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nucleotide sequence of the 3'-terminal two-thirds of the grapevine leafroll-associated virus-3 genome reveals a typical monopartite closterovirus.the rna genome of grapevine leafroll-associated closterovirus-3 (glrav-3) was cloned as a cdna generated from glrav-3-specific dsrna, and a partial genome sequence of 13154 nucleotides (nt) including the 3' terminus was determined. the sequenced portion contained 13 open reading frames (orfs) potentially encoding, in the 5'-3' direction, proteins of > 77 kda (orf1a; helicase, hel), 61 kda (orf1b; rna-dependent rna polymerase, rdrp), 6 kda (orf2), 5 kda (orf3, small transmembrane protein), 59 kda ...19989603346
nucleotide sequence and organization of ten open reading frames in the genome of grapevine leafroll-associated virus 1 and identification of three subgenomic rnas.the genome of grapevine leafroll-associated virus 1 (glrav-1) was cloned and the sequence of 12394 nts determined. it contains 10 major open reading frames (orfs) and a 3'-non-coding region lacking a poly(a) tract. the first orf (orf 1a) encodes a putative rna helicase at the c-terminal portion of an apparently larger protein. the downstream orf, 1b, overlaps orf 1a and lacks an initiation codon. this orf encodes an rna-dependent rna polymerase of m(r) 59276. orf 2 encodes a small hydrophobic pr ...200010675398
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/ ...200011011079
nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2.the genome of pineapple mealybug wilt-associated closterovirus-2 (pmwav-2) was cloned from double-stranded rna isolated from diseased pineapple and its sequence determined. the 3'-terminal 14861 nt of the single-stranded rna genome contains ten open reading frames (orfs) which, from 5' to 3', potentially encode a >204 kda polyprotein containing papain-like protease, methyltransferase and helicase domains (orf1a), a 65 kda rna-dependent rna polymerase (orf1b), a 5 kda hydrophobic protein (orf2), ...200111125151
complete nucleotide sequence and genome organization of grapevine leafroll-associated virus 3, type member of the genus ampelovirus.this study reports on the complete genome sequence of grapevine leafroll-associated virus 3, the type member of the genus ampelovirus. the genome is 17 919 nt in size and contains 13 open reading frames (orfs). previously, the sequence of 13 154 nt of the 3'-terminal of the genome was reported. the newly sequenced portion contains a 158 nt 5' utr, a single papain-like protease and a methyltransferase-like (mt) domain. orf1a encodes a large polypeptide with a molecular mass of 245 kda. with a pre ...200415218195
little cherry virus-2: sequence and genomic organization of an unusual member of the closteroviridae.the complete genomic sequence of variant usa6b of little cherry virus-2 (lchv-2), has been determined and is 15045 nucleotides in length, coding for 11 open reading frames (orfs). the sequence shares 77.2% identity with a previously published, ca. 6 kb partial replicase sequence of lchv-2 (variant usa6a). both lchv-2/usa6a and lchv-2/usa6b were obtained from the same tree infected with little cherry disease, and would suggest a mixed infection. lchv-2/usa6b is more closely related to the partial ...200515449137
genetic variability and population structure of grapevine leafroll-associated virus 3 isolates.the genetic variability and population structure of a collection of 45 grapevine leafroll-associated virus 3 (glrav-3) isolates were studied by single-stranded conformation polymorphism (sscp) and sequence analysis of the rna-dependent rna polymerase (rdrp), heat-shock protein 70 homologue (hsp-70) and coat protein (cp) genes. the distribution of sscp profiles was not correlated with the geographical origin of the isolates, indicating that glrav-3 is a single, undifferentiated population. the ma ...200515604449
comparison of three techniques for detection of grapevine leafroll-associated virus 1.thirty seven plants of grapevine from the research station of viticulture, karlstejn was examined for the presence of leafroll viruses. grapevine leafroll-associated virus 1 (glrav-1) was detected in the grapevines plants tested using double-antibody sandwich elisa (das-elisa), rt-pcr and molecular hybridization with non-radioactive rna probes. both molecular methods were based on a detection of the glrav-1 heat-shock protein 70 (hsp70) gene and showed a higher sensitivity in the detection of gl ...200515929397
the symptomless leaf infection with grapevine leafroll associated virus 3 in grown in vitro plants as a simple model system for investigation of viral effects on photosynthesis.the photosynthetic changes evaluated by oxygen evolution, chlorophyll fluorescence, photoacoustics, and delayed fluorescence (df) were studied in leaves of grown in vitro for 8 weeks grapevine plants (vitis vinifera) infected by grapevine leafroll-associated virus 3 (glrav-3). the infected leaves were characterized during the viral infection without visible disease symptoms. the symptomless infection led to a decrease in plant biomass. the non-photochemical fluorescence quenching, qn, declined, ...200716716452
gene expression associated with compatible viral diseases in grapevine cultivars.viral diseases affect grapevine cultures without inducing any resistance response. thus, these plants develop systemic diseases and are chronically infected. molecular events associated with viral compatible infections responsible for disease establishment and symptoms development are poorly understood. in this study, we surveyed viral infection in grapevines at a transcriptional level. gene expression in the vitis vinifera red wine cultivars carménère and cabernet-sauvignon naturally infected w ...200716775684
