Targeted metagenomic sequencing for detection of vertebrate viruses in wastewater for public health surveillance

Abstract

Viruses of concern for quantitative wastewater monitoring are usually selected as a result of an outbreak and subsequent detection in wastewater. However, targeted metagenomics could proactively identify viruses of concern when used as an initial screening tool. To evaluate the utility of targeted metagenomics for wastewater screening, we used ViroCap, a panel of probes designed to target all known vertebrate viruses. Untreated wastewater was collected from wastewater treatment plants (WWTPs) and building-level manholes associated with vulnerable populations in Houston, TX. We evaluated differences in vertebrate virus detection between WWTP and building-level samples, classified human viruses in wastewater, and performed phylogenetic analysis on astrovirus sequencing reads to evaluate targeted metagenomics for subspecies level classification. Vertebrate viruses varied widely across building-level samples. Rarely detected and abundant viruses were identified in WWTP and building-level samples, including enteric, respiratory, and bloodborne viruses. Furthermore, full length genomes were assembled from astrovirus reads and two human astrovirus serotypes were classified in wastewater samples. This study demonstrates the utility of targeted metagenomics as an initial screening step for public health surveillance.

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

“…Beyond the concentration and extraction method, the sensitivity of probe-capture sequencing will likely vary with the probe panel selected and the seasonal fluctuations in wastewater microbial composition. In the present study and other studies using broad virus capture panels, 22,49 human virus sequences were predominantly enteric virus targets present in the panel, such as mamastrovirus, and the detection of SARS-CoV-2 was limited. However, several studies using the narrower RVOP panel found remarkably high coverages of SARS-CoV-2, surpassing other respiratory human viruses included in the panel.…”

“…McCall et al compared methods with very different sampling volumes (300 μL for direct extraction and 50 mL for HA filtration) and suggested that direct extraction may yield a lower equivalent volume of viruses in the final extracted nucleic acid compared to prefiltered samples. 22 Spurbeck et al indirectly compared five wastewater virus concentration/extraction methods, but each was applied to wastewater samples from a different location(s). They found that Innovaprep ultrafiltration yielded the highest virus sequence recovery in untargeted RNA sequencing, although most sequences corresponded to bacteriophage.…”

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

“…23 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. 24,[28][29][30]41 Although differences in samples, sample processing, and bioinformatics make direct comparisons impossible (Table 1), these results point to trade-offs in probe panel selection.…”

“…17 with probe capture provides an opportunity to surveil a broad range of viruses in wastewater, capturing disease dynamics for pathogens lacking comprehensive clinical surveillance. 20 Several targeted virus sequencing panels have recently been applied to wastewater samples, including Illumina Respiratory Virus (RVOP), 21−23 Virus Surveillance (VSP), 20,24 and Respiratory Pathogen/AMR (RPIP), 23,25 VirCapSeq-VERT, 26 ViroCap, 27,28 Twist Comprehensive Virus Capture Panel, 29 and Qiagen xHyb Adventitious. 30 When compared to untargeted sequencing, probe capture enrichment was found to successfully increase target virus relative abundance by orders of magnitude (Table 1).…”

Evaluation of the impact of concentration and extraction methods on the targeted sequencing of human viruses from wastewater

“…These methods included size separation (e.g., Innovaprep ultrafiltration pipette, centrifugal ultrafiltration), capture based on virus surface characteristics (e.g., Nanotrap beads, electron-negative HA membrane), and direct nucleic acid extraction (e.g., Promega Wizard Enviro large-volume extraction, or extraction of wastewater solids after centrifugation). These routine monitoring methods were also used to obtain SARS-CoV-2 RNA for sequencing, with varying success 19–22 and later extended for detection of a wider spectrum of viruses 14, 15, 23–25 . To date, few studies have directly compared the effects of different methods on the success of virus probe-capture enrichment sequencing.…”