Abstract
PCR amplicon next generation sequencing (NGS) analysis offers a broadly applicable and targeted approach to detect populations of both high- or low-frequency virus variants in one or more plant samples. In this study, amplicon NGS was used to explore the diversity of the tripartite genome virus, Prunus necrotic ringspot virus (PNRSV) from 53 PNRSV-infected trees using amplicons from conserved gene regions of each of PNRSV RNA1, RNA2 and RNA3. Sequencing of the amplicons from 53 PNRSV-infected trees revealed differing levels of polymorphism across the three different components of the PNRSV genome with a total number of 5040, 2083 and 5486 sequence variants observed for RNA1, RNA2 and RNA3 respectively. The RNA2 had the lowest diversity of sequences compared to RNA1 and RNA3, reflecting the lack of flexibility tolerated by the replicase gene that is encoded by this RNA component. Distinct PNRSV phylo-groups, consisting of closely related clusters of sequence variants, were observed in each of PNRSV RNA1, RNA2 and RNA3. Most plant samples had a single phylo-group for each RNA component. Haplotype network analysis showed that smaller clusters of PNRSV sequence variants were genetically connected to the largest sequence variant cluster within a phylo-group of each RNA component. Some plant samples had sequence variants occurring in multiple PNRSV phylo-groups in at least one of each RNA and these phylo-groups formed distinct clades that represent PNRSV genetic strains. Variants within the same phylo-group of each Prunus plant sample had ≥97% similarity and phylo-groups within a Prunus plant sample and between samples had less ≤97% similarity. Based on the analysis of diversity, a definition of a PNRSV genetic strain was proposed. The proposed definition was applied to determine the number of PNRSV genetic strains in each of the plant samples and the complexity in defining genetic strains in multipartite genome viruses was explored.
Highlights
RNA viruses are genetically heterogeneous and their replication mechanism results in mutants or variants that may differ genetically from the original virus [1]
The total number of raw reads was 3,431,678 when the next generation sequencing (NGS) amplicon data for Prunus necrotic ringspot virus (PNRSV) MT, RNA dependent RNA polymerase (RdRp) and coat protein (CP) gene segments of RNA1, RNA2 and RNA3 respectively, which were amplified from all 53 plant samples, were combined
This study describes a novel use of amplicon NGS to investigate the diversity of a tripartite genome virus, PNRSV, by focusing on conserved regions within the MT, RdRp and CP genes of each RNA component of the genome
Summary
RNA viruses are genetically heterogeneous and their replication mechanism results in mutants or variants that may differ genetically from the original virus [1]. A single virus isolate (representing a single specific sample of virus) does not consist of a single RNA sequence, rather, it consists of a population of related sequence variants, often referred to as ‘quasi-species’, which are generated due to the error-prone nature of the viral RNA-dependent RNA polymerase and rapid replication of virus genomes [2,3,4] These populations of sequence variants are significant in defining strains that make up virus species and have been shown to have implications in the emergence of new and fitter virus strains that result from changing selection pressures [2] and could complicate their diagnosis. The polymorphisms could lead to an accumulation of less virulent strains that are more persistent in the population due to their lack of symptom expression and latent infection status
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