our Department‟s Resistance Databank. Their RT regions were amplified, cloned and verified with sequencing. Site-directed mutagenesis was used to induce mutations at 103 amino acid position in some of these clones to generate more standards with either one of the three codons (AAA, AAC and AAT). The two assays were optimized and validated, and a standard curve was generated for each assay using 10-fold serial dilution (5x107-5x100 DNA copy/μL) of a K103N-mutant plasmid standard. The optimized and validated SPCR assays were used to screen 40 nested PCR products of previously genotyped patient samples for minor K103N variants. Results: Two sensitive and reproducible selective real-time PCR (SPCR) assays, with cut-offs of 8.23 and 10.33 and a detection limit of 0.01% for the K103N resistance variants, were successfully developed. The assays detected a prevalence of 25.64-46.15% for the K103N resistance mutation in 39 patient samples. The genotyping (population sequencing) missed 40-53.85% of these variants. Conclusion: In conclusion, sensitive and reliable selective real-time PCR assays to detect and quantify minor K103N variants of HIV-1 in nested PCR products were successfully developed. The assay had a lower detection limit of 0.01%.
Dried plasma spot testing – the answer for making blood transfusion testing safer in africa?
(Stellenbosch : Stellenbosch University, 2018-12) Pistorius, Charlotte; Preiser, Wolfgang; Bird, Arthur; Stellenbosch University. Faculty of Medicine and health Sciences. Dept. of Pathology. Medical Virology.
ENGLISH ABSTRACT: Sub-Saharan Africa (SSA) has a unique set of challenges pertaining to blood transfusion. Two of the largest contributing factors are: (1) the most common disease states in SSA require large amounts of blood as a lifesaving intervention e.g. malaria, and (2) the highest burden of infectious diseases transmissible through transfusion (Tapko, Toure, & Sambo, 2014) is found in SSA. This has often led to the dichotomous donor base that exists in SSA, consisting of Voluntary Non-remunerated Blood donors (VNBD) and family or replacement donors (FRD), since transfusion centres are unable to supply the demand when relying only on VNBD. VNBD are the safest blood donors as they have no monetary incentives and are under no direct social pressure to donate. Monetary incentives have been shown to entice individuals that know or suspect themselves to be infected with a blood borne agent to donate blood. Nucleic Acid Testing (NAT) in conjunction with serological testing is the gold standard for testing, however the vast distances and high temperatures of SSA makes transport of traditional plasma samples a logistical nightmare. Many publications evaluating the stability, suitability and ease of use of dried blood spot (DBS) and dried plasma spot (DPS) for NAT have been published. Generally results have been shown to be comparable to traditional plasma samples. DBS are being used successfully in the early infant diagnosis (EID) programs for HIV by means of PCR testing especially in Africa. Ethical approval was obtained to conduct a study to determine whether DBS and/or DPS testing would be suitable for use in a resource limited setting for blood screening. Two cohorts were included. Cohort A consisted of 900 de-identified negative new donor samples. Cohort B consisted of 100 de-identified confirmed positive donor samples, 9 procured reactive samples, and a contamination panel. After routine donor testing was completed at Western Province Blood Transfusion Service, one DBS sample and one DPS sample for each blood donor was prepared and analysed with the Ultrio Elite Assay on the Panther analyser (Hologic Inc., USA). Logistically DBS/DPS is well suited for the resource-poor countries as samples are: a.Easy to obtain (fingerpick samples could be used). b.Transport is simplified as samples will not leak or haemolyse due to high temperatures. c.Samples can be stored at room temperature. DBS/DPS samples demonstrated superb specificity. DPS samples would be suited for screening blood and reduced cost involved in NAT testing provided that the HBV sensitivity is increased. Further detailed economic viability and large-scale studies need to be performed to determine sensitivity and specificity within a specific population.
Erythrocyte apoptosis (erythroptosis) and anaemia in chronic HIV-1 infection : relationship with immune activation and viraemia
(Stellenbosch : Stellenbosch University, 2013-12) Loots, Stanley; Glashoff, Richard H.; Ipp, Hayley; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology, Medical Virology.
ENGLISH ABSTRACT: Chronic HIV-1 infection is characterized by extensive inflammation/immune activation and also by anaemia. Macrophages and neutrophils produce reactive oxygen species (ROS) which can cause damage to surrounding cells, including erythrocytes. Damaged erythrocytes may die by apoptosis (erythroptosis) or be tagged for clearance by monocytes/ macrophages. In this study we investigated HIV-1-associated anaemia and erythroptosis in asymptomatic, untreated HIV-1 infected individuals and how it relates to oxidative stress and immune activation.
