Wastewater Sequencing Reveals Persistent Circulation and Rising Prevalence of Several Oncogenic Viruses Across Texas

Summary Background Oncogenic viruses cause high-risk cancers in humans and are responsible for nearly 20% of all cancer cases worldwide. Currently, very limited data exists in the realm of wastewater-based viral epidemiology (WBE) of cancer-causing viruses, with existing studies using targeted approaches (i.e PCR-based approaches) which lack scalability. Our study aims to carry out WBE with hybrid-capture probes to detect and track multiple oncogenic viruses simultaneously in wastewater across Texas, USA, overcoming the drawbacks associated with targeted approaches. Methods Here, we used a hybrid-capture approach to detect, filter and sequence oncogenic virus signals from wastewater samples collected over a duration of three years, from May 2022 to May 2025. Once viral reads were sequenced, we utilized established computational tools to characterize reads into their respective virus of origin. Next, viral abundances of each characterized oncogenic virus were tracked over time and read coverage across their genomes was measured using read mapping techniques. Findings We detected six known oncogenic viruses, along with three suspected oncogenic viruses across all sampling locations within Texas. Over three years, viral abundance gradually increased, with distinct peaks and dips over the summer and winter months. The prevalence of high-risk viruses such as HPV and EBV rose sharply, with increases in abundance observed post-2024. We also obtained nearly 100% genome coverage with viral reads captured using a hybrid-capture technique for almost all oncogenic viruses and their types.] Interpretations Our study shows that a hybrid-capture method can efficiently overcome the challenges faced with using targeted approaches for WBE. Using this method, we get broader read coverage, coupled with concurrent and consistent real-time tracking dynamics of multiple oncogenic viruses. Our findings also emphasize the persistent circulation and rising prevalence of high-risk cancer-causing viruses, underscoring the need for sustained public health interventions to protect communities and assess viral prevalence in high-risk populations. Funding This work was supported by S.B. 1780, 87th Legislature, 2021 Reg. Sess. (Texas 2021), the Baylor College of Medicine and the Alkek Foundation Seed Funds. Research in context Evidence before this study Cancer-causing viruses are of major clinical significance, responsible for nearly 20% of all recorded cancer incidences in humans worldwide. With some of these viruses causing high-risk cancers such as cervical cancer, there is a need for improved detection, tracking and control of oncogenic viruses across the globe. During the SARS-CoV-2 pandemic, wastewater-based viral epidemiology (WBE) rose to the forefront of virus tracking, utilizing non-invasive methods to detect and monitor the prevalence of medically relevant viruses of concern. Today, WBE has been utilized to accelerate the surveillance of numerous viruses such as SARS-CoV-2, mpox, influenza and more. To reinforce our prior knowledge on current trends in WBE of oncogenic viruses, we searched for research articles in PubMed and Google Scholar containing keywords “wastewater” and “oncogenic viruses” or “tumor viruses”. Further, to look specifically at oncogenic viruses of clinical concern, we searched for studies containing the keywords “wastewater” along with each of the 9 oncogenic viruses—Human papillomavirus, Hepatitis B virus, Hepatitis C virus, BK virus, Epstein-Barr virus, Merkel cell polyomavirus, Human polyomavirus, Kaposi Sarcoma-associated virus and Human T-cell lymphotropic virus type 1. On assessment of the search results, we found that specific oncogenic viruses had been detected in the wastewater of numerous countries including Egypt, Uruguay, Canada, Catalonia, Italy, India and Australia. These studies primarily focused on using PCR-based techniques to sequence and obtain viral read sequences from wastewater, measuring viral RNA concentrations in the process. While most of these studies focused on sole detection of oncogenic viruses, some included the analysis of prevalence of viral load over time. However, we found no other studies showcasing concurrent tracking dynamics of multiple oncogenic viruses in the United States over a span of three years. Added value of this study Considering the medical significance of oncogenic viruses, we set out to utilize Texas Wastewater and Environmental Biomonitoring’s (TexWEB’s) hybrid-capture approach to detect and track these viruses in wastewater. Current WBE approaches utilize PCR-based techniques, which have drawbacks such as limited specificity to a single target. Our study highlights the value in using a hybrid-capture method to bring forth a greatly improved WBE approach, providing near real-time tracking of all oncogenic viruses. To our knowledge, our work is the first comprehensive WBE approach which uses a sequencing-based method to detect all known oncogenic viruses concurrently. Here, we present the tracking of viral abundances over the course of three years for all viruses, analyzing seasonal variations in the process. Further, we also showcase the ability to identify genomic regions on viral reference genomes from which sequenced reads originate. This information can be an invaluable tool towards understanding the dynamics of the prevalence of cancer-causing viruses in the general population, their relationship to cancer incidences in humans, and their mechanisms of viral evolution. Implications of all the available evidence Our study highlights the advantages of using a hybrid-capture approach for WBE of oncogenic viruses. This approach can be used to detect a complete panel of multiple selected viruses and provides viral abundance and prevalence information, overcoming the drawback of high specificity to single targets that come with PCR-based approaches. We also highlight the scope of this approach in tracking and monitoring multiple relevant oncogenic viruses concurrently at regular sampling intervals. Data generated using this technique can be used by public health departments to identify viruses of immediate concern, set up pandemic preparedness and intervention programs, as well as promote vaccination drives while spreading general public health awareness among communities.

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