Nyaga, Martin MuneneGoedhals, DominiqueBester, Philip ArmandSondlane, Hlengiwe2024-07-192024-07-192023http://hdl.handle.net/11660/12695Dissertation (M.Med.Sc.(Virology))--University of the Free State, 2023Respiratory syncytial virus (RSV) is a highly contagious virus that is responsible for most infant hospitalisations in developed nations and accounts for a significant childhood mortality in low- and middle-income countries (LMICs). The RSV has two main subtypes, namely A and B, which co-circulate and exhibit a predominance during different RSV epidemic seasons. This virus exhibits a high degree of genetic diversity, resulting in the emergence of various genotypes. The regulation of viral replication is primarily governed by two major RSV glycoproteins, namely the attachment (G) protein and the fusion (F) protein. These proteins possess potential glycosylation sites, although the variability is more pronounced in the G protein, while the F protein remains relatively conserved across different virus isolates. Following the advent of severe acute respiratory syndrome coronavirus-2 and the onset of the coronavirus disease 2019 (COVID-19) era, a significant decline in RSV activity was observed, aligning with the enforcement of non-pharmaceutical interventions (NPI). Consequently, the COVID-19 pandemic brought about a significant shift in the seasonality and epidemiology of RSV, resulting in an out of season RSV outbreak. Therefore, this study aimed to investigate the genetic diversity of the circulating strains in South Africa before and during the COVID-19 pandemic. In this study, 69 nasopharyngeal swabs were collected from children who presented with respiratory distress (n=50) and severe acute respiratory infection (SARI) (n=19) and required hospital admission in different participating hospitals in the Free State province, Bloemfontein, South Africa. The samples were then subjected to multiplex panels to detect RSV. The viral RNA was extracted from RSV positive samples and overlapping fragments of the RSVA/B genomes were amplified using the Superscript IV One-Step RT-PCR kit. Libraries were prepared with QIAseq-FX single-cell RNA library preparation kit. The RSV whole genome was sequenced using the Illumina MiSeq, platform, and the obtained data was then analysed using Genome Detective and Geneious software. The virus genotypes were identified through phylogenetic analysis utilising a Nextclade typing tool. The phylodynamics of RSV, genetic diversity, and evolutionary patterns were analysed using IQ Tree and BEAST software. The sequence phylogenetic analysis revealed a notable level of genetic diversity among the RSV strains in South Africa. In this study, the analysis showed that all study strains belonged to the GA2.3.5 and GB5.0.5a genotypes, which were predominant during the whole study period. These genotypes have been characterised previously and are circulating globally as the predominating genotypes. The phylogenetic analysis of the G-gene showed that strains responsible for the South African epidemic observed during the COVID-19 resurgence in 2020-2021 formed distinct clusters with contemporary strains from various geographic origins, suggesting continuous minor introduction within South Africa. Furthermore, the estimated mean evolutionary rates for RSV-A and RSV-B were found to be 1.48 × 10⁻³ and 1.92 × 10⁻³ nucleotide substitutions/site/year, respectively. The genetic diversity patterns in GA2.3.5 and GB5.0.5a genotypes were similar according to the Bayesian Skyride plot analysis. Our results suggest that the COVID-19 lockdown in South Africa caused a minor bottleneck suggesting other strains ceased to circulate, then followed by increased diversity as restrictions were lifted, indicating increased circulation of this variants. The relaxation of COVID-19 restrictions in South Africa led to a surge in the off-season RSV epidemic among children. This increase in cases can be attributed to the presence of pre-existing strains, which may have been able to spread in the paediatric population that is immunologically naïve or have waned immunity. Therefore, it is crucial to conduct genomic sequencing to gain insights into the circulation patterns of RSV and the diversity of RSV strains. Given the need to comprehensively understand the RSV evolutionary patterns and its impact on vaccine effectiveness, it is imperative for genomic surveillance strategies to focus on and prioritize the use of whole genome sequencing.enOff-season RSV epidemicCOVID-19whole genome sequencingnon-pharmaceutical interventionspaediatric, virusesnasopharyngeal swabsrespiratory infectionrespiratory distressDissertationUniversity of the Free State