Publications

nucleotide sequence and genome structure of grapevine rupestris stem pitting associated virus-1 reveal similarities to apple stem pitting virus.	rupestris stem pitting (rsp), a component of the rugose wood complex, is one of the most widespread graft-transmissible diseases of grapevines. here we report on the consistent association of a high molecular mass dsrna (ca. 8.7 kbp) with rsp. the dsrna was reverse-transcribed and cdnas generated were cloned into lambda zap ii. sequence analysis of the cdna clones showed that the dsrna was of viral origin and the putative virus was designated rupestris stem pitting associated virus-1 (rspav-1). ...	1998	9714258
nucleotide sequence and rt-pcr detection of a virus associated with grapevine rupestris stem-pitting disease.	abstract grapevine rupestris stem pitting (rsp) is a graft-transmissible disease of unknown etiology. we have characterized a virus associated with this disease. the entire genomic sequence (genbank accession number af026278) consisted of 8,725 nucleotides excluding a poly(a) tail. six open reading frames (orf) were found. orf1 potentially encodes a polypeptide with a methyltransferase domain, a papain-like proteinase domain, a helicase domain, and a rna-dependent rna polymerase domain; orf2, or ...	1998	18944859
rupestris stem pitting associated virus-1 consists of a family of sequence variants.	rupestris stem pitting (rsp) seems to be one of the most widespread virus diseases of grapevines. a virus, designated as rupestris stem pitting associated virus-1 (rspav-1), is consistently associated with, and likely to be the causative agent of rsp. sequence analyses of cdna clones derived from several rsp-affected grapevines suggested that a family of sequence variants of rspav-1 was associated with rsp. the genome structure of the sequence variants is identical to that of rspav-1 in that the ...	1999	10603163
a strategy for rapid cdna cloning from double-stranded rna templates isolated from plants infected with rna viruses by using taq dna polymerase.	a fast and efficient cdna cloning procedure for plant rna viruses was developed. in this procedure, double-stranded rna (dsrna) was used as a template source. standard cdna synthesis reagents and random hexamers were then used for making cdnas. taq dna polymerase was used to add additional (a) at the ends of cdnas, a ta cloning kit to ligate the cdnas to vectors, and an electroporator to transform the dnas to e. coli cells. dsrnas were extracted from grapevine tissues infected with four differen ...	2000	10644087
improved rna extraction and one-tube rt-pcr assay for simultaneous detection of control plant rna plus several viruses in plant extracts.	a procedure was developed for simultaneous detection of plant rna viruses and of plant rna, as a control. rt-pcr amplification with primers designed for the detection of the plant mrnas encoding malate dehydrogenase (mdh) and the large subunit of ribulose bisphosphate carboxylase oxygenase (rubiscol) was used for the development of a plant extraction procedure that consistently yields extracts that can be amplified. the control amplification was used successfully on extracts from cane, leaf and/ ...	2000	11011079
complete genome sequences of two new variants of grapevine rupestris stem pitting-associated virus and comparative analyses.	grapevine rupestris stem pitting-associated virus (grspav), a member of the genus foveavirus within the family flexiviridae, is the putative causal agent of the disease rupestris stem pitting (rsp) of grapevines. grspav comprises a family of variants whose pathological characteristics have not been determined. recently, many of the indicator "st george" plants (vitis rupestris) used throughout the world to index rsp tested positive for grspav. this finding questions the validity of past biologic ...	2005	15831969
rupestris stem pitting associated virus isolates are composed by mixtures of genomic variants which share a highly conserved coat protein.	broad spectrum primers were used to amplify a fragment comprising the cp gene and putative orf6 by rt-pcr from ds-rna templates originating from 46 portuguese varieties, totalling 190 samples, including some wild vitis ssp sylvestris vines, and 2 vines from slovenia. sscp analysis was used as a preliminary screen to avoid cloning and sequencing very similar variants. four groups of variants were recognized. in pair wise comparisons between nucleotide sequences the minimal homology found was 81%. ...	2006	16132183
