Mavhandu-Ramarumo, Lufuno GraceTebit, Denis MangaBessong, Pascal ObongTambe, Lisa Arrah Mbang2025-06-192025-06-192025-05-16Tambe, L. A. M. 2025. Studies on severe acute respiratory syndrome coronavirus type -2 in Northern South Africa. Thohoyandou, South Africa.<https://univendspace.univen.ac.za/handle/11602/2831>.https://univendspace.univen.ac.za/handle/11602/2831PhD (Microbiology)Department of Biochemistry and MicrobiologyBackground: The last three decades have been characterized by the re-emergence of the Coronaviridae family into the human population, causing severe respiratory disease with increased morbidity rates. A dearth of information exists on human coronavirus (HCoV) molecular epidemiology, and circulation in different populations in Africa. As the COVID-19 pandemic progressed across the globe, wastewater-based epidemiology (WBE) was proposed as an alternative tool for assessing and monitoring the occurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the community level. Additionally, through wastewater-based genomic surveillance of SARS-CoV-2, the evolutionary patterns and distribution of viral types at the population level can be comprehensively characterized. This study systematically reviewed literature published prior to the SARS-CoV-2 outbreak to investigate the prevalence and molecular epidemiology of HCoVs circulating in Africa. Secondly, this study established a wastewater-based surveillance (WBS) system to track the trends of SARS-CoV-2, investigate SARS-CoV-2 variants of concern (VOC) circulating in the population, and determine the prevalence of people infected in the Vhembe and Mopani districts. Thirdly, through WBS, to describe the molecular epidemiology of SARS-CoV-2 and document the respiratory viruses occurring in the Vhembe and Mopani districts. Methodology: A systematic literature review was conducted according to the PRISMA guidelines, to understand the prevalence and molecular epidemiology of HCoVs in Africa. For the second and third objectives, wastewater influents from seven wastewater treatment plants (WWTPs) and one waste sedimentation pond (WSP) were collected weekly from January 2021 to June 2022. Out of a total of 487 samples collected, about 75% (365/487) were positive for SARS-CoV-2 RNA by qRT-PCR. Of these, 80 met the criteria for allele-specific genotyping (ASG). Positive SARS-CoV-2 RNA detected throughout the surveillance period were compared to 7-day moving average (7D-MA) of clinical cases reported per sub-district. Next, SARS-CoV-2 RNA detected during the surveillance period was normalized using the flow rate and population size. The Spearman’s correlation coefficient was used to determine the relationship between non-normalized, and normalized SARS-CoV-2 RNA data when compared to the reported clinical cases. Finally, positive SARS-CoV-2 RNA copies with a standard deviation of less than one (SD <1) were used to predict the prevalence of people infected in the study sites using the Monte Carlo simulation model. This predicted prevalence was also compared to the 7D-MA clinical cases reported per sub-district, and the correlation between them was determined. Subsequently, samples positive for SARS-CoV-2 were subjected to whole genome sequences (WGS) using the ATOPlex next-generation sequencing method and analyzed for lineage and clade assignment using the Pangolin and Nextclade tool. Relatedness of identified sequences was determined by phylogenetic analysis. VOC was analyzed for prevalence and geographical distribution. Concordance for VOC between ASG and WGS analyses was determined. Results: Findings from the systematic literature review showed that thirteen out of 54 (24%) African countries had published data on HCoV prevalence and/or genomic epidemiology, from hospitals, clinics, homes, community gatherings, farms, and individuals at airports. The first published data on HCoV was from South Africa in 2008. There was heterogeneity in the type of tests used in determining HCoV prevalence. Two studies reported that risk factors for HCoV include exposure to infected animals or humans. The second objective, establishing a wastewater-based surveillance system was achieved. Briefly, SARS-CoV-2 viral load was detected in wastewater one week prior to increased infection cases reported at the district level during the third and fourth waves, thus serving as an early warning system. Of interest, towards the end of the surveillance period, increased SARS-CoV-2 viral load detected in wastewater were not reflected in the reported clinical cases. Comparing the reported number of cases per district to the predicted prevalence revealed more cases in the Vhembe District than in the Mopani District. Third, SARS-CoV-2 molecular epidemiology and the distribution of respiratory virus were described. A total of 60 SARS-CoV-2 full genomes were analyzed. Delta and Omicron variants were detected as early as January and February 2021, respectively, while the Beta variant was detected in July 2021. Delta variant was significantly predominant at a prevalence of 45%, followed by Omicron (32%), and Beta (5%). Eighteen percent (11/60) of the sequences were assigned a lineage by Pangolin tool, but not a specific WHO variant name. Upon phylogenetic analysis, some of these sequences were seen clustering with the Alpha (2/11) and Delta (2/11) variants, while the remaining sequences clustered with each other. Mutations in the receptor-binding domain (RBD) of the Spike protein (S-protein) were investigated, with some peculiarities observed such as mutation E484K absent in all Beta variant study sequences. Three previously undescribed mutations (A631S, V327I, D427Y) were