antiviral activity of tiazofurin and mycophenolic acid against grapevine leafroll-associated virus 3 in vitis vinifera explants.the ability to control plant viral diseases with chemicals has great potential value for agriculture, but few chemicals are available to date due to the difficulty in obtaining effective drugs. imp dehydrogenase is an enzyme which catalyzes the conversion of inosine 5'-monophosphate to xanthosine 5'-monophosphate in the de novo purine nucleotide synthetic pathway, and is considered a sensitive target for antiviral drugs. two impdh inhibitors, tiazofurin (tr) and mycophenolic acid (mpa), were tes ...200717125850
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 ...200717129630
senescence-associated genes induced during compatible viral interactions with grapevine and arabidopsis.the senescence process is the last stage in leaf development and is characterized by dramatic changes in cellular metabolism and the degeneration of cellular structures. several reports of senescence-associated genes (sags) have appeared, and an overlap in some of the genes induced during senescence and pathogen infections has been observed. for example, the enhanced expression of sags in response to diseases caused by fungi, bacteria, and viruses that trigger the hypersensitive response (hr) or ...200717761729
complete nucleotide sequence of a south african isolate of grapevine leafroll-associated virus 3 reveals a 5'utr of 737 nucleotides. 200818274697
generation and characterization of a recombinant antibody fragment that binds to the coat protein of grapevine leafroll-associated virus 3.pathogen-specific recombinant antibodies have been used to characterize pathogen infections and to engineer resistance in crops. we selected a single-chain antibody fragment (scfvlr3cp-1) specific for the coat protein of grapevine leafroll-associated virus 3 (glrav-3), one of the agents of grapevine leafroll (glr) disease, from a phage display library. the antibody binds specifically to the entire length of glrav-3 particles and has a high binding affinity value (k(d)) of 42 nm. the amino acid m ...200818478181
genome analysis and detection of a chilean isolate of grapevine leafroll associated virus-3.the complete genome of the chilean isolate cl-766 of grapevine leafroll-associated virus-3 (glrav-3) has been sequenced. this is the first genome sequence obtained from a glrav-3 isolate of the southern hemisphere. the genomic rna of 17,919 nucleotides contains 13 open reading frames (orfs) with 5' and 3' untranslated regions (utr) of 158 and 277 nucleotides, respectively. comparison with ny1, the only isolate with complete genomic sequence available today, shows 97.6% nucleotide identity betwee ...200818498048
detection and partial characterization of a second closterovirus associated with little cherry disease, little cherry virus-2.abstract little cherry disease (lchd) is a complex and serious viral disease of cherry. although originally described almost 70 years ago, there has been little progress in identifying the causal agent of the disease due to the difficulty in obtaining purified virus from infected trees. this problem was partially overcome in 1997 when the compete sequence of a closterovirus associated with lchd, little cherry virus (lchv), was published. this virus could be associated with some, but not all, tre ...200118943345
partial characterization of a closterovirus associated with apple mealybug-transmitted little cherry disease in north america.abstract little cherry disease (lchd) is a serious economic problem of sweet cherry production in western north america where apple mealybug is the principle vector. lchd is associated with a distinct species of double-stranded (ds) rna. in this study, filamentous virus particles were purified from lchd-infected trees and shown to contain single-stranded rna corresponding to the previously reported dsrna isolated from infected trees. the virus particles were characterized and were similar to mon ...200118943346
partial genome organization, identification of the coat protein gene, and detection of grapevine leafroll-associated virus-5.abstract the genome of grapevine leafroll-associated virus-5 (glrav-5) was cloned, and the sequence of 4766 nt was determined. degenerate oligonucleotide primers designed from the conserved closterovirus heat shock 70 protein (hsp 70) homologue were used to obtain viral-specific sequences to anchor the cloning of the viral rna with a genomic walking approach. the partial nucleotide (nt) sequence of glrav-5 showed the presence of four open reading frames (orf a through d), potentially coding for ...200118943347
transmission of grapevine leafroll-associated virus 3 by the vine mealybug (planococcus ficus).grapevine leafroll disease is caused by grapevine leafroll-associated viruses (glravs). within this virus complex, glrav-3 is the predominant species in the world. several glravs have been shown to be transmitted from vine to vine by mealybugs although a detailed characterization of transmission biology is lacking. the introduction of the vine mealybug (planococcus ficus) in california and other regions of the world may result in increasing disease incidence of established glravs. we studied the ...200818943455
an assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody.grapevine leafroll-associated virus 3 (glrav-3) is a major pathogen of grapevine. a previously described single-chain fragment variable (scfv) antibody (scfvlr3), directed against the coat protein (cp) of glrav-3, was expressed in escherichia coli and used to develop a diagnostic elisa kit. the antibody was fused to the light chain constant domain of human immunoglobulin to create the bivalent reagent c(l)-lr3, which was purified from the periplasmic fraction, giving a yield of ~5 mg/l. the sens ...200919082687