Genotyping respiratory viruses in sudden unexpected death in infancy cases (SUDI) at Tygerberg Medico-legal Mortuary
(Stellenbosch : Stellenbosch University, 2023-03) Vanmali, Hameer Deepak; De Beer, Corena; Claassen, Mathilda; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.
ENGLISH SUMMARY: Background: Infant mortality remains a major global concern. Sudden unexpected death in infancy (SUDI) is reported globally and accounted for 40% of infant deaths between 2012 and 2016 in the Western Cape. No standardised investigation protocol exists for SUDI cases in South Africa, and research is poorly funded. Previous research highlight the burden of respiratory viruses in infants and SUDI cases, however molecular typing of respiratory viruses in SUDI cases is lacking. Methods: HRV and RSV polymerase chain reaction (PCR)-positive trachea and lung swab samples of SUDI cases admitted to Tygerberg Medico-legal Mortuary between 2015 and 2019 were identified and included in this study. The Allplex™ RV Essential Assay was repeated on these samples to confirm that only PCR-positive samples were selected and included in this study. Positive samples underwent automated nucleic acid extraction, one-step, nested RT-PCR, and confirmed for amplification by gel electrophoresis before sequencing. Sequencing results were aligned and underwent phylogenetic analysis. Results: A total of 116 SUDI cases were HRV and/or RSV PCR-positive and included in the study, with a median age of SUDI of 10.9 weeks. More cases were female (53.4%), of African ethnicity (51.7%), and from informal housing (54.3%). Of the infants that were included in this study, 35.4% died during the autumn and winter, compared to summer and spring. According to the information provided by parents or caregivers, most infants bed-shared (94.8%) and when compared to prone and supine positions, most infants were placed to sleep on their side (49.14%) and were found in this position (40.05%). Human rhinovirus (HRV) detected in trachea and respiratory syncytial virus (RSV) detected in the lung were significantly associated with sex and all four seasons (p<0.05). HRV samples fell within three distinct species , HRV-A (n=28), followed by -C (n=11), and -B (n=4). In total, eight HRV-A (A28, A80, A10, A82, A43, A56, A11, and A63), one -B (B84), and seven -C (C22, C19, C24, C35, C29, C38, and C2) genotypes were identified. Two RSV groups were identified, RSV-A (n=5) and -B (n=5). No RSV-A sequences were assigned as ON1, and two samples were assigned BA9 after amino acid alignment indicating characteristic of 20 amino acid duplication, as well as K218T, L223P, K225I, V251T, D253N, S267P, H287Y, S288L, and E292G substitutions. Conclusion: These study results were comparable to SUDI trends within the East Metropole of Cape Town, as well as globally. Respiratory viruses, especially in combination with sociodemographic and meteorological factors, remain key contributors to SUDI. The phylogenetic analysis describes the first molecular characterisation of respiratory viruses in SUDI cases in Africa. Without prospective sampling the current burden and characterisation of circulating HRV and RSV types and associations with clinical disease severity are unknown.
HIV-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.
Implementation, evaluation and use of methods to identify SARS-CoV-2 genetic variants of significance
(Stellenbosch : Stellenbosch University, 2023-03) Karabo, Phadu; Wolfgang, Preiser; Tongai, Maponga; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.