detected in Delta variant sequences. Concordance in variant assignments between allele-specific genotyping and WGS was seen in 51.2% of the study sequences. Respiratory virus surveillance revealed year-round circulation of human Adenoviruses (HAdVs), while HCoVs, influenza viruses and human parainfluenza viruses (HPIVs) were mostly detected in winter. Influenza A and B viruses (IAV and IBV) detected in the study site in 2021 were remarkably different from those reported in circulation nationwide by the NICD. Specifically, IAV (H5N1)/Guandong, a highly pathogenic influenza virus, was detected, although at a low frequency. Discussion and Conclusion: The systematic review revealed that despite the outbreaks of SARS in Southeast Asia in 2002 and MERS in 2012 in the Middle East, the quantum of virologic investigations on HCoV on the African continent was scanty. Pandemic preparedness requires cognizance of disease outbreaks in other continents, establishment of test and surveillance protocols, and infrastructure for eventualities. Regarding the establishment of a wastewater-based surveillance system for SARS-CoV-2 monitoring, this study demonstrates effective surveillance over an extended period in rural settings. This is important because most reports about the application of WBE for monitoring SARS-CoV-2 and circulating variants are predominantly from more urbanized regions in South Africa and other parts of the world. Thus, it reveals applicability of monitoring pathogens in rural areas, despite challenges encountered such as poor or non-existent sewerage systems. Such challenges are common in the African continent, highlighting the need for more of such investigations to strengthen pandemic preparedness measures. The presence of Delta and Omicron VOCs observed prior to other reports in South Africa highlights the importance of population-based approaches in genomic surveillance over approaches that rely on individual samples. Again, it also emphasizes the need for pandemic preparedness efforts to be extended to all geographic regions. Wastewater is known to potentially capture more viral diversity, including SARS-CoV-2 genetic diversity, and could reveal new viruses and VOCs in circulation before they emerge in the wider human population. Thus, continuous surveillance is necessary for documentation of cryptic lineages, which may contribute towards improving vaccine.1 online resource (enUniversity of VendaHCov prevalence and distributionUCTDAfricapandemic preparednessCOVID-19SARS-Cov-2WBEReceptor binding domainViral revolutionRespiratory virusesEarly warninggenomic surveillanceVOC'sMolecular epidemiologyThesisTambe LAM. Studies on severe acute respiratory syndrome coronavirus type -2 in Northern South Africa. []. , 2025 [cited yyyy month dd]. Available from:Tambe, L. A. M. (2025). <i>Studies on severe acute respiratory syndrome coronavirus type -2 in Northern South Africa</i>. (). . Retrieved fromTambe, Lisa Arrah Mbang. <i>"Studies on severe acute respiratory syndrome coronavirus type -2 in Northern South Africa."</i> ., , 2025.TY - Thesis AU - Tambe, Lisa Arrah Mbang AB - Background: The last three decades have been characterized by the re-emergence of the Coronaviridae family into the human population, causing severe respiratory disease with increased morbidity rates. A dearth of information exists on human coronavirus (HCoV) molecular epidemiology, and circulation in different populations in Africa. As the COVID-19 pandemic progressed across the globe, wastewater-based epidemiology (WBE) was proposed as an alternative tool for assessing and monitoring the occurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the community level. Additionally, through wastewater-based genomic surveillance of SARS-CoV-2, the evolutionary patterns and distribution of viral types at the population level can be comprehensively characterized. This study systematically reviewed literature published prior to the SARS-CoV-2 outbreak to investigate the prevalence and molecular epidemiology of HCoVs circulating in Africa. Secondly, this study established a wastewater-based surveillance (WBS) system to track the trends of SARS-CoV-2, investigate SARS-CoV-2 variants of concern (VOC) circulating in the population, and determine the prevalence of people infected in the Vhembe and Mopani districts. Thirdly, through WBS, to describe the molecular epidemiology of SARS-CoV-2 and document the respiratory viruses occurring in the Vhembe and Mopani districts. Methodology: A systematic literature review was conducted according to the PRISMA guidelines, to understand the prevalence and molecular epidemiology of HCoVs in Africa. For the second and third objectives, wastewater influents from seven wastewater treatment plants (WWTPs) and one waste sedimentation pond (WSP) were collected weekly from January 2021 to June 2022. Out of a total of 487 samples collected, about 75% (365/487) were positive for SARS-CoV-2 RNA by qRT-PCR. Of these, 80 met the criteria for allele-specific genotyping (ASG). Positive SARS-CoV-2 RNA detected throughout the surveillance period were compared to 7-day moving average (7D-MA) of clinical cases reported per sub-district. Next, SARS-CoV-2 RNA detected during the surveillance period was normalized using the flow rate and population size. The Spearman’s correlation coefficient was used to determine the relationship between non-normalized, and normalized SARS-CoV-2 RNA data when compared to the reported clinical cases. Finally, positive SARS-CoV-2 RNA copies with a standard deviation of less than one (SD <1) were used to predict the prevalence of people infected in the study sites using