Metagenomic Insights into Human Wastewater Virosphere and the Viability Profiles of Common Gastroenteric Pathogens

The integration of next-generation sequencing (NGS) methods with wastewater-based surveillance has fundamentally revolutionized how we track pathogens. This study determines clinically relevant human viruses in wastewater samples from the Durban area of South Africa to understand the prevalence pattern, treatment reduction, and potential infectiousness post-disinfection. During April–June 2025, 9 influent and 9 post-chlorinated effluent samples collected from three differently configured wastewater treatment plants (WWTPs) were analyzed using Illumina shotgun sequencing. A viability assay for some viral targets was undertaken through in vitro cell culture integrated with real-time quantitative polymerase chain reaction (ICC-qPCR). In the combined sample matrices, the genus Lentivirus predominated, followed by mastadenovirus, Orthobunyavirus, Orthopoxvirus, Parapoxvirus, and Betapolyomavirus. Despite treatment reduction in viral concentrations, these viruses, including Betacoronavirus, Alphacoronavirus, Alphapolyomavirus, Gammapapillomavirus, Orthonairovirus, and Orthohantavirus, persisted in effluent samples, as they were notably present in ≥90% of each WWTP. The comparative ICC-qPCR results with qPCR indicated incomplete post-treatment decay (0–66.7% vs 33.3–100%) for high-priority gastroenteric viruses. Our metagenomic profiles uncovered several viral genera, which included clinically relevant species in the area. The viral persistence observed during treatment and the viability status of enteropathogenic viruses in postchlorinated effluents underscore the need for integration of advanced disinfection stages to achieve effluents of desirable virologic quality.