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

Abstract

Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant pathogen that infects important Solanaceae 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.

Keywords: ARTIC amplicon sequencing; Tomato Brown Rugose Fruit Virus (ToBRFV); lineage abundance estimation; wastewater.

Figure 1

ToBRFV-specific primer binding sites and resulting amplicons mapped to the ToBRFV MK133093 genome sequence. In silico visualization of primer binding sites; forward and reverse primers are denoted by the upward and downward pointing triangles, respectively, and each resulting amplicon is numbered and denoted by blue and orange flags [48]. Pool 1 primers and amplicons are blue, pool 2 primers and amplicons are orange. Nucleotide positions are labeled at 1000 bp intervals.

Figure 2

Taxonomic Classification of RNA Shotgun and ToBRFV-Seq Read Pairs at the Domain- and Species-level. Kraken2 classified read pair counts at the domain level for (A) RNA shotgun library prepared samples and (B) ToBRFV-Seq prepared samples [39]. At the domain level, categories included are viral, archaea, bacterial, eukaryotic, or other reads, where other includes unclassified reads, plasmids, adapters, linkers, and/or primers. (C) The percentage of reads represented by each domain for both prep methods. Samples A–E were used to calculate the average, the blank was excluded. (D,E) Percentage of total reads classified as ToBRFV (T), other tobamoviruses (OT), and the remainder of virus classified reads (RR) (F). Counts of tobamovirus classified read pairs at the species level (G). (A,B,D,E) Reads are represented on a log scale to magnify and visualize low abundance reads. Two-tailed tests were used to compare the mean read counts of RNA shotgun and ToBRFV-Seq classified reads, respectively. (D) RNA shotgun samples, p < 0.05, n = 5, df = 4, t-values, (T-OT) 3.569, (T-RR)-1.015, and (RR-OT)-1.181, critical t-value ± 2.776. (E) ToBRFV-Seq samples, p < 0.05, n = 5, df = 4, t-values, (T-OT) 12.523, (T-RR) 12.523, and (RR-OT) 2.449, critical t-value ± 2.776. Thus, there is a statistically significant difference between the number of ToBRFV and other tobamovirus reads produced by RNA shotgun sequencing, whereas there is a statistically significant difference between the number of ToBRFV reads produced compared to both other tobamovirus reads and the read remainder.

Figure 3

ToBRFV Clade-Abundance Estimates of Synthetic Read Datasets and WWI samples with Altob. Heatmaps display Altob clade abundance estimates of (A) datasets containing 1000, 10,000, or 100,000 synthetic reads from eight ToBRFV genomes representative of each clade. (B) Datasets combining 100,000 synthetic reads from either all eight genomes, four genomes, or two genomes. (C) Reads from WWI samples prepared with RNA shotgun (Shotgun) or our ToBRFV-Seq assay (ToBRFV) (A–E).

Figure 4

Evaluation of virus-species abundance in WWI samples using counts of viral reads taxonomically classified by Kraken2. (A) Percentage of virus paired read counts of the seven most abundant virus species found in each sample and count of all other species. (B) Percentage of virus species classified reads assigned to the most abundant species in each WWI sample prepared by RNA shotgun and a blank.