genome organization and phylogenetic relationship of pineapple mealybug wilt associated virus-3 with family closteroviridae members.the nucleotide sequence of pineapple mealybug wilt associated virus-3 (pmwav-3) (closteroviridae: ampelovirus), spanning seven open reading frames (orfs) and the untranslatable region of the 3' end was determined. based on the amino acid identities with orthologous orfs of pmwav-1 (54%-73%) and pmwav-2 (13%-35%), we propose pmwav-3 is a new species in the pmwav complex. pmwav-3 lacks an intergenic region between orf1b and orf2, encodes a relatively small, 28.8 kda, coat protein, and lacks a coat ...200919225875
diversity of ampeloviruses in mealybug and soft scale vectors and in grapevine hosts from leafroll-affected vineyards.the occurrence and diversity of grapevine leafroll-associated virus 1 (glrav-1) and grapevine leafroll-associated virus 3 (glrav-3) in the soft scales parthenolecanium corni and pulvinaria innumerabilis and in the mealybug pseudococcus maritimus was determined in leafroll-affected vineyards in the finger lakes region of new york. groups of 1 to 4 specimens were collected under loose grapevine bark and tested by reverse-transcription polymerase chain reaction (rt-pcr) for segments of the second d ...200919740031
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 ...201020172578
mapping of the 5' terminal nucleotides of grapevine leafroll-associated virus 3 sgrnas.grapevine leafroll-associated virus 3 (glrav-3) is a positive sense, single stranded rna virus that has a detrimental effect on wine and table grapes worldwide. previous studies have shown that glrav-3, like other closteroviruses produces subgenomic (sg) rnas during replication and that these sgrnas are deployed for the expression of the orfs on the 3' half of the genome. in this study a dsrna blot confirmed the presence of three, 3' co-terminal sgrnas species [sgrna(orf3/4), sgrna(orf5) and sgr ...201020561952
citrus mealybug (hemiptera: pseudococcidae) movement and population dynamics in an arbor-trained vineyard.the citrus mealybug, planococcus citri (risso) (hemiptera: pseudococcidae), is the main grapevine pest in vineyards in some countries, such as spain and brazil. in galician vineyards (northwestern spain), mealybug population levels are low because the accumulated degree-days are lower than in other grapevine-growing areas. the main problem caused by mealybugs is the transmission of viruses, even at low infestation levels. the active period of citrus mealybug in the study vineyard lasted from jul ...201020568606
mealybug transmission of grapevine leafroll viruses: an analysis of virus-vector specificity.to understand ecological factors mediating the spread of insect-borne plant pathogens, vector species for these pathogens need to be identified. grapevine leafroll disease is caused by a complex of phylogenetically related closteroviruses, some of which are transmitted by insect vectors; however, the specificities of these complex virus-vector interactions are poorly understood thus far. through biological assays and phylogenetic analyses, we studied the role of vector-pathogen specificity in th ...201020626287
3'-coterminal subgenomic rnas and putative cis-acting elements of grapevine leafroll-associated virus 3 reveals 'unique' features of gene expression strategy in the genus ampelovirus.the family closteroviridae comprises genera with monopartite genomes, closterovirus and ampelovirus, and with bipartite and tripartite genomes, crinivirus. by contrast to closteroviruses in the genera closterovirus and crinivirus, much less is known about the molecular biology of viruses in the genus ampelovirus, although they cause serious diseases in agriculturally important perennial crops like grapevines, pineapple, cherries and plums.201020682046
modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (vitis vinifera l.) leaves.symptoms of grapevine leafroll disease (glrd) in red-fruited wine grape (vitis vinifera l.) cultivars consist of green veins and red and reddish-purple discoloration of inter-veinal areas of leaves. the reddish-purple color of symptomatic leaves may be due to the accumulation of anthocyanins and could reflect an up-regulation of genes involved in their biosynthesis.201020731850
three genetic grapevine leafroll-associated virus 3 variants identified from south african vineyards show high variability in their 5'utr.three genetic variants of grapevine leafroll-associated virus 3 (glrav-3) were identified in vineyards of the western cape, south africa. the glrav-3 variants were identified by single-strand conformation polymorphism (sscp) profiles generated from a region amplified in orf5. orf5 sequence data confirmed the three genetic variant groups, and a specific sscp profile was assigned to each variant group. the results of sscp analysis of this region in orf5 showed that this method gives a fast and rel ...201020830600
five phylogenetic groups identified in the coat protein gene of grapevine leafroll-associated virus 3 obtained from portuguese grapevine varieties.the genetic variability and population structure of grapevine leafroll-associated virus 3 (glrav-3) variants were updated by examining the diversity within the viral coat protein (cp) gene among 174 isolates belonging to a collection of vitis vinifera representing most of the portuguese varieties. phylogenetic analysis revealed the existence of five well-defined clusters. three of these correspond to previously defined groups, another corresponds to variants from chile for which only one sequenc ...201021153745
