Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains

Abstract

In recent years, high throughput sequencing (HTS) has brought new possibilities to the study of the diversity and complexity of plant viromes. Mixed infection of a single plant with several viruses is frequently observed in such studies. We analyzed the virome of 10 tomato and sweet pepper samples from Slovakia, all showing the presence of potato virus Y (PVY) infection. Most datasets allow the determination of the nearly complete sequence of a single-variant PVY genome, belonging to one of the PVY recombinant strains (N-Wi, NTNa, or NTNb). However, in three to-mato samples (T1, T40, and T62) the presence of N-type and O-type sequences spanning the same genome region was documented, indicative of mixed infections involving different PVY strains variants, hampering the automated assembly of PVY genomes present in the sample. The N- and O-type in silico data were further confirmed by specific RT-PCR assays targeting UTR-P1 and NIa genomic parts. Although full genomes could not be de novo assembled directly in this situation, their deep coverage by relatively long paired reads allowed their manual re-assembly using very stringent mapping parameters. These results highlight the complexity of PVY infection of some host plants and the challenges that can be met when trying to precisely identify the PVY isolates involved in mixed infection.

Phylogrouping and pathogrouping of Potato virus Y isolates from pepper plants in Turkey

“…RDP5 analysis of the P1 regions of three pepper PVY isolates (DB57, DB59 and KB4) together with three O, three N and three C phylogroup homologous sequences chosen at random from the alignment of 634 sequences (see below) indicated that the N-terminal portion of the pepper P1 genes have an O phylogroup region with a cross-over point around 310 nts with an average P-value (eight methods) of having occurred by chance of 1.20x10 -21 (Figure 3). This con rms that they are from phylogroup R1, and, in particular, from its N-Wi cluster as reported by Glasa et al, (2021).…”

“…These have a major O parent and a minor N group minor parent of their 5' terminal region, but have a 3' terminal region that was either from an O lineage parent (R1) or an N lineage parent (R2). Glasa et al (2021) have now also shown that both recombinant lineages have major sublineages de ned by the 5' termini of their P1 regions, which is either N-lineage (pathogroups N:O and NTNa) or N lineage with a 5' O lineage cap of 310 nts (pathogroups N-Wi, NTNb). Our three pepper PVY recombinant isolates have the O+N 5' terminal O region con rming that they are from the N-Wi pathogroup, which is part of the R1 phylogroup.…”

“…The recombinant complexity of the PVY population was partly resolved when it was shown (Gibbs et al, 2017;Fuentes et al, 2019), that the current world PVY population has, in addition to the O, C and N lineages, two major recombinant lineages, R1 and R2, that have a major 'parent' of PVY-O, and a minor PVY-N parent that provided the 5'-terminal region of the genome in R1 isolates, or a N parent of both 5'and 3'-termini in R2 isolates. Furthermore, Glasa et al (2021) showed that the P1 gene of some R1 and R2 isolates had a N 'parent' and some had an O+N 'parent'. In addition to these four major recombinant lineages, further individual recombination events are found in all lineages, so that more than half of collected isolates are recombinants, although it is likely that the R1 and R2 recombinants are preferentially sampled as they cause 'potato tuber necrotic ringspot disease', drawing the attention of eld pathologists.…”

High-Throughput Sequencing Discloses the Cucumber Mosaic Virus (CMV) Diversity in Slovakia and Reveals New Hosts of CMV from the Papaveraceae Family

“…Moreover, in previous works analyzing viral populations in Slovakia, mixed infections involving different variants of the same virus species were noted, both for annual (PVY/tomato [45]) or perennial hosts (CVA/cherry [46]; GRSPaV/grapevine [47]). In our experiments, only a single-variant CMV population was detected in each sample (as supported by analysis of de novo contig assemblies and visual inspection of reads), enabling the accurate reconstruction of nearly full-length CMV genomes.…”

Identification of Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein-Banding Virus (CYVBV) in Cocoa (Theobroma cacao) Germplasm

“…Recent advances in next-generation sequencing (NGS) have led to the unprecedented generation of genomic and transcriptome data that are now available in public data depositories. These data have brought new possibilities to identify coinfecting known and novel virus genomes [25,26]. Therefore, we screened selected genomic datasets from three projects that report sequencing data for cocoa accessions from Brazil, Puerto Rico or Trinidad with the aim of detecting the Trinidad viruses.…”