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• Testing protocols for DNA (mpox) and RNA (norovirus, rotavirus) viruses in wastewater were developed and validated. • Wastewater surveillance effectively tracked the diarrhea viruses' activity in the community. • The mpox virus signal was detectable in wastewater at a low prevalence area. • Four key components were provided to increase the values of wastewater surveillance network. The wastewater surveillance network successfully established for COVID-19 showed great potential to monitor other infectious viruses, such as norovirus, rotavirus and mpox virus. In this study, we established and validated detection methods for these viruses in wastewater. We developed a supernatant-based method to detect RNA viruses from wastewater samples and applied it to the monthly diarrhea viruses (norovirus genogroup I & II, and rotavirus) surveillance in wastewater treatment plants (WWTPs) at a city-wide level for 16 months. Significant correlations were observed between the diarrhea viruses concentrations in wastewater and detection rates in faecal specimens by clinical surveillance. The highest norovirus concentration in wastewater was obtained in winter, consistent with the seasonal pattern of norovirus outbreak in Hong Kong. Additionally, we established a pellet-based method to monitor DNA viruses in wastewater and detected weak signals for mpox virus in wastewater from a WWTP serving approximately 16,700 people, when the first mpox patient in Hong Kong was admitted to the hospital within the catchment area. Genomic sequencing provided confirmatory evidence for the validity of the results. Our findings emphasized the efficacy of the wastewater surveillance network in WWTPs as a cost-effective tool to track the transmission trend of diarrhea viruses and to provide sensitive detection of novel emerging viruses such as mpox virus in low-prevalence areas. The developed methods and surveillance results provide confidence for establishing robust wastewater surveillance programs to control infectious diseases in the post-pandemic era. [Display omitted] [ABSTRACT FROM AUTHOR]

• Two preanalytical methods (supernatant-based and pellet-based) were established for influenza A/B wastewater surveillance. • Sanger sequencing confirmed the presence of influenza A/B RNA in wastewater. • Wastewater influenza A virus concentration effectively reflected influenza activity in the community. • H1pdm virus was the dominant subtype in the community at the time of this study. • Wastewater surveillance can provide virus types and subtypes information for public health actions in the future. Wastewater surveillance is considered as a powerful tool in providing cost-effective, population-wide and near real-time surveillance results for controlling infectious diseases (i.e., SARS-CoV-2, influenza virus), complementary to clinical surveillance. To facilitate the utility of this emerging tool, we developed two preanalytical protocols (supernatant-based and pellet-based) for influenza A/B virus (IAV/IBV) wastewater surveillance and applied them to the established wastewater surveillance network for large-scale longitudinal monitoring in Hong Kong. We tested 724 wastewater samples from 24 stationary sites for weekly surveillance for 8 months and 458 wastewater samples from 11 wastewater treatment plants (WWTPs) for more frequent (three times per week) city-wide surveillance for 4 months when influenza season commenced. We found the city-wide IAV virus concentration in wastewater were associated with the detection rate and influenza-like illness plus rates (ILI+) of clinical respiratory specimens and increased significantly after the cancelling of mask mandate that was in place for COVID-19. IBV was at low detection rates and low virus concentration levels, consistent with the low detection rates observed by clinical surveillance. In addition, we conducted virus subtype identification in selected wastewater samples, and observed the H1pdm was the major circulation subtype. Moreover, the obtained virus signals were confirmed by Sanger sequencing of PCR products, suggesting the feasibility and applicability of established methods for rapid detection of influenza virus types and subtypes in wastewater surveillance. This study demonstrates the applicability of IAV/IBV wastewater surveillance to current wastewater infrastructures and it could be used as a rapid and cost-effective surveillance strategy to track virus transmission patterns in the community for timely public health actions in the future. [ABSTRACT FROM AUTHOR]