quantitation of grapevine leafroll associated virus-1 and -3, grapevine virus a, grapevine fanleaf virus and grapevine fleck virus in field-collected vitis vinifera l. 'nebbiolo' by real-time reverse transcription-pcr.taqman one-step real-time qrt-pcr assays were developed for the quantitation of grapevine leafroll associated virus-1 and -3 (glrav-1 and -3), grapevine virus a (gva), grapevine fanleaf virus (gflv) and grapevine fleck virus (gfkv) in vitis vinifera l. virus load in the progenies of three 'nebbiolo' clones planted in two experimental vineyards in piemonte (northwestern italy), and carrying the viruses in different combinations, was evaluated. quantitation primers were designed on the rna-depende ...201021167205
genome-wide analysis of the set domain group family in grapevine.the set domain group (sdg) proteins represent an evolutionarily-conserved family of epigenetic regulators present in eukaryotes and are putative candidates for the catalysis of lysine methylation in histones. plant genomes analyses of this family have been performed in arabidopsis, maize, and rice and functional studies have shown that sdg genes are involved in the control of plant development. in this work, we describe the identification and structural characterization of sdg genes in the vitis ...201121293861
genetic diversity in the 3' terminal 4.7-kb region of grapevine leafroll-associated virus 3.grapevine leafroll-associated virus 3 (glrav-3; ampelovirus, closteroviridae), associated with grapevine leafroll disease, is an important pathogen found across all major grape-growing regions of the world. the genetic diversity of glrav-3 in napa valley, ca, was studied by sequencing 4.7 kb in the 3' terminal region of 50 isolates obtained from vitis vinifera 'merlot'. glrav-3 isolates were subdivided into four distinct phylogenetic clades. no evidence of positive selection was observed in the ...201121391825
compatible glrav-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in vitis vinifera.virus infections in grapevine cause important economic losses and affect fruit quality worldwide. although the phenotypic symptoms associated to viral infections have been described, the molecular plant response triggered by virus infection is still poorly understood in vitis vinifera. as a first step to understand the fruit changes and mechanisms involved in the compatible grapevine-virus interaction, we analyzed the berry transcriptome in two stages of development in the red wine cultivar cabe ...201121786204
occurrence of grapevine leafroll-associated virus complex in napa valley.grapevine leafroll disease (gld) is caused by a complex of several virus species (grapevine leafroll-associated viruses, glrav) in the family closteroviridae. because of its increasing importance, it is critical to determine which species of glrav is predominant in each region where this disease is occurring. a structured sampling design, utilizing a combination of rt-pcr based testing and sequencing methods, was used to survey glravs in napa valley (california, usa) vineyards (n = 36). of the 2 ...201122039446
genomic analysis of grapevine leafroll associated virus-5 and related viruses.the grapevine leafroll-associated viruses (glravs) (closteroviridae) represent an emerging threat to world grape production. one group of glravs within the genus ampelovirus, the glrav-4-like viruses (glrav-4lvs), contains a fragmented collection of seven viruses only two of which (glrav-pr and glracv) are fully sequenced. here in reporting the sequence of glrav-5, a member of glrav-4lvs, we identify genomic elements common to the glrav-4lv group. exclusive properties include a highly conserved ...201121893115
Molecular and histological characterisation of primary (ß-proteobacteria) and secondary (?-proteobacteria) endosymbionts of three species of mealybugs (Pseudococcus longispinus, Pseudococcus calceolariae, Pseudococcus viburni) (Pseudococcidae, Hemiptera) and prediction of interactions of their GroEL homologs with Grape Leaf Roll-associated Virus-3.Microscopic localization of endosymbiotic bacteria in three species of mealybug (Pseudococcus longispinus, long-tailed mealybug; Pseudococcus calceolariae, citrophilus mealybug; and Pseudococcus. viburni, obscure mealybug) showed these organisms were confined to bacteriocyte cells within a bacteriome centrally located within the haemocoel. Two species of bacteria were present, with the secondary endosymbiont, in all cases, living within the primary endosymbiont. DNA from the dissected bacteriome ...201122156418
effect of host plant tissue on the vector transmission of grapevine leafroll-associated virus 3.many biotic and abiotic factors affect the transmission efficiency of vector-borne plant pathogens. insect vector within-plant distribution and host tissue preference are known to affect pathogen acquisition and inoculation rates. in this study, we first investigated whether feeding tissue affects the transmission of grapevine leafroll-associated virus 3 by planococcus ficus (signoret) (hemiptera: pseudococcidae) and the effect of mealybug within-plant distribution on virus transmission under gr ...201122066175
real-time multiplex rt-pcr for the simultaneous detection of the five main grapevine viruses.a real-time multiplex rt-pcr has been developed for the simultaneous detection and identification of the major rna viruses that infect grapevines (grapevine fanleaf virus, arabis mosaic virus, grapevine leafroll-associated virus 1, grapevine leafroll-associated virus 3 and grapevine fleck virus). serial dilutions of infected plant extracts were tested using the new method, and the results were compared with those obtained using a commercially available elisa and real-time singleplex rt-pcr. the ...201323219809
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 ...201424957718
