Accumulated data and results from the recent study of dsRNA isolated from grapevines used in experiments of insect and graft transmission of 'Shiraz' disease

RT-PCR amplicons of dsRNA isolated from various grapevines, which were used in the experiments of transmission of 'Shiraz' disease (SD) from 'Cinsaut Blanc' clone P163/12 to SD-susceptible 'Merlot' and 'Shiraz' using mealybug Planococcus ficus and grafting were investigated. The amplicons were generated in RT-PCR based on virus-specific or random hexamers oligonucleotide primers. Standard molecular techniques and high-throughput sequencing (HTS), respectively, were applied. The results supported the hypothesis that GVA M5v variant present in 'Cinsaut Blanc' P163/12, which is a member of group II of GVA variants associated with SD, is crucial for developing this disease. HTS data did not reveal any other grapevine viruses besides GLRaV-3 and GVA in SD-affected grapevines, except for GVE which, however, was not present in all diseased plants

Genome Sequences and Structures of Two Biologically Distinct Strains of Grapevine leafroll - associated virus 2 and Sequence Analysis

Grapevine leafroll-associated virus 2 (GLRaV-2), a member of the genus Closterovirus within Closteroviridae, is implicated in several important diseases of grapevines including "leafroll”, "graft-incompatibility”, and "quick decline” worldwide. Several GLRaV-2 isolates have been detected from different grapevine genotypes. However, the genomes of these isolates were not sequenced or only partially sequenced. Consequently, the relationship of these viral isolates at the molecular level has not been determined. Here, we group the various GLRaV-2 isolates into four strains based on their coat protein gene sequences. We show that isolates "PN” (originated from Vitis vinifera cv. "Pinot noir”), "Sem” (from V. vinifera cv. "Semillon”) and "94/970” (from V. vinifera cv. "Muscat of Alexandria”) belong to the same strain, "93/955” (from hybrid "LN-33”) and "H4” (from V. rupestris "St. George”) each represents a distinct strain, while Grapevine rootstock stem lesion-associated viru

Two classes of subgenomic RNA of grapevine virus A produced by internal controller elements

AbstractGrapevine virus A (GVA), a species of the recently established genus Vitivirus, consists of an ∼7.3-kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). The virus, which is closely associated with the grapevine rugose wood disease complex, has been poorly investigated genetically. We explored the production of viral RNAs in a GVA-infected Nicotiana benthamiana herbaceous host and characterized one nested set of three 5′-terminal sgRNAs of 5.1, 5.5, and 6.0 kb, and another, of three 3′-terminal sgRNAs of 2.2, 1.8, and 1.0 kb that could serve for expression of ORFs 2–3, respectively. Neither 3′- nor 5′-terminal sgRNAs, which would correspond to ORF5, was detected, suggesting that expression of this ORF occurs via a bi- or polycistronic mRNA. The 5′-terminal sgRNAs were abundant in dsRNA-enriched extracts. Cloning and sequence analysis of the 3′ end of 5.5-kb 5′-terminal sgRNA and the 5′ end of the 1.8-kb 3′-terminal sgRNA suggested that a mechanism other than specific cleavage was involved in production of these sgRNAs. Apparently, the production of the 5′- and 3′-terminal sgRNAs was controlled by sequences upstream of the 5′-terminus of each of ORFs 2–4. Detection of both plus and minus strands of the 5′- and 3′-terminal sgRNAs, though in different levels of accumulation, suggested that each of these cis-acting elements is involved in production of four RNAs: a 3′-terminal plus-strand sgRNA which could act as an mRNA, the corresponding 3′-terminal minus-strand RNA, a 5′-terminal plus-strand sgRNA, and the corresponding 5′-terminal minus-strand RNA