Longitudinal Wastewater-Based Epidemiology Reveals the Spatiotemporal Dynamics and Genotype Diversity of Diarrheal Viruses in Urban Guangdong, China.

Following the normalization of the COVID-19 pandemic, the focus of wastewater-based epidemiology (WBE) must be broadened from SARS-CoV-2 to encompass surveillance of other major infectious diseases, particularly for pathogens where conventional clinical monitoring systems exhibit inherent surveillance gaps. In this study, we conducted a continuous two-year WBE study (January 2023 to December 2024) across three high-population-density cities in Guangdong, China to establish epidemiological baselines for enteric diarrheal viruses. We analyzed monthly raw wastewater samples from major treatment plants using advanced molecular methods, including digital PCR (ddPCR) for viral load quantification and targeted high-throughput sequencing (tNGS) for genotypic analysis. Our findings revealed diverse circulation patterns among the monitored enteric viruses. Astrovirus (AstV) had the highest detection rate (100%), reflecting its broad endemic distribution, while Norovirus genogroup II (NoV GII) exhibited relatively high viral loads (median 4 × 104 copies/mL) and presented explosive seasonal peaks (significant upward trend in spring.), highlighting its epidemic potential. Furthermore, distinct spatiotemporal patterns were observed, with Sapovirus showing a significant summer peak in Foshan city, contrasting with the winter/spring peaks in the other cities. The tNGS results demonstrated similar sensitivity to RT-PCR in virus detection, and sequencing analyses uncovered the co-circulation and periodic shifts in dominant viral genotypes, such as the emergence of multiple NoV and AstV lineages. This longitudinal WBE surveillance successfully established critical baseline data and demonstrated significant regional heterogeneity in viral circulation, providing essential, complementary data to inform public health strategies for preventing diarrheal outbreaks in urban settings.