efficient methods for sample processing and cdna synthesis by rt-pcr for the detection of grapevine viruses and viroids.	template preparation is important in reverse-transcription polymerase chain reaction (rt-pcr)-based detection methods. a tissuelyser with tungsten carbide beads was used for simultaneous processing of up to 48 samples under the same conditions in the development of a simple and rapid procedure to prepare a plant extract for rt reaction. a sandpaper method was also developed by which wood tissue of dormant cuttings could be macerated easily to process with minimal time and effort. it was also dem ...	2006	16510197
molecular analysis of a california strain of rupestris stem pitting-associated virus isolated from declining syrah grapevines.	the sequence of the genome of a rupestris stem pitting-associated virus (rspav) isolated from a declining syrah grapevine in california, designated the syrah strain (rspav-sy) was determined. the genome of this strain had an overall nucleotide identity of 77% in comparison with rspav sequences in genbank; the coat protein was the most conserved gene among rspav sequences and the replicase was the least conserved gene. phylogenetic analysis of partial coat protein and replicase gene sequences sho ...	2006	16547827
genetic diversity analyses of grapevine rupestris stem pitting-associated virus reveal distinct population structures in scion versus rootstock varieties.	grapevine rupestris stem pitting-associated virus (grspav) is a member of the genus foveavirus within the family flexiviridae. grspav is closely associated with the disease rupestris stem pitting and is frequently detected in grapevines worldwide. previous research in several laboratories suggests that grspav consists of a family of sequence variants. however, the genetic composition of grspav variants in viral isolates from scion and rootstock varieties has not been studied extensively. in this ...	2006	16690939
simultaneous detection of nine grapevine viruses by multiplex reverse transcription-polymerase chain reaction with coamplification of a plant rna as internal control.	abstract a multiplex reverse transcription-polymerase chain reaction (mrt-pcr) was developed for simultaneous detection of nine grapevine viruses: arabis mosaic virus, grapevine fanleaf virus, grapevine virus a, grapevine virus b, rupestris stem pitting-associated virus, grapevine fleck virus, grapevine leafroll-associated virus-1, -2, and -3, in combination with a plant rna internal control used as an indicator of the effectiveness of rna extraction and rt-pcr. primers were designed from conser ...	2006	18943959
molecular analysis of double-stranded rnas reveals complex infection of grapevines with multiple viruses.	the table grape variety "waltham cross" was infected with leafroll and shiraz disease. to reveal specific viruses that are associated with the diseased plants, we used an rt-pcr-based strategy to determine partial genome sequences of these viruses. upon cloning and sequencing of the rt-pcr products, we detected seven groups of viral variants that are related to four species of the closteroviridae: grapevine leafroll-associated virus 1, 2, 3, and 5, in addition to rupestris stem pitting-associate ...	2007	17129630
subcellular localization of the triple gene block proteins encoded by a foveavirus infecting grapevines.	grapevine rupestris stem pitting-associated virus (grspav; foveavirus; flexiviridae) contains a positive-sense, ssrna genome. grspav occurs worldwide in grapes and is involved in the rugose wood disease complex. the grspav genome contains the triple gene block (tgb), a genetic module present in several genera of plant rna viruses. tgb encodes three proteins (tgbp1, tgbp2 and tgbp3) that are believed to work together to achieve intra- and inter-cellular transport of virions in infected plants. to ...	2008	18804498
real-time rt-pcr (taqman) assays for the detection of viruses associated with rugose wood complex of grapevine.	real-time taqman rt-pcr (taqman rt-pcr) assays were developed to detect the viruses associated with rugose wood complex of grapevines. the viruses detected were rupestris stem pitting-associated virus in the genus foveavirus, grapevine virus a, grapevine virus b and grapevine virus d in the genus vitivirus. the coat protein was found to be the most conserved gene within the viral species, therefore, the primers and probes for taqman rt-pcr assays were designed from the multiple alignment of the ...	2008	18848580
analysis of multiple virus-infected grapevine plant reveals persistence but uneven virus distribution.	ln33 grapevine plants were artificially inoculated with budwoods originating from a field-cultivated traminer grapevine which was naturally infected with grapevine leafroll-associated virus 1 (glrav-1), grapevine virus a (gva), grapevine virus b (gvb), rupestris stem pitting-associated virus (rspav), and an unclassified tymovirus. four years after inoculation, a comparison of the cane weights between healthy and infected grapevines did not show any significant difference. corky bark symptoms or ...	2009	19941393