evidence of grapevine leafroll associated virus-1-3, grapevine fleck virus and grapevine virus b occurring in himachal pradesh, india.during a survey conducted in the grapevine orchards of himachal pradesh, variety of symptoms ranging from leaf yellowing, vein greening, reduced leaf size, downward rolling/cup shaped leaves to reduced fruit bearing were observed. symptomatic leaf samples were collected and analyzed by serological (das-elisa) and molecular methods (rt-pcr, pcr) for viruses and phytoplasma known worldwide on grapevine. das-elisa was used for detection of grapevine leafroll associated virus 1, 2 and 3 (glrav-1, 2 ...201324426260
ferrisia gilli (hemiptera: pseudococcidae) transmits grapevine leafroll-associated viruses.several mealybug species are vectors of grapevine leafroll-associated viruses (glrav), which cause the economically important grapevine leafroll disease in grape-producing regions worldwide. the mealybug ferrisia gilli gullan is a new pest of grapevines in el dorado county, located in the sierra foothill wine-growing region of california. glrav species 1, 2, 3, and 4lv have been detected in vineyards with symptomatic vines in the sierra foothills. we conducted controlled virus acquisition and tr ...201627329635
transmission of grapevine virus a and grapevine leafroll-associated virus 1 and 3 by heliococcus bohemicus (hemiptera: pseudococcidae) nymphs from plants with mixed infections.mealybugs (hemiptera: pseudococcidae) represent a serious threat for viticulture as vectors of phloem-restricted viruses associated with the grapevine rugose wood and leafroll diseases. heliococcus bohemicus (šulc) is known to be involved in the spread of these two viral diseases, being a vector of the grapevine virus a (gva) and the grapevine leafroll-associated virus 1 and 3 (glrav-1 and glrav-3). this study investigated the acquisition and transmission efficiency of h. bohemicus fed on mixed- ...201627329628
slowing the spread of grapevine leafroll-associated viruses in commercial vineyards with insecticide control of the vector, pseudococcus maritimus (hemiptera: pseudococcidae).vineyards were surveyed for grapevine leafroll-associated viruses and their insect vectors in new york state's finger lakes region in 2006-2008. grape mealybug, pseudococcus maritimus (erhorn) (hemiptera: pseudococcidae), european fruit lecanium, parthenolecanium corni (bouche), and cottony maple scale, pulvinaria acericola (walsh and riley) (hemiptera: coccidae) were identified as vector species in this region. an increase in the incidence of grapevine leafroll-associated virus 1 (glrav-1) and ...201526223949
in vivo inhibition of trans-plasma membrane electron transport by antiviral drugs in grapevine.electrophysiological techniques were applied to investigate the action of antiviral drugs during trans-plasma events in in vivo grapevine cells infected by glrav-1 and glrav-3. carbon fiber microelectrodes and redox-sensitive dyes were used to measure trans-plasma membrane electron transport (t-pmet) activity in healthy and infected samples treated with ribavirin, tiazofurin and oseltamivir. each drug caused a reduction in oxidation current (expressed as δ[fe(2+)]) in healthy samples, indicating ...201323774971
combined effect of virus infection and water stress on water flow and water economy in grapevines.water limitation is one of the major threats affecting grapevine production. thus, improving water-use efficiency (wue) is crucial for a sustainable viticulture industry in mediterranean regions. under field conditions, water stress (ws) is often combined with viral infections as those are present in major grape-growing areas worldwide. grapevine leafroll-associated virus 3 (glrav-3) is one of the most important viruses affecting grapevines. indeed, the optimization of water use in a real contex ...201728044321
differential expression of mirnas and associated gene targets in grapevine leafroll-associated virus 3-infected plants.micrornas (mirnas) are a class of endogenous small non-coding rnas (srna) that play an essential role in the regulation of target mrnas expressed during plant development and in response to stress. microrna expression profiling has helped to identify mirnas that regulate a range of processes, including the plant's defence response to pathogens. in this study, differential mirna expression in own-rooted vitis vinifera cv. cabernet sauvignon plants infected with grapevine leafroll-associated virus ...201728025711
a synoptic analysis of the temporal and spatial aspects of grapevine leafroll disease in a historic napa vineyard and experimental vine blocks.five grapevine leafroll-associated virus 3 (glrav-3) epidemics were analyzed utilizing a standardized approach to robustly characterize the temporal and spatial parameters. published data included in the analysis are from spain, new zealand, and napa valley, ca together with new data from a historic vineyard in napa valley, ca. linear regression analyses of logit-transformed incidence data indicated a maximum average increase of 11% per year in disease incidence, with considerable variation amon ...201727938242
harbin: a quantitation pcr analysis tool.to enable analysis and comparisons of different relative quantitation experiments, a web-browser application called harbin was created that uses a quantile-based scoring system for the comparison of samples at different time points and between experiments.201727695982
