Masters Degrees (Medical Virology)

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    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.
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    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.
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    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.
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    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.
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    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.