RNA Viromics of Southern California Wastewater and Detection of SARS-CoV-2 Single-Nucleotide Variants

Abstract

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early-warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the COVID-19 pandemic to track disease burden through analyses of SARS-CoV-2 RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the healthcare system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven treatment plants (WTPs), collected August 2020 – January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically-important SARS-CoV-2 variants, along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. Importance: Wastewater based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the COVID-19 pandemic to track the spread of SARS-CoV-2 without adding burden to healthcare systems. In this study, we used metatranscriptomics and RT-ddPCR to assay RNA viruses across Southern California wastewater from August 2020 – January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single nucleotide variants through sequencing. Likewise, WTPs harbored different viromes, and we detected other human pathogens such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

Making waves: Wastewater surveillance of SARS-CoV-2 in an endemic future

“…We anticipate that technologies that have been developed for variant monitoring in wastewater will continue to play an important role for monitoring the evolution of SARS-CoV-2 in the endemic phase, when clinical testing and sequencing will probably decrease. WBS, coupled with genomic sequencing, would play a prominent role in maintaining a watchful eye on the evolution and emergence of SARS-CoV-2 variants circulating in the community as has previously been demonstrated ( Crits-Christoph et al, 2021 ; Rothman et al, 2021 ; Smyth et al, 2022 ). Quantitative, VOC-specific PCR-based assays ( Lee et al, 2022 , 2021b , 2021a ), RT-ddPCR ( Gering et al, 2021 ; Heijnen et al, 2021 ) and nested RT-PCR ( La Rosa et al, 2021 ) will continue to provide a cost-effective means for identifying and quantifying the distribution of VOCs in populations.…”

“…To this end, WBS coupled with next generation sequencing represents a highly promising tool. As wastewater is a complex matrix containing a myriad of microorganisms, the detection of the pathogens requires either amplification or metagenomics with an implemented viral nucleotide acid enrichment ( Crits-Christoph et al, 2021 ; Rothman et al, 2021 ), both of which rely on, and are biased by, a priori knowledge of genomic information on those biological agents. Pathogen detection using molecular methods independent of prior knowledge need to be developed for early warning surveillance of novel viruses with risk to human health.…”

Applications of de Bruijn graphs in microbiome research

“…The identification of genetic variants between microbes, like single‐nucleotide variants (SNVs) and indels, has broad applications in biomedical and ecological studies [34], monitoring outbreaks of pathogens [35,36], and differentiating microbial populations at the strain level [37]. Many of the standard approaches used for variant discovery utilize mapping to a reference genome or sequence, which can be computationally costly and may not always be possible when references are not available or are too divergent to be used for accurate comparisons.…”

Monitoring of SARS-CoV-2 Variants by Wastewater-Based Surveillance as a Sustainable and Pragmatic Approach—A Case Study of Jaipur (India)

“…As a result, finding variants in wastewater could be a more efficient, quicker, and practical way to track the appearance and spread of new variants in a community. Several research works have attempted to sequence the SARS-CoV-2 genome and detected genetic variations from wastewater samples in several locations, including Montana, USA [16], California, USA [17], Switzerland [18], London [19], Canada [20], and India [21], among others. To effectively control the COVID-19 pandemic in a coordinated manner, it is critical to promote this unique strategy on a much larger scale globally and to build a repository of dominant variants in circulation.…”