Tracking viral RNA loads during in-sewer transport: insights from real-world field experiments

Wastewater-based surveillance (WBS) is widely used to monitor respiratory viruses, yet uncertainties remain regarding how viral RNA concentrations in wastewater reflect infection dynamics. Specifically, diurnal variation in shedding and RNA losses during in-sewer transport can impact measured signals. We conducted a field study in a 5-km trunk sewer (travel time of one hour). Wastewater was sampled at the sewer inlet and outlet using autosamplers collecting time-proportional one-hour composite samples over 24 hours. The one-hour composite samples were analyzed for assessing intra-daily fluctuations, and 24-hour composites for signal change. Biofilms from the sewer-pipe walls were collected at three locations. Nucleic acids were extracted, and SARS-CoV-2, Influenza A/B, and Respiratory Syncytial Virus (RSV) RNA were quantified using a multiplex digital PCR assay. All viruses showed pronounced diurnal variation, with consistent morning load peaks. Viral RNA in the bulk liquid decreased during in-sewer transport, with modelled changes ranging from 15% to 72% across pathogens. Biofilms served as minor reservoirs of viral RNA; for SARS-CoV-2, sequencing revealed similarity between biofilm and bulk liquid RNA. Our study provides a full-scale assessment of in-sewer transport effects on viral RNA and highlights the need to account for complex in-sewer dynamics when interpreting WBS data.