Analysis of intra-host genetic diversity of Prunus necrotic ringspot virus (PNRSV) using amplicon next generation sequencing

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.

The population structure of Rose rosette virus in the USA

“…Studies on the genetic diversity of plant viruses play a significant role in understanding virus evolution as well as the development and/or improvement of virus detection protocols [20,34,35]. Due to the occurrence of quasi-species, recombination and reassortment it is not always easy to define the population for viruses with multipartite genomes [14].…”

“…This has broadened current knowledge on the mechanisms that generate genetic diversity and the evolutionary forces that shape the genetic structure and dynamics of virus populations [36]. HTS has allowed for the discovery of novel viruses across a vast array of sample types, but due to large numbers of host/non-viral reads, gaining adequate coverage of full viral genomes within a sample in order to look at population structure is difficult [14]. In order to increase the viral relative to host sequence, we used a combination of targeted reverse transcription to ensure that all seven segments were amplified, followed by virus -specific Hi-Plex PCR, and finally HTS sequencing.…”

“…While these isolates are a consensus of the quasi-species population within a sample, this method allows for rapid acquisition of sequence data from a large number of samples, which is time-consuming and difficult using traditional Sanger sequencing. Although Hi-Plex and similar technologies have been applied broadly in human viruses and genes related to cancer studies [18,37], only a handful have been reported on plant viruses so far [14]. These studies focused on the mutational profile of a virus isolate, specifically, identifying single-nucleotide polymorphisms (SNPs) across the genome [10,14,38,39].…”

“…In plant virology, HTS is mostly applied in virus detection and discovery [6][7][8][9][10], but a major drawback in the methodology is the output of a high percentage of non-viral sequence, limiting sequencing depth and identification of virus variants [11,12]. HTS of PCR amplicons minimizes host sequence interference and has been used successfully to study the population structure of fungal and animal viruses [13][14][15][16]. Accurate variant calling relies on the identification of mutations appearing in multiple amplicon reads, thus permitting stringent filtering of sequencing chemistryinduced errors [17,18].…”

“…Quasi-species can have significant impact on virus evolution, as changes in selection pressure can give rise to new strains and species [14,[19][20][21]. Micro (quasi-species) and macroevolution (recombination/reassortment) may also lead to the emergence of highly virulent strains and wide host range [14,22]. Equally, these could lead to an accumulation of less virulent strains that are persistent in the population due to their optimal interaction with their hosts [23].…”