The molecular characterization of South African isolates of Grapevine Rupestris Stem Pitting-associated virus (GRSPaV)

[摘要] ENGLISH ABSTRACT: The first aim of this study was to reliably and rapidly detect Grapevine rupestris stem pittingassociatedvirus (GRSPaV) in grapevine. This was achieved by screening 94 grapevinesusing crude plant extracts in both quantitative and conventional reverse transcriptionpolymerase chain reaction (RT-PCR). The second aim was to establish a technique capable ofdifferentiating GRSPaV sequence variants. The application of this technique is for the largescalescreening of diseased vines to associate sequence variants of GRSPaV with diseasesymptoms. Nested quantitative polymerase chain reaction and high resolution melting assays(qPCR-HRM) were developed for three regions of the GRSPaV genome (coat protein, RNAdependantRNA-polymerase and triple gene block movement protein). The qPCR-HRMtechnique using the high saturation dye, EvaGreen™, and the Rotor-Gene™ 6000 analyzerwas validated with a panel of sixteen sequence-characterized viral isolates. Diluted RT-PCRproducts and cloned cDNA gave the most consistent amplification plots and dissociationprofiles. RT-PCR products generated from total RNA extracts were used as template forqPCR-HRM assays and for direct sequencing of sixteen samples in the three aforementionedregions. The average amplification efficiency for qPCR was 1.52±0.04. Auto-calling of userdefinegenotypes was performed at a confidence interval of 70%. Phylogenetic analysis of thethree regions of the GRSPaV genome was performed with published GenBank sequences toconfirm the HRM data. The dominant sequence variants found in the South African sampleset radiated with Group II, reference full-length variant GRSPaV-SG1. GRSPaV-infectedsamples can in future be subjected to qPCR-HRM assays developed during this study. Thiscan be performed to establish similarity to known genotypes and therefore phylogeneticgroups. Mixed infection of sequence variants and quasi-species were a common occurrence.The assay will be useful in establishing correlation of specific genotypes to differentphenotypical expression of viral disease. This could provide insight into the etiology ofdiseases associated with GRSPaV.

[发布机构] Stellenbosch University