responses of in vitro-grown plantlets (vitis vinifera) to grapevine leafroll-associated virus-3 and peg-induced drought stress.stresses caused by viral diseases and drought have long threatened sustainable production of grapevine. these two stresses frequently occur simultaneously in many of grapevine growing regions of the world. we studied responses of in vitro-grown plantlets (vitis vinifera) to grapevine leafroll associated virus-3 (glrav-3) and peg-induced drought stress. results showed that stress induced by either virus infection or drought had negative effects on vegetative growth, caused significant decreases a ...201627313542
molecular characterization of a grapevine leafroll-associated virus 4 from slovenian vineyards.during a survey conducted in vineyards in slovenia, variety of grapevine leafroll disease symptoms were observed. mixed infection with grapevine leafroll-associated viruses 3 and 4 (glrav-3, -4) in two grapevines from a vineyard in south-western part of slovenia was confirmed by das-elisa in 2010. the 3'final 1769 nucleotides of the slovenian glrav-4 isolate were assembled from amplicons obtained by ic rt-pcr. the complete coat protein (cp) and p23 gene sequences were compared with other glrav-4 ...201627265467
next-generation sequencing for virus detection: covering all the bases.the use of next-generation sequencing has become an established method for virus detection. efficient study design for accurate detection relies on the optimal amount of data representing a significant portion of a virus genome.201627250973
sybr(®) green-based real-time quantitative reverse-transcription pcr for detection and discrimination of grapevine viruses.a sybr(®) green-based real-time quantitative reverse transcription pcr (qrt-pcr) assay in combination with melt-curve analysis (mca) was optimized for the detection of nine grapevine viruses. the detection limits for simplex qrt-pcr for all nine grapevine viruses were estimated to be in the range of 214-1112 copies of the virus genome. amplicons with melting temperatures (tm) separated by at least 2°c in the mca could differentiate two viruses in the same reaction. therefore, eight of the nine v ...201627246908
interactive effects of grapevine leafroll-associated virus 3 (glrav-3) and water stress on the physiology of vitis vinifera l. cv. malvasia de banyalbufar and giro-ros.among several biotic and abiotic stress combinations, interaction between drought and pathogen is one of the most studied combinations in some crops but still not in grapevine. in the present work, we focused on the interaction effects of biotic (glrav-3) and abiotic (drought) stresses on grapevine photosynthetic metabolism on two cultivars (cvs. 'malvasia de banyalbufar and giro-ros'). non-infected and glrav-3 infected potted plants were compared under water stress conditions (ws) and well-wate ...201627153513
alterations in primary and secondary metabolism in vitis vinifera 'malvasía de banyalbufar' upon infection with grapevine leafroll-associated virus 3.plant defense mechanisms against pathogens result in differential regulation of various processes of primary and secondary metabolism. imaging techniques, such as fluorescence imaging and thermography, are very valuable tools providing spatial and temporal information about these processes. in this study, effects of grapevine leafroll-associated virus 3 (glrav-3) on grapevine physiology were analyzed in pot-grown asymptomatic plants of the white cultivar malvasía de banyalbufar. the virus trigge ...201626926417
impacts of grapevine leafroll disease on fruit yield and grape and wine chemistry in a wine grape (vitis vinifera l.) cultivar.grapevine leafroll disease (gld) is an economically important virus disease affecting wine grapes (vitis vinifera l.), but little is known about its effect on wine chemistry and sensory composition of wines. in this study, impacts of gld on fruit yield, berry quality and wine chemistry and sensory features were investigated in a red wine grape cultivar planted in a commercial vineyard. own-rooted merlot vines showing gld symptoms and tested positive for grapevine leafroll-associated virus 3 and ...201626919614
differential distribution and titre of selected grapevine leafroll-associated virus 3 genetic variants within grapevine rootstocks.in this study of three grapevine leafroll-associated virus 3 (glrav-3) genetic variants in two grapevine rootstock hosts, glrav-3 detection was shown to be affected by the virus distribution, titre, and the genetic variant. group vi and nz2 glrav-3 variants had reduced detectability compared with the group i variant. differences in the genomic and subgenomic rna (sgrna) expression levels, and differences in the level of expression between the genetic variants were also observed. the observed dif ...201626906692
modeling the accuracy of three detection methods of grapevine leafroll-associated virus 3 during the dormant period using a bayesian approach.grapevine leafroll-associated virus 3 (glrav-3) has a worldwide distribution and is the most economically important virus that causes grapevine leafroll disease. reliable, sensitive, and specific methods are required for the detection of the pathogen in order to assure the production of healthy plant material and control of the disease. although different serological and nucleic acid-based methods have been developed for the detection of glrav-3, diagnostic parameters have not been established, ...201626780435
effects of grapevine leafroll associated virus 3 (glrav-3) and duration of infection on fruit composition and wine chemical profile of vitis vinifera l. cv. sauvignon blanc.in order to determine the effects of grapevine leafroll associated virus 3 (glrav-3) on fruit composition and chemical profile of juice and wine from vitis vinifera l. cv. sauvignon blanc grown in new zealand, composition variables were measured on fruit from vines either infected with glrav-3 (established or recent infections) or uninfected vines. physiological ripeness (20.4°brix) was the criterion established to determine the harvest date for each of the three treatments. date of grape ripene ...201626675855
