Detection, persistence, and rising prevalence of oncogenic viruses revealed by wastewater metagenomics.

Oncogenic viruses cause high-risk cancers in humans and are responsible for nearly 20% of all cancer cases worldwide. Currently, very limited data exist in the realm of wastewater-based viral epidemiology (WBE) for cancer-causing viruses, with existing studies using targeted approaches (i.e., PCR-based approaches) that lack genomic resolution. In this study, we used a hybrid-capture approach to detect, filter, and sequence all known oncogenic virus signals from wastewater samples collected over 3 years (May 2022-May 2025) in 16 Texas cities, covering nearly 25% of the state's population. Once sequenced, we used custom computational tools designed for wastewater metagenomics to assign reads into their respective virus of origin, estimate viral abundances over time, and measure genomic read coverage. Our data indicate that we successfully detected oncogenic viruses, including six known oncogenic viruses, and three suspected oncogenic viruses, across all sampling locations within Texas. We observed a gradual increase in the viral abundance of oncogenic viruses over 3 years, with distinct peaks and dips over the summer and winter months. The prevalence of high-risk viruses such as human papillomavirus (HPV) and Epstein-Barr virus (EBV) rose, with sharp increases in viral abundance observed post-2024. We also obtained nearly 100% genome coverage with viral reads captured using this hybrid-capture technique for nearly all oncogenic viruses, with resolution down to the species and type taxonomic levels in some cases, such as that of HPV. Our study showcases the utility of hybrid-capture techniques to detect and track multiple oncogenic viruses simultaneously.IMPORTANCECancer-causing viruses are of major clinical significance, responsible for nearly 20% of all recorded cancer incidences in humans worldwide. There is a need for improved detection, tracking, and control of oncogenic viruses across the globe. To our knowledge, this work is the first comprehensive WBE approach used to detect all known oncogenic viruses concurrently, demonstrating the feasibility of monitoring the presence and levels of cancer-causing viruses and enabling the possibility of public health interventions in the future. Using this method, we obtain broad genomic coverage at strong depth and specificity, coupled with consistent real-time tracking dynamics of multiple oncogenic viruses. Furthermore, we showcase the ability to identify genomic regions on viral reference genomes from which sequenced reads originate. This information can be an invaluable tool toward understanding the viral prevalence dynamics in general populations, their relationship to cancer incidences in humans, and their mechanisms of viral evolution, including mutations.