ENGLISH SUMMARY: Background: The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants, designated as variants of concern (VOCs) by the World Health Organization (WHO) continue to threaten measures in place to contain the spread of the virus. Consequently, efforts have been made to intensify genomic surveillance to track and monitor the evolution and spread of SARS-CoV-2 VOCs to help inform public health interventions in a timely manner. However, whole genome sequencing (WGS) is expensive, has a longer turnaround time and requires expert bioinformatics analysis, making it unfeasible for near real-time monitoring and reporting of SARS-CoV-2 variants. There is a need for affordable variant screening methods for the rapid detection and differentiation of SARS-CoV-2 VOCs. Methods: Ten SARS-CoV-2 variant screening assays targeting SARS-CoV-2 Alpha, Beta, Delta and Omicron VOCs were used. The analytical sensitivity of the assays was assessed using RNA extracted from cell culture supernatants of isolates of the Beta and Delta VOCs and the limit of detection (LOD) and linearity of the assays were determined. Furthermore, the assays were used to screen for SARS-CoV-2 VOCs in 898 patient samples diagnosed with infection between November 2020 and January 2022. Screening results were validated against WGS to determine the clinical sensitivity, and specificity of variant screening assays, and the agreement between assays (using WGS as the gold standard) was reported as the kappa value. Lastly, the TaqMan SNP genotyping panel, TaqPath COVID-19, and ModularDx assays were implemented to estimate the proportion of SARS-CoV-2 VOCs that circulated between November 2020 and January 2022. Results: The SARS-COV-2 variant screening assay had a correlation coefficient between 0.98 and 0.99, indicating good linearity between the dilution and the corresponding average cycle threshold values (Ct-values) at each standard dilution. Of the 10 screening assays, the Smartchek B.1.351 assay had the lowest clinical sensitivity (86.4%) followed by the Allplex Variants I assay (95.5%). The sensitivity of the ModularDx assay and the TaqPath COVID-19 assays was 98.6% and 99.7%, respectively. Furthermore, the TaqMan mutation panel, Allplex Variants II, IV and Master assays were 100% sensitive. All assays demonstrated 100% specificity and moderate to high concordance in comparison with WGS. The predominant circulating variant before and during May 2021 was the Beta VOC, constituting 86.7%, 100% and 79.4% of samples screened in November and December 2020 and May 2021 respectively. The Delta VOC rapidly spread, displacing the Beta VOC in May 2021 and dominating from June until October 2021. In November, December 2021, and January 2022 the Omicron VOC predominated, with the proportion of samples resulting in failure of the S gene target to amplify, SGTF rising from 87.7% in November to 99.5% in December. Conclusion: This study demonstrated that SARS-CoV-2 variant screening assays can be used as rapid and affordable tools to monitor signature mutations in SARS-CoV-2 VOCs. In addition, they can be useful tools for scaling up SARS-CoV-2 genomic surveillance. However, they need to be regularly updated and cannot replace traditional sequencing methods, but rather serve as tools to complement sequencing in monitoring circulating SARS-CoV-2 VOCs.
Improved methods to recover HIV-1 integrated proviruses and integration sites
(Stellenbosch : Stellenbosch University, 2020-12) Delaney, Kayla Eileen; Van Zyl, Gert Uves; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Background Long-lived HIV-1 infected cells that form part of the latent HIV-1 reservoir represent a major barrier to HIV-1 cure despite antiretroviral therapy (ART). The majority of these cells contribute to a persistent HIV-1 infection through clonal expansion. Many methods exist to study clonal expansion by identifying identical integration sites in different cells. However, the majority of proviruses are defective and until recently, methods did not exist to enable the simultaneous characterisation of integration sites and integrated proviruses, to identify which HIV-1 infected cell clones harbour intact proviruses. As recent methods are laborious and expensive, more efficient methods of linking intact proviral sequences and their cognate integration site are required. In addition, third-generation sequencing platforms allow for real-time long read-length sequence data which is ideal to characterise proviruses. These methods provide faster and simpler genome assembly than short read length second-generation sequencing platforms. Therefore, the overall aim of this study was to contribute to the HIV cure agenda, by improving our understanding of true HIV reservoirs by developing methods that would improve the characterisation of HIV-1 infected cell clones that harbour intact HIV genomes. Methods Intact proviral genomes are rare and attempts to enrich and link these proviral sequences to their integration site were investigated. The Integration Site Loop Amplification (ISLA) assay published by Wagner et al. (2014) was adapted for HIV-1 subtype C. Verification by Sanger sequencing showed that no integration sites were recovered and the “Premium whole genome amplification” method from Oxford Nanopore Technologies’ (ONT) third-generation sequencing technology was attempted as an alternative method. To investigate the utility of ONT sequencing, the “Amplicons by Ligation” method was utilised to sequence 9 near-full-length provisionally intact HIV-1 proviral sequences, previously sequenced by Illumina MiSeq. The consensus sequences for ONT sequencing were generated through a custom bioinformatic pipeline and compared to the Illumina MiSeq consensus sequences. Results The modified ISLA approach for HIV-1 subtype C or Premium whole genome amplification method did not succeed in recovering the HIV-1 proviral integration sites, of rare intact HIV-1 genomes. For near-full-length HIV genome sequencing, the “Amplicons by Ligation” protocol ONT sequencing achieved high coverage across the HIV genome and apart from hypervariable HIV-1 envelope there was a near perfect concordance between the consensus sequences generated with ONT and the previous Illumina MiSeq sequences, with an overall concordance of >99% for 8 out of 9 samples. Conclusion HIV-1 integration sites were not recovered in this study and efficient methods for simultaneous and efficient identification of intact proviral genomes and their integration sites remain unavailable. ONT sequencing allows for efficient and accurate sequencing of long fragments in real-time which may overcome technical barriers and eliminate laborious, time-consuming and expensive methods currently used for integration site identification and near-full-length HIV-1 genome sequencing. As part of the research towards future possible HIV cures, it remains a priority to investigate the persistence of cells harbouring intact HIV-1 genomes, and the role of clonal cellular proliferation in their survival.
