Abstract
The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS) of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp) gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV) was the most frequently detected Ilarvirus, occurring in 48 of the 61 Ilarvirus-positive trees and Prune dwarf virus (PDV) and Apple mosaic virus (ApMV) were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV) was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus-like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus-like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus-like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples, and the need for a standardized approach to accurately determine what constitutes an active, viable virus infection after detection by molecular based methods.
Highlights
The genus Ilarvirus is the largest in the Bromoviridae family, includes more than 20 recognized and tentative Ilarvirus species which are divided into six subgroups, and are characterized by a positive-sense, single-stranded tripartite RNA genome (Bujarski et al, 2012)
BLASTn analyses of the clustered RNA2 amplicon sequence variants revealed the presence of four Ilarvirus species (PNRSV, Prune dwarf virus (PDV), American plum line pattern virus (APLPV), and Apple mosaic virus (ApMV)) that have been previously reported in Prunus species and two potentially new Ilarvirus species amongst the 61 Prunus samples (Table 4; Table S2)
next generation sequencing (NGS) of Ilarvirus genus-specific PCR amplicons was used in this study to identify the diversity of Ilarvirus species detected in 61 Prunus trees in Australia
Summary
The genus Ilarvirus is the largest in the Bromoviridae family, includes more than 20 recognized and tentative Ilarvirus species which are divided into six subgroups, and are characterized by a positive-sense, single-stranded tripartite RNA genome (Bujarski et al, 2012). Genomic RNA1 and RNA2 of all Ilarvirus species harbor genes that encode conserved proteins involved in viral replication. Genes on RNA3 encodes a movement protein (MP) and a coat protein (CP), which is expressed via sub-genomic RNA4 (Codoner and Elena, 2008; Pallas et al, 2012a). Several Ilarvirus species infect a wide plant host range within the family Rosaceae, including Prunus species, and can cause diseases of economic importance (Card et al, 2007; Pallas et al, 2012b). The infection of some plant hosts by several Ilarvirus species complicates species identification and necessitates the use of several different specific RT-PCR tests for detection. Sequence diversity exists within Ilarvirus species (Kinoti et al, 2017), which can make the design of specific primers difficult and impact their detection by RT-PCR tests
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