deep sequencing analysis of rnas from a grapevine showing syrah decline symptoms reveals a multiple virus infection that includes a novel virus.	in a search for viruses associated with decline symptoms of syrah grapevines, we have undertaken an analysis of total plant rna sequences using life sciences 454 high-throughput sequencing. 67.5 megabases of sequence data were derived from reverse-transcribed cdna fragments, and screened for sequences of viral or viroid origin. the data revealed that a vine showing decline symptoms supported a mixed infection that included seven different rna genomes. fragments identified as derived from viruses ...	2009	19304303
sequence diversity, population genetics and potential recombination events in grapevine rupestris stem pitting-associated virus in pacific north-west vineyards.	grapevine rupestris stem pitting-associated virus (grspav; genus foveavirus, family flexiviridae) is present in many grape-growing regions of the world. a total of 84 full-length coat protein (cp) sequences and 57 sequences representing the helicase-encoding region (hr) of the rna-dependent rna polymerase were obtained from wine grape cultivars grown in the pacific north-west (pnw) of the united states and their molecular diversity was compared with corresponding sequences previously reported fr ...	2010	19759241
a diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.	at least 58 viruses have been reported to infect grapevines causing economic damage globally. conventional detection strategies based on serological assays, biological indexing and rt-pcr targeting one or few viruses in each assay are widely used. grapevines are prone to contain mixed infections of several viruses, making the use of these techniques time-consuming. a 70-mer oligonucleotide microarray able to detect simultaneously a broad spectrum of known viruses as well as new viruses by cross- ...	2010	19914293
deep sequencing analysis of viruses infecting grapevines: virome of a vineyard.	double stranded rna, isolated from 44 pooled randomly selected vines from a diseased south african vineyard, has been used in a deep sequencing analysis to build a census of the viral population. the dsrna was sequenced in an unbiased manner using the sequencing-by-synthesis technology offered by the illumina genome analyzer ii and yielded 837 megabases of metagenomic sequence data. four known viral pathogens were identified. it was found that grapevine leafroll-associated virus 3 (glrav-3) is t ...	2010	20172578
rugose wood-associated viruses do not appear to be involved in shiraz (syrah) decline in south africa.	the presence of rugose-wood-associated viruses of the genera foveavirus and vitivirus in the family betaflexiviridae was investigated in various clones of own-rooted and grafted vitis vinifera cv. shiraz that were affected, or not, by shiraz decline, and in rootstocks. rt nested-pcr amplification of double-stranded rna using degenerate primers for the simultaneous detection of foveaviruses and vitiviruses (dovas ci, katis ni in j virol meth 170:99-106, 2003), cloning of dna amplicons, sscp analy ...	2010	20549265
the capsid protein of grapevine rupestris stem pitting-associated virus contains a typical nuclear localization signal and targets to the nucleus.	grapevine rupestris stem pitting-associated virus (grspav) is a positive strand, ssrna virus of the genus foveavirus (family betaflexiviridae; order tymovirales). grspav is distributed in table and wine grapes worldwide and comprises a large family of sequence variants. as a newly discovered virus, mechanisms of virus replication and movement of grspav have not been elucidated. we recently revealed the subcellular localization of the proteins encoded by the triple gene block of grspav (rebelo et ...	2010	20708048
deep sequencing analysis of viral short rnas from an infected pinot noir grapevine.	virus-derived short interfering rnas (vsirnas) isolated from grapevine v. vinifera pinot noir clone entav 115 were analyzed by high-throughput sequencing using the illumina solexa platform. we identified and characterized vsirnas derived from grapevine field plants naturally infected with different viruses belonging to the genera foveavirus, maculavirus, marafivirus and nepovirus. these vsirnas were mainly of 21 and 22 nucleotides (nt) in size and were discontinuously distributed throughout grap ...	2010	20875658
