COS 92-1 - Large-scale virus detection and variant sequencing across Southern California’s wastewater.

Abstract: Wastewater based epidemiology (WBE) is a useful method to detect pathogen prevalence and may serve to effectively monitor diseases and water contamination at a broad scale. For example, WBE has been used throughout the COVID-19 pandemic to track viral genomic diversity and the emergence of potential SARS-CoV-2 variants. Unlike sequencing SARS-CoV-2 in healthcare settings, WBE has been used to sequence virus without adding additional testing or supplies burden to the healthcare system and has detected other viruses without the need to collect infected specimens.

Here, we present a study in which we sequenced RNA extracted from sewage influent samples obtained from eight wastewater treatment plants representing 16 million people in Southern California over April 2020 – August 2021. We present multiple use cases for viral sequencing: SARS-CoV-2 eight members of the genus Tobamovirus, and the detection of several animal-infecting viruses such as Norovirus and other Coronaviruses along with animal-specific bacteriophages. We sequenced the resulting viral RNA in three separate ways: We sequenced SARS-CoV-2 with Illumina Respiratory Virus Enrichment and metatranscriptomic sequencing (N = 275), and QIAseq SARS-CoV-2 tiled amplicon sequencing (N = 96), while we used untargeted RNA-seq (N = 275) for tobamoviruses and animal-infecting viruses/bacteriophages.

We were able to assign clades to SARS-CoV-2 reads, which largely mirrored emergent SARS-CoV-2 clades isolated from healthcare settings and identified single nucleotide variants (SNVs) of the virus that often corresponded to variants of interest and/or concern. Through our analyses, we also detected a large diversity of putatively novel SARS-CoV-2 SNVs, and SNVs of unknown potential function and prevalence.

For tobamoviruses, we assembled 1,083 high-quality genomes, and detected thousands of single nucleotide variants with amino acid-altering consequences along with synonymous mutations across the eight viral species, which represents potential evolution with functional consequences in genomes of these viruses.

Regarding animal-infecting viruses, we detected several different Coronaviruses, Norovirus, and notable counts of arthropod-infectious permutetra-related viruses along with crAssphage (a bacteriophage commonly found in human samples).

Our study shows the potential of WBE to sequence RNA viruses across large wastewater catchments in urban and suburban areas and to track strain diversity and SNVs for important pathogens of humans, other animals, and plants. Due to the ubiquity of several detected viruses, we suggest that alternative markers for human fecal contamination and microbial water quality be considered for use such as the Tomato Brown Rugose Fruit Virus or crAssphage.