the Monte Carlo simulation model. This predicted prevalence was also compared to the 7D-MA clinical cases reported per sub-district, and the correlation between them was determined. Subsequently, samples positive for SARS-CoV-2 were subjected to whole genome sequences (WGS) using the ATOPlex next-generation sequencing method and analyzed for lineage and clade assignment using the Pangolin and Nextclade tool. Relatedness of identified sequences was determined by phylogenetic analysis. VOC was analyzed for prevalence and geographical distribution. Concordance for VOC between ASG and WGS analyses was determined. Results: Findings from the systematic literature review showed that thirteen out of 54 (24%) African countries had published data on HCoV prevalence and/or genomic epidemiology, from hospitals, clinics, homes, community gatherings, farms, and individuals at airports. The first published data on HCoV was from South Africa in 2008. There was heterogeneity in the type of tests used in determining HCoV prevalence. Two studies reported that risk factors for HCoV include exposure to infected animals or humans. The second objective, establishing a wastewater-based surveillance system was achieved. Briefly, SARS-CoV-2 viral load was detected in wastewater one week prior to increased infection cases reported at the district level during the third and fourth waves, thus serving as an early warning system. Of interest, towards the end of the surveillance period, increased SARS-CoV-2 viral load detected in wastewater were not reflected in the reported clinical cases. Comparing the reported number of cases per district to the predicted prevalence revealed more cases in the Vhembe District than in the Mopani District. Third, SARS-CoV-2 molecular epidemiology and the distribution of respiratory virus were described. A total of 60 SARS-CoV-2 full genomes were analyzed. Delta and Omicron variants were detected as early as January and February 2021, respectively, while the Beta variant was detected in July 2021. Delta variant was significantly predominant at a prevalence of 45%, followed by Omicron (32%), and Beta (5%). Eighteen percent (11/60) of the sequences were assigned a lineage by Pangolin tool, but not a specific WHO variant name. Upon phylogenetic analysis, some of these sequences were seen clustering with the Alpha (2/11) and Delta (2/11) variants, while the remaining sequences clustered with each other. Mutations in the receptor-binding domain (RBD) of the Spike protein (S-protein) were investigated, with some peculiarities observed such as mutation E484K absent in all Beta variant study sequences. Three previously undescribed mutations (A631S, V327I, D427Y) were detected in Delta variant sequences. Concordance in variant assignments between allele-specific genotyping and WGS was seen in 51.2% of the study sequences. Respiratory virus surveillance revealed year-round circulation of human Adenoviruses (HAdVs), while HCoVs, influenza viruses and human parainfluenza viruses (HPIVs) were mostly detected in winter. Influenza A and B viruses (IAV and IBV) detected in the study site in 2021 were remarkably different from those reported in circulation nationwide by the NICD. Specifically, IAV (H5N1)/Guandong, a highly pathogenic influenza virus, was detected, although at a low frequency. Discussion and Conclusion: The systematic review revealed that despite the outbreaks of SARS in Southeast Asia in 2002 and MERS in 2012 in the Middle East, the quantum of virologic investigations on HCoV on the African continent was scanty. Pandemic preparedness requires cognizance of disease outbreaks in other continents, establishment of test and surveillance protocols, and infrastructure for eventualities. Regarding the establishment of a wastewater-based surveillance system for SARS-CoV-2 monitoring, this study demonstrates effective surveillance over an extended period in rural settings. This is important because most reports about the application of WBE for monitoring SARS-CoV-2 and circulating variants are predominantly from more urbanized regions in South Africa and other parts of the world. Thus, it reveals applicability of monitoring pathogens in rural areas, despite challenges encountered such as poor or non-existent sewerage systems. Such challenges are common in the African continent, highlighting the need for more of such investigations to strengthen pandemic preparedness measures. The presence of Delta and Omicron VOCs observed prior to other reports in South Africa highlights the importance of population-based approaches in genomic surveillance over approaches that rely on individual samples. Again, it also emphasizes the need for pandemic preparedness efforts to be extended to all geographic regions. Wastewater is known to potentially capture more viral diversity, including SARS-CoV-2 genetic diversity, and could reveal new viruses and VOCs in circulation before they emerge in the wider human population. Thus, continuous surveillance is necessary for documentation of cryptic lineages, which may contribute towards improving vaccine. DA - 2025-05-16 DB - ResearchSpace DP - Univen KW - HCov prevalence and distribution KW - Africa KW - pandemic preparedness KW - COVID-19 KW - SARS-Cov-2 KW - WBE KW - Receptor binding domain KW - Viral revolution KW - Respiratory viruses KW - Early warning KW - genomic surveillance KW - VOC's KW - Molecular epidemiology LK - https://univendspace.univen.ac.za PY - 2025 T1 - Studies on severe acute respiratory syndrome coronavirus type -2 in Northern South Africa TI - Studies on severe acute respiratory syndrome coronavirus type -2 in Northern South Africa UR - ER -