complete nucleotide sequence of a new variant of grapevine rupestris stem pitting-associated virus from southern italy.		2011	21305326
a multidisciplinary study on the effects of phloem-limited viruses on the agronomical performance and berry quality of vitis vinifera cv. nebbiolo.	viral infections are known to have a detrimental effect on grapevine yield and performance, but there is still a lack of knowledge about their effect on the quality and safety of end products. vines of vitis vinifera cv. nebbiolo clone 308, affected simultaneously by grapevine leafroll-associated virus 1 (glrav-1), grapevine virus a (gva), and rupestris stem pitting associated virus (rspav), were subjected to integrated analyses of agronomical performance, grape berry characteristics, instrument ...	2011	21856458
a new grapevine virus discovered by deep sequencing of virus- and viroid-derived small rnas in cv pinot gris.	field symptoms of chlorotic mottling and leaf deformations were observed on the cv pinot gris (pg) in the trentino region (italy). extensive assays excluded the presence of widely distributed nepo-, ampelo- and vitiviruses. an analysis of small rna populations from two pg grapevines showing or not symptoms was carried out by illumina high throughput sequencing. the study disclosed the virus and viroids contents of the two vines that was composed by grapevine rupestris stem pitting-associated vir ...	2011	22036729
co-evolution between grapevine rupestris stem pitting-associated virus and vitis vinifera l. leads to decreased defence responses and increased transcription of genes related to photosynthesis.	grapevine rupestris stem pitting-associated virus (grspav) is a widespread virus infecting vitis spp. although it has established a compatible viral interaction in vitis vinifera without the development of phenotypic alterations, it can occur as distinct variants that show different symptoms in diverse vitis species. the changes induced by grspav in v. vinifera cv 'bosco', an italian white grape variety, were investigated by combining agronomic, physiological, and molecular approaches, in order ...	2012	22987838
construction and biological activities of the first infectious cdna clones of the genus foveavirus.	grapevine rupestris stem pitting-associated virus (grspav, genus foveavirus, family betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and syrah decline. little is known about the biology and pathological properties of grspav. in this work, we engineered a full-length infectious cdna clone for grspav and a gfp-tagged variant, both under the transcriptional control of cauliflower mosaic virus 3 ...	2013	23099206
the triple gene block movement proteins of a grape virus in the genus foveavirus confer limited cell-to-cell spread of a mutant potato virus x.	grapevine rupestris stem pitting-associated virus (grspav) is a member of the genus foveavirus in the family betaflexiviridae. the genome of grspav encodes five proteins, among which are three movement proteins designated the triple gene block (tgb) proteins. the tgb proteins of grspav are highly similar to their counterparts in potato virus x (pvx), as reflected in size, modular structure, conservation of critical amino acid sequence motifs, as well as similar cellular localization. based on th ...	2013	23543158
genome-wide identification of viral and host transcripts targeted by viral sirnas in vitis vinifera.	in plants, rna silencing is a surveillance mechanism against invading viruses. it involves the production of virus-derived small interfering rnas (vsirnas), which guide the rna-induced silencing complex (risc) to inactivate viruses. vsirnas may also promote the silencing of host mrnas in a sequence-specific manner. in this work, vsirnas derived from two grapevine-infecting viruses (grapevine fleck virus and grapevine rupestris stem pitting-associated virus) were selected from cdna libraries of s ...	2013	22947170
a leafhopper-transmissible dna virus with novel evolutionary lineage in the family geminiviridae implicated in grapevine redleaf disease by next-generation sequencing.	a graft-transmissible disease displaying red veins, red blotches and total reddening of leaves in red-berried wine grape (vitis vinifera l.) cultivars was observed in commercial vineyards. next-generation sequencing technology was used to identify etiological agent(s) associated with this emerging disease, designated as grapevine redleaf disease (grd). high quality rna extracted from leaves of grape cultivars merlot and cabernet franc with and without grd symptoms was used to prepare cdna librar ...	2013	23755117
