A Novel Tiled-Amplicon Sequencing Assay Targeting theTomato Brown Rugose Fruit Virus(ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada

Abstract

AbstractTomato Brown Rugose Fruit Virus(ToBRFV) is a plant pathogen that infects importantSolanaceaecrop species and can dramatically reduce tomato crop yields. TheToBRFVhas rapidly spread around the globe due to its ability to escape detection by antiviral host genes, most notablyTm-22, which are used to confer resistance to otherTobamovirusesin tomato plants. Development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detectedToBRFVin municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution ofToBRFVin WWI throughout Ontario, Canada. To aid in globalToBRFVsurveillance efforts, we developed a tiled-amplicon approach to sequence and track the evolution ofToBRFVgenomes in municipal WWI. Our assay recovers 97.5% of the 6393 bpToBRFVRefSeq genome, omitting the terminal 5’ and 3’ ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recoveringToBRFVgenomes. OurToBRFVassay was developed using existing ARTIC Network resources, which includes genome specific primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance ofToBRFVclades in samples.