Detection of multiple sequence variants of Grapevine rupestris stem pitting-associated virus using primers targeting the polymerase domain and partial genome sequencing of a novel variant

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus within the new family Betaflexiviridae. GRSPaV is distributed among grapevines worldwide and is implicated in the disease rupestris stem pitting (RSP) of the rugose wood complex and two other disorders. GRSPaV is composed of a wide range of sequence variants, and so far, the complete genomes of five sequence variants have been sequenced. Quick and reliable detection of different GRSPaV variants is a critical step in the elimination and control of GRSPaV. Previously, primers designed from various genomic regions have been used in RT-PCR for the detection of GRSPaV variants. The efficiency of RT-PCR varied widely depending on the spectrum of the primers that were used. In this study, we designed a pair of degenerate primers based on the consensus sequence of the genomic region encoding the highly conserved RNA-dependent RNA polymerase domain from five reference isolates of GRSPaV for which the genome sequence are available. We demonstrate that this set of primers is comparable, if not superior, to the broad-spectrum primers RSP13&14 in detecting multiple GRSPaV variants. Using these degenerate primers, we identified two new and distinct sequence variants. The 3′ terminal genomic region of one of the new variants, GRSPaV-ML, spanning the 3′ part of ORF1, through the entire open reading frames 2-4, and the 5′ region of ORF5 were sequenced. Sequence comparison demonstrates that GRSPaV-ML is distinct from each of the five reference isolates. © 2011 Association of Applied Biologists.