detection and molecular characterization of egyptian isolates of grapevine viruses.	selected commercial and/or local vineyards and nurseries in three different governorates of egypt (alexandria, el-beheira and el-menofia) were surveyed for symptoms indicative of infection by grapevine viruses. leaf samples from red-fruited and white-fruited vitis vinefera were tested for grapevine leafroll associated viruses (glrav-1, glrav-2, and glrav-3), grapevine viruses a and b (gva, gvb), grapevine rupestris stem pitting virus (grspav), grapevine fanleaf virus (gflv), and grapevine fleck ...	2014	24957718
effect of temperature on symptom expression and sequence polymorphism of grapevine yellow speckle viroid 1 in grapevine.	symptom expression of yellow speckle disease was studied in a row of 32 individual vitis vinifera cv. chardonnay vines in the warmer summer of 2009/2010 as compared with the cooler summer of 2011/2012 in south australia. rt-pcr analysis showed that all these vines were positive for hop stunt viroid, grapevine yellow speckle viroid 1 (gysvd-1) and grapevine rupestris stem pitting-associated virus. four vines named vines 1, 8, 11 and 15 were selected for further analyses. vines 1 and 8 had never s ...	2014	24905287
molecular characterizations of two grapevine rupestris stem pitting-associated virus isolates from china.	the complete nucleotide sequences of two isolates of grapevine rupestris stem pitting-associated virus (lsl and jf) collected from grapevine of xingcheng in liaoning province, china, were determined. the genomes of both lsl and jf were found to contain five open reading frames (orfs). sequence alignments showed that the genomic sequences of jf were 76.1 %-83.5 % identical to the other ten grspav isolates that have been reported previously and that the nucleotide sequence identity of isolate lsl ...	2015	26215445
subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family betaflexiviridae.	as a member of the newly established betaflexiviridae family, grapevine rupestris stem pitting-associated virus (grspav) has an rna genome containing five orfs. orf1 encodes a putative replicase polyprotein typical of the alphavirus superfamily of positive-strand ssrna viruses. several viruses of this superfamily have been demonstrated to replicate in structures designated viral replication complexes associated with intracellular membranes. however, structure and cellular localization of the rep ...	2015	25502653
multiplex rt-pcr method for the simultaneous detection of nine grapevine viruses.	viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. arabis mosaic virus, grapevine fanleaf virus, grapevine virus a, grapevine virus b, grapevine rupestris stem pitting-associated virus, grapevine fleck virus, and grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. a multiple ...	2015	25287494
effect of repeated ribavirin treatment on grapevine viruses.	the effect of ribavirin treatment for the chemotherapy of several grapevine viruses was evaluated. four grapevine cultivars were repeatedly treated with ribavirin in two different concentrations and with three different lengths of treatment. repeating the ribavirin treatment always had a significant effect on the number of healthy grapevine plants obtained. ribavirin concentration and length of exposure showed a significant difference in sanitation of the grapevine rupestris stem pitting-associa ...	2017	27928920
analysis of grapevine rupestris stem pitting-associated virus in slovakia reveals differences in intra-host population diversity and naturally occurring recombination events.	grapevine rupestris stem pitting-associated virus (grspav) is a worldwide-distributed pathogen in grapevines with a high genetic variability. our study revealed differences in the complexity of grspav population in a single host. a single-variant grspav infection was detected from the sk30 grapevine plant. on the contrary, sk704 grapevine was infected by three different grspav variants. variant-specific rt-pcr detection protocols have been developed in this work to study distribution of the thre ...	2017	28167886
comparative detection of a large population of grapevine viruses by taqman(®) rt-qpcr and elisa.	grapevine (vitis spp.) can be infected by numerous viruses that are often widespread and of great economic importance. reliable detection methods are necessary for sanitary selection which is the only way to partly control grapevine virus diseases. biological indexing and elisa are currently the standard methods for screening propagation material, and pcr-methods are becoming increasingly popular. due to the diversity of virus isolates, it is essential to verify that the tests allow the detectio ...	2017	27923589