Genetic diversity and viability of human norovirus strains detected in selected wastewater samples from Tshwane district, South Africa

Abstract

Norovirus, a leading cause of acute gastroenteritis worldwide, is characterised by its substantial genetic diversity and periodic emergence of novel variants, necessitating continuous monitoring to inform public health interventions. This study aimed to assess norovirus diversity and viability in wastewater from Pretoria, South Africa (SA), using next-generation sequencing (NGS) and advanced molecular assays. Between June 2018 and August 2020, 200 samples comprising raw sewage and final effluent were collected bi-weekly from two wastewater treatment works (WWTWs). Viruses were concentrated using skimmed milk flocculation and glass wool adsorption- elution methods. Noroviruses were detected in 81% of samples, with a higher positivity rate in raw sewage (89%) compared to final effluent (73%). Amplicon-based NGS targeting the norovirus BC genotyping region revealed 89 distinct genogroup (G) I and GII RNA-dependant RNA polymerase-capsid combinations, including 51 putative novel recombinants and one emerging novel strain, emphasizing the significant genetic variability present in wastewater. Complementing this, archived stool samples from paediatric gastroenteritis patients confirmed positive for GII norovirus underwent semi- nested PCR using in-house designed primers, followed by sequencing on the Illumina NextSeq 2000 platform. Complete genomes were successfully obtained for 56% of samples, uncovering different GII.4 variants and a GII.3[PNA] strain harbouring a putative novel P-type, which had 96.4% nucleotide similarity to a 2010 South African strain. Additionally, this study presented the first near-complete genome of GII.13[PNA8], which has only been reported in SA so far. Wastewater samples were analysed using both targeted and non-targeted NGS approaches. While the targeted method confirmed the presence of various GII strains, the non-targeted approach faced challenges with non-viral sequences predominating. The identification of strains such as GII.4 San Francisco[P31], previously observed in the United Kingdom, the United States of America, Gabon and India, underlines the global distribution and potential spread of this GII.4 variant. The detection of less common strains such as GII.7[P7] points to possible subclinical circulation in the population. A separate analysis focused on the viability of noroviruses in final effluent samples using viability RT-qPCR (vPCR) with PMAxx™- Triton X-100. While significant reductions in potentially viable noroviruses were observed post-treatment at both WWTWs, concerning levels of viable noroviruses persisted. At WWTW1, half of GI-positive and 64.9% of GII-positive final effluent samples showed no detectable RNA after vPCR, while WWTW2 achieved a 100% RNA reduction in 92.6% of GII-positive samples. These findings suggest that despite treatment processes, the discharge of viable noroviruses remains a public health concern due to their high initial load and low infectious dose. Overall, this comprehensive surveillance using NGS and vPCR highlights the genetic diversity, emergence of novel recombinants, and challenges in assessing viral viability in treated wastewater. Such data are crucial for enhancing wastewater-based epidemiology and guiding strategies to mitigate the spread of noroviruses in both environmental and clinical settings.