Enhanced wastewater surveillance of respiratory viruses: Unraveling partitioning behavior and demixing co-circulating lineages

Wastewater-based epidemiology (WBE) enables community-level pathogen surveillance, yet incomplete understanding of viral partitioning behavior complicates strategy optimization and data interpretation. We demonstrated that pH and surfactants critically govern viral distribution between wastewater phases. Despite high solid–liquid partition coefficients, viruses predominantly reside in the liquid phase due to wastewater’s low solid content (10²-10³ mg/L). Leveraging these insights, we conducted one-year RT-qPCR surveillance across Sichuan University and Chengdu, capturing two SARS-CoV-2 waves and an influenza A outbreak. To address lineage demixing challenges from suboptimal sequencing, we developed NextVpower, a deconvolution tool resolving co-circulating lineages while tracking critical mutations for clinical early-warning. Notably, NextVpower achieved the first demixing of both HA- and NA-lineages for H3N2 influenza A virus in wastewater. This advances WBE’s capacity to link wastewater-derived mutations to epidemiological trends, enhancing real-time outbreak preparedness through optimized viral recovery and lineage-resolved analytics.