interactions within susceptible hosts drive establishment of genetically distinct variants of an insect-borne pathogen.coinfections are common, leading to pathogen interactions during transmission and establishment in a host. however, few studies have tested the relative strengths of pathogen interactions in vectors and hosts that determine the outcome of infection. we tested interactions between two genetically distinct variants of the mealybug-transmitted grapevine leafroll-associated virus 3. the transmission efficiency of each variant in single variant inoculations by two vector species was determined. the e ...201526470292
phylogenomic analysis reveals deep divergence and recombination in an economically important grapevine virus.the evolutionary history of the exclusively grapevine (vitis spp.) infecting, grapevine leafroll-associated virus 3 (glrav-3) has not been studied extensively, partly due to limited available sequence data. in this study we trace the evolutionary history of glrav-3, focussing on isolate gh24, a newly discovered variant. gh24 was discovered through the use of next-generation sequencing (ngs) and the whole genome sequence determined and validated with sanger sequencing. we assembled an alignment o ...201525992606
relative quantitation goes viral: an rt-qpcr assay for a grapevine virus.accurate detection and quantitation of viruses can be beneficial to plant-virus interaction studies. in this study, three sybr green real-time rt-pcr assays were developed to quantitate grapevine leafroll-associated virus 3 (glrav-3) in infected vines. three genomic regions (orf1a, coat protein and 3'utr) were targeted to quantitate glrav-3 relative to three stably expressed reference genes (actin, gapdh and α-tubulin). these assays were able to detect all known variant groups of glrav-3, includ ...201425286180
pseudococcus maritimus (hemiptera: pseudococcidae) and parthenolecanium corni (hemiptera: coccidae) are capable of transmitting grapevine leafroll-associated virus 3 between vitis x labruscana and vitis vinifera.the grape mealybug, pseudococcus maritimus (ehrhorn), and european fruit lecanium scale, parthenolecanium corni (bouché), are the predominant species of coccoidea in washington state vineyards. the grape mealybug has been established as a vector of grapevine leafroll-associated virus 3 (glrav-3) between wine grape (vitis vinifera l.) cultivars, elevating its pest status. the objective of this study was to determine if glrav-3 could be transmitted between vitis x labruscana l. and v. vinifera by ...201324468559
molecular characterization and population structure of blackberry vein banding associated virus, new ampelovirus associated with yellow vein disease.blackberry yellow vein disease is the most important viral disease of blackberry in the united states. experiments were conducted to characterize a new virus identified in symptomatic plants. molecular analysis revealed a genome organization resembling grapevine leafroll-associated virus 3, the type species of the genus ampelovirus in the family closteroviridae. the genome of the virus, provisionally named blackberry vein banding associated virus (bvbav), consists of 18,643 nucleotides and conta ...201324126200
rapid detection of grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method.grapevine leafroll disease (gld) is the most important disease of grapevines in south africa. grapevine leafroll-associated virus type 3 (glrav-3) has a close association with the disease and is prevalent in south african vineyards. gld can be controlled using a combination of virus-free planting material, systemic insecticides to control vector populations and removal of infected vines by roguing. infected vines are identified each autumn using either symptom display (in red cultivars) or elisa ...201324025344
ecology and management of grapevine leafroll disease.grapevine leafroll disease (gld) is caused by a complex of vector-borne virus species in the family closteroviridae. gld is present in all grape-growing regions of the world, primarily affecting wine grape varieties. the disease has emerged in the last two decades as one of the major factors affecting grape fruit quality, leading to research efforts aimed at reducing its economic impact. most research has focused on the pathogens themselves, such as improved detection protocols, with limited wor ...201323630520
grapevine leafroll-associated virus 3.grapevine leafroll disease (gld) is one of the most important grapevine viral diseases affecting grapevines worldwide. the impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. several viruses from the family closteroviridae are associated with gld. however, grapevine leafroll-associated virus 3 (glrav-3), the ...201323596440
molecular characterisation of two divergent variants of grapevine leafroll-associated virus 3 in new zealand.partial genomic sequences of two divergent grapevine leafroll-associated virus 3 (glrav-3) variants, nz1-b and nz2, from new zealand were determined and analysed (11,827 nt and 7,612 nt, respectively). at the nucleotide level, both variants are more than 20 % different from the previously published glrav-3 sequences, from phylogenetic groups 1 to 5. phylogenetic analysis indicated that nz1-b is a variant of the previously identified divergent nz-1, while nz2 is a novel sequence with only 76 % nu ...201323408128
