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

• Published in: Viruses Vol. 16; no. 3; p. 460
• Main Authors: Nash, Delaney, Ellmen, Isaac, Knapp, Jennifer J, Menon, Ria, Overton, Alyssa K, Cheng, Jiujun, Lynch, Michael D J, Nissimov, Jozef I, Charles, Trevor C
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01-03-2024; MDPI
• Subjects: ARTIC amplicon sequencing; Biochemical assays; Contamination; Crop diseases; Crop yield; Crops; Diseases and pests; Distribution; DNA sequencing; Ebola virus; Epidemics; Flowers & plants; Fruit; Genetic aspects; Genomes; Humans; Identification and classification; Infections; lineage abundance estimation; Methods; Municipal wastewater; Mutation; Nucleotide sequencing; Ontario; Pathogens; Phylogenetics; Plant viruses; Proteins; RNA polymerase; Severe acute respiratory syndrome coronavirus 2; Solanum lycopersicum; Tobamovirus - genetics; Tomato Brown Rugose Fruit Virus (ToBRFV); Tomatoes; Virus diseases of plants; Viruses; Wastewater; Wastewater treatment; Water Purification; Water treatment; Ontario; Canada; Ontario Canada; lineage abundance estimation; wastewater; ARTIC amplicon sequencing; Tomato Brown Rugose Fruit Virus (ToBRFV)
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v16030460

Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant pathogen that infects important
crop species and can dramatically reduce tomato crop yields. The ToBRFV has rapidly spread around the globe due to its ability to escape detection by antiviral host genes which confer resistance to other tobamoviruses in tomato plants. The development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detected ToBRFV in municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution of ToBRFV in WWI throughout Ontario, Canada. To aid in global ToBRFV surveillance efforts, we developed a tiled amplicon approach to sequence and track the evolution of ToBRFV genomes in municipal WWI. Our assay recovers 95.7% of the 6393 bp ToBRFV RefSeq genome, omitting the terminal 5' and 3' ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recovering ToBRFV genomes. Our ToBRFV assay was developed using existing ARTIC Network resources, including primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance of ToBRFV clades in samples.