In vitro and in vivo characterisation of SARS-CoV-2 wildtype, Beta, Delta and Omicron variants of concern : growth kinetics and viral shedding
(Stellenbosch : Stellenbosch University, 2023-03) Sutherland, Andrew David; Wolfgang, Preiser; Tasnim, Suliman; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.
ENGLISH SUMMARY: Background and Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that emerged in Wuhan, China, in December 2019. By October 2022, SARS-CoV-2 had caused more than 626 million known cases worldwide, resulting in over 6.5 million deaths. South Africa reported over 4 million cases and over 100 000 deaths, experiencing five epidemic waves each caused by a novel variant of concern (VOC), apart from the first pandemic wave. The first wave was caused by the SARS-CoV-2 wild-type (WT), the second SARS-CoV-2 Beta VOC, the third SARS-CoV-2 Delta VOC, the fourth SARS-CoV-2 Omicron VOC and the fifth wave by SARS-CoV-2 Omicron subvariants. Each VOC possessed a unique set of mutations that resulted in augmented transmissibility and infectivity. These augmented characteristics can be impacted by multiple factors, including individual patient viral shedding dynamics and replicative fitness. Infectiousness has been inferred from the RNA concentration in a sample, whilst useful, multiple studies have revealed that the ratio between RNA and infectious virus particles varies greatly during active infection. Therefore, a patient’s true infectiousness can only be determined through virus isolation. Augmented replication of a particular VOC can translate into a competitive advantage. Replicative fitness can be assessed through experiments designed to compare the rate of change of the number of virus particles over time, known as replication kinetics. Understanding these two factors can provide information on why different VOC were able to transmit differently and provide further insight into interpreting clinical results. Aim and objectives: This study had two main aims, firstly, to investigate the replicative fitness of SARS-CoV-2 WT, Beta and Delta. Secondly, to characterize the viral load and infectious particle shedding in a group of vaccinated and boosted German tourists that presented with breakthrough infections, referring to infections diagnosed at least 14 days after the last dose of a full vaccination course and/or a booster vaccination, with SARS-CoV-2 Omicron VOC. This was to be accomplished by isolating all virus variants circulating in South Africa, producing a virus stock of each of these and performing replication kinetic experiments, and by monitoring viral RNA concentrations and successful isolation outcomes over time amongst the breakthrough infections cohort. Methods: Replication-competent SARS-CoV-2 was isolated from PCR-positive patient samples submitted to the National Health Laboratory Service (NHLS), Tygerberg Business Unit, Cape Town, South Africa. Samples were used to inoculate Vero E6 and H1299-E3 cells, after which the cell cultures were grown at 37°C and monitored daily for cytopathic effects (CPE). When >80% CPE was observed the supernatant was harvested and sub-cultured to produce a working stock. Replication kinetic experiments were performed on Vero E6 cells, and samples for analysis were taken at different time points post-infection (p.i.). RNA and infectious particle concentrations were determined. To characterize the infectious period, samples were collected daily, up to day eight of symptom onset, from a cohort of 11 German tourists who were fully vaccinated that presented with breakthrough Omicron infections. RNA concentrations were determined using RT-PCR and the infectiousness of the patients was assessed by attempting virus isolation from each sample. Results: Virus isolation was attempted from 136 SARS-CoV-2 positive patient samples and yielded 17 virus isolates - three SARS-CoV-2 WT, five SARS-CoV-2 Beta VOC, four SARS-CoV-2 Delta VOC, one SARS-CoV-2 Alpha VOC and three SARS-CoV-2 Omicron BA.1 VOC. Attempts to isolate Omicron sub-lineages BA.2, BA.4 and BA.5 as well as SARS-CoV-2 C.1.2 were unsuccessful. Beta showed a similar growth curve when compared to WT, indicating no significant replicative advantage, whereas Delta had a significantly steeper growth curve, indicating an enhanced replication rate for Delta. Characterisation of viral loads amongst the study cohort, despite a high level of heterogeneity between patients, showed an increase up to day three followed by decreasing RNA concentrations thereafter. Virus isolation was more likely to be successful before day five from symptom onset and associated with higher viral loads, whilst also showing high levels of heterogeneity between patients. Conclusion: In line with the aims and objectives of this study at least one virus stock was successfully produced from the SARS-CoV-2 VOC that circulated in South Africa, apart from Omicron BA.2, BA.4 and BA.5. Analysis of VOC growth kinetics using Vero E6 cells revealed that Beta did not differ from the WT. Increased transmission associated with Beta is therefore likely due to other factors, such as immune escape and receptor affinity. Delta showed a significant replication advantage over Beta and WT, which likely played a role in its increased transmissibility. Results from the breakthrough infection cohort emphasised a high level of heterogeneity in RNA concentrations and infectious particle shedding between patients. Patients appeared to be more infectious closer to symptom onset and when RNA concentrations were higher, in line with other studies. This research highlights the importance of virus culture techniques in understanding the in vitro characteristics of viruses.
