Wastewater sequencing reveals community and variant dynamics of the collective human virome

Abstract

Wastewater is a discarded human by-product, but its analysis may help us understand the health of populations. Epidemiologists first analyzed wastewater to track outbreaks of poliovirus decades ago, but so-called wastewater-based epidemiology was reinvigorated to monitor SARS-CoV-2 levels while bypassing the difficulties and pit falls of individual testing. Current approaches overlook the activity of most human viruses and preclude a deeper understanding of human virome community dynamics. Here, we conduct a comprehensive sequencing-based analysis of 363 longitudinal wastewater samples from ten distinct sites in two major cities. Critical to detection is the use of a viral probe capture set targeting thousands of viral species or variants. Over 450 distinct pathogenic viruses from 28 viral families are observed, most of which have never been detected in such samples. Sequencing reads of established pathogens and emerging viruses correlate to clinical data sets of SARS-CoV-2, influenza virus, and monkeypox viruses, outlining the public health utility of this approach. Viral communities are tightly organized by space and time. Finally, the most abundant human viruses yield sequence variant information consistent with regional spread and evolution. We reveal the viral landscape of human wastewater and its potential to improve our understanding of outbreaks, transmission, and its effects on overall population health.

Virome Sequencing Identifies H5N1 Avian Influenza in Wastewater from Nine Cities

“…SARS-CoV-2, Influenza, and Mpox) correlate to clinical case data. 17 Seasonal influenza serotypes H3N2 and H1N1 are routinely detected in TEPHI wastewater samples, and levels have corresponded to clinical case loads from May 2022 through the beginning of March 2024. Until that point, serotype H5N1 was never detected (0 out of 1,337 wastewater samples).…”

“…All collection, processing, and sequencing methods have been described in an earlier publication 17 and were followed precisely. To restate, between 100–500 mL of raw wastewater influent was collected into 500 ml leak-proof prelabeled sample bottles at 23 wastewater treatment plants in 10 Texas cities, the identities of these cities are masked on the request of local public health officials and water utilities.…”

Considerations and Opportunities for Probe Capture Enrichment Sequencing of Emerging Viruses from Wastewater

“…Opportunities for technical advancement include (1) increasing the enrichment factor via changes to hybridization conditions or new chemistry that strengthens hybridization, (2) eliminating probes that capture highly abundant off-target sequences, both during in silico panel design and during panel testing specifically on wastewater samples, (3) balancing the number of probes specific for prevalent viruses and rare viruses based on empirical data from wastewater monitoring, and/or designing custom panels that exclude high-abundance targets, (4) enabling transparency and optimization by embracing open source rather than proprietary panel designs, using available bioinformatic tools. 16,45 (e.g., Kraken2, 46 Centrifuge 47 ), combined mapping and assembly/consensus-building tools (e.g., EsViritu, 29 IDSeq, 48 and DRAGEN, 49 among others), and de novo assembly followed by homology searches (e.g., BLAST, Hidden Markov Model searches (HMMs)). The diversity of approaches reflects the many different goals of wastewater virus sequencing ranging from fast classification of reads to variant tracking to the discovery of novel viruses.…”

“…Child et al applied both RVOP and RPIP in parallel and reported lower coverage of SARS-CoV-2 with RPIP, likely due to competition for sequencing with other targets . Several other studies applied very broad panels covering major groups of human viruses and reported that the predominant human virus was Mamastrovirus followed by other enteric viruses, while respiratory viruses were at lower relative abundance sometimes by orders of magnitude. ,− , Although differences in samples, sample processing, and bioinformatics make direct comparisons impossible (Table ), these results point to trade-offs in probe panel selection.…”

“…Several pan-viral probe panels designed prior to the COVID-19 pandemic (i.e., panels lacking probes for SARS-CoV-2) were able to recover partial SARS-CoV-2 genomic sequences from clinical samples with high viral concentrations. − Additionally, the high-level multiplexing available with probe capture provides an opportunity to surveil a broad range of viruses in wastewater, capturing disease dynamics for pathogens lacking comprehensive clinical surveillance . Several targeted virus sequencing panels have recently been applied to wastewater samples, including Illumina Respiratory Virus (RVOP), − Virus Surveillance (VSP), , and Respiratory Pathogen/AMR (RPIP), , VirCapSeq-VERT, ViroCap, , Twist Comprehensive Virus Capture Panel, and Qiagen xHyb Adventitious . When compared to untargeted sequencing, probe capture enrichment was found to successfully increase target virus relative abundance by orders of magnitude (Table ).…”