Browsing by Author "Sutherland, Andrew David"

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    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.