Hybrid-capture-enabled longitudinal metagenomics allows strain-resolved human-associated virus surveillance in wastewater

ABSTRACT

Background: Targeted PCR panels dominate wastewater virus surveillance but require predefined targets and provide limited strain resolution. Hybrid-capture metagenomics can expand breadth compared to PCR panels. We assessed whether a hybrid-capture workflow can support long-term, strain-resolved wastewater virome surveillance and how these signals relate to clinical indicators. Methods: We conducted a 31-month longitudinal analysis (April 2022-December 2024) of 127 weekly 24-h composite wastewater samples from Leuven, Belgium. Samples were enriched using a comprehensive viral hybrid-capture panel (>3,000 viral species) and sequenced. We compared viral abundance and genomic composition with national clinical time series using dynamic time warping to quantify lead-lag relationships and analyzed genotype turnover for key pathogens, including Rotavirus A, adenovirus, and norovirus. Findings: We recovered a strain-resolved virome comprising 339 viral species and 715 strains. Virus composition showed clear seasonal trends and sufficient genome completeness for genotyping and subtyping across many viruses. Rotavirus A abundance in wastewater correlated with national rotavirus A activity and typically preceded clinical indicators by about one week. While aggregate adenovirus patterns correlated poorly with clinical data due to bias, species-specific analysis revealed strong concordance. A shift from norovirus GII.4 to GII.17 became evident from January 2024. Moreover, viral metagenomics detected viruses that are not systematically surveilled in wastewater, including Parvovirus B19 surge starting in June 2023, 14 months prior to the national public health alert. Sporadic detections of measles, mpox virus and repeated detections of unusual, zoonotic rotavirus A segment genotypes with consistent co-occurrence patterns were observed. Interpretation: Longitudinal hybrid-capture wastewater metagenomics can provide comprehensive, strain-resolved surveillance that complements targeted PCR by improving breadth and enabling genomic tracking across pathogens. A more systematic integration of wastewater genomics with clinical data could be a highly valuable complementary approach, allowing early detection and characterization of (asymptomatically) circulating virus variants and facilitating targeted follow-up of viruses of interest.