In-house genotypic antiretroviral resistance test : optimisation and validation for use in research and diagnostics
(Stellenbosch : University of Stellenbosch, 2011-03) Claassen, Mathilda; Van Zyl, Gert Uves; Engelbrecht, Susan; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
It is estimated that 32.8 million people are living with Human Immunodeficiency Virus (HIV) globally with the number of people receiving antiretroviral therapy in low- and middle- income counties increasing to more than 5 million people in 2009. These successes are threatened by treatment failure and the development of resistance to treatment. With an estimated 3.7% patients failing first line treatment after 2 years and 17.9% after 4 years on treatment there is a need for a practical and cheap in-house drug resistance assay that can be used to provide drug resistance data to clinicians and to use as a research tool to investigate drug resistance. In this study we attempted to optimize and validate an in-house drug resistance assay, adapted from Jacobs et al, 2008, to be used as a diagnostic tool and to study the presence of antiretroviral resistance in patients on the Western Cape Mother-To-Child-Transmission (MTCT) regimen. Quality control samples were received from The National Institute of Communicable Diseases AIDS Virus Research Unit, The Round Robin HIV-1 genotyping assessment system from the University of Würzburg and the QCMD assessment system were used for the optimization and validation of an in-house drug resistance assay. The ViroSeq™ HIV-1 Genotyping System was used for comparison of sample and mutation detection. It was possible to optimise and validate a genotyping assay for diagnostic testing and research use by comparison with the ViroSeq™ HIV-1 Genotyping System and evaluation with external quality assessment systems. This assay could subsequently be used to determine the development of genotypic-antiretroviral resistance in patients treated according to the provincial prevention of mother-to-child-transmission (PMTCT) protocol in the Western Cape (single dose nevirapine (sd-NVP), combined with a short course Zidovudine (AZT)). Patient samples were collected from pregnant women who took part in the Western Cape PMTCT program and visited the Tygerberg Obstetrics Clinic and Delft Community Hospital. EDTA blood was obtained to measure CD4-cell count, viral load, and to do genotyping for viral subtype and the presence of resistance mutations. Information on prior exposure to antiretroviral therapy was also collected. A detected resistance rate of 17.1% in this predominantly HIV-1 subtype C population is lower than previously recorded when sd-NVP was administered to HIV-1 subtype C positive patients in PMTCT programs. This could indicate that a dual PMTCT regimen including AZT and NVP reduces the risk of resistance to NVP relative to a regimen that uses sd-NVP. The genotyping assay uses four primers to amplify the PR and the RT gene separately to obtain PCR products, of 487 and 804 base pairs respectively for sequencing. The two PCR products were sequenced with three and five primers respectively to sequence the complete PR and approximately 250 amino acids of the RT gene. The sequences generated, thus, are analysed and aligned with the Sequencer V4.7 software to obtain a consensus sequence of approximately 1200 base pairs for analysis of resistance mutations in the protease and reverse transcriptase genes. The developed assay was hence further simplified and improved, by combining the PR and RT assay into one, which was optimised and validated for use in the routine diagnostic setting. The final genotyping assay uses 8 primers for sequencing to obtain a 1200 bp sequence for genotyping that contains the protease and the 5’ of the reverse transcriptase genes in which antiretroviral resistance associated mutations are found. The assay was accredited by SANAS in 2008.

Browse

Browsing Masters Degrees (Medical Virology) by browse.metadata.type "Thesis"

Results Per Page

Sort Options