generic and sequence-variant specific molecular assays for the detection of the highly variable grapevine leafroll-associated virus 3.grapevine leafroll-associated virus 3 (glrav-3) is an economically important virus, which is found in all grapevine growing regions worldwide. its accurate detection in nursery and field samples is of high importance for certification schemes and disease management programmes. to reduce false negatives that can be caused by sequence variability, a new universal primer pair was designed against a divergent sequence data set, targeting the open reading frame 4 (heat shock protein 70 homologue gene ...201323313884
genetic diversity analyses reveal novel recombination events in grapevine leafroll-associated virus 3 in china.grapevine leafroll-associated virus 3 (glrav-3) is the most prevalent causal agent of grapevine leafroll disease (gld). of the 75 grapevine samples collected from three regions in china, 46.7% and 94.7% of samples tested positive for glrav-3 in reverse transcription-pcr (rt-pcr) and reverse transcription nested pcr (rt-npcr), respectively. the sscp analysis for the clones of complete cp gene from 16 glrav-3 isolates showed that 15 isolates contained one predominant haplotype and one isolate had ...201323085611
a divergent variant of grapevine leafroll-associated virus 3 is present in california.grapevine leafroll-associated viruses are a problem for grape production globally. symptoms are caused by a number of distinct viral species. during a survey of napa valley vineyards (california, usa), we found evidence of a new variant of grapevine leafroll-associated virus 3 (glrav-3). we isolated its genome from a symptomatic greenhouse-raised plant and fully sequenced it.201223062082
real-time rt-pcr high-resolution melting curve analysis and multiplex rt-pcr to detect and differentiate grapevine leafroll-associated virus 3 variant groups i, ii, iii and vi.grapevine leafroll-associated virus 3 (glrav-3) is the main contributing agent of leafroll disease worldwide. four of the six glrav-3 variant groups known have been found in south africa, but their individual contribution to leafroll disease is unknown. in order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of glrav-3.201223016734
complete nucleotide sequence of a chinese isolate of grapevine leafroll-associated virus 3 reveals a 5' utr of 802 nucleotides.grapevine leafroll-associated virus 3 (glrav-3) is widely spread in china. here we report, for the first time, the complete nucleotide sequence of the chinese isolate (ln) of glrav-3. the 18,563-nt genomic rna is the largest of the glrav-3 genomes reported to date, with a 5' untranslated region of 802 nt. its sequence shares 87.99-98.15 % identity with those of previously reported isolates, and phylogenetic analysis suggested placing isolate ln in group 3, together with another fully sequenced i ...201323011775
high-throughput sequence analysis of small rnas in grapevine (vitis vinifera l.) affected by grapevine leafroll disease.grapevine leafroll disease (glrd) is one of the most economically important virus diseases of grapevine (vitis spp.) worldwide. in this study, we used high-throughput sequencing of cdna libraries made from small rnas (srnas) to compare profiles of srna populations recovered from own-rooted merlot grapevines with and without glrd symptoms. the data revealed the presence of srnas specific to grapevine leafroll-associated virus 3, hop stunt viroid (hpsvd), grapevine yellow speckle viroid 1 (gysvd-1 ...201222827483
the p19.7 rna silencing suppressor from grapevine leafroll-associated virus 3 shows different levels of activity across phylogenetic groups.at least five phylogenetic groups have been reported for grapevine leafroll-associated virus 3 (glrav-3). the p19.7 protein encoded by the glrav-3 was previously identified as an rna silencing suppressor. in this study, five constructs of p19.7 belonging to different groups were compared for their suppressing activity. for each p19.7 variant, the accumulation level of green fluorescent protein mrna and specific sirnas were determined using co-infiltration assays in transgenic 16c nicotiana benth ...201222714284
complete nucleotide sequence of a new strain of grapevine leafroll-associated virus 3 in south africa.the complete genome sequences of two newly identified genetic variants of grapevine leafroll-associated virus 3 (glrav-3) are described. two isolates, gh11 (18671 nt) and gh30 (18576 nt) were sequenced and found to be less than 70 % similar to other south african glrav-3 variants. the genome organization of gh11 and gh30 is similar to that of previously described glrav-3 isolates. however, no corresponding open reading frame 2 (orf2) could be identified. phylogenetic analysis indicates that gh11 ...201222614811
letter to the editor: characterization of a grapevine leafroll-associated virus 3 from india showing incongruence in its phylogeny.survey conducted during 2010-2011 in the vineyards of nashik and pune regions of india revealed the association of an ampelovirus antigenically related to grapevine leafroll-associated virus 3 (glrav-3) with seven cultivars of grapevine. upon sequencing of the coat protein (cp) and partial heat shock protein 70 homologue (hsp70h) genes of glrav-3 present in the cultivar cabernet souvignon showed distinct grouping behaviour. it clustered in group 2 and group 1 on the basis of cp and hsp70h sequen ...201222562226
infection and colonization of nicotiana benthamiana by grapevine leafroll-associated virus 3.grapevine leafroll disease is an increasing problem in all grape-growing regions of the world. the most widespread agent of the disease, grapevine leafroll-associated virus 3 (glrav-3), has never been shown to infect species outside of the genus vitis. virus transmission to several plant species used as model systems was tested using the vine mealybug, planococcus ficus. we show that glrav-3 is able to infect nicotiana benthamiana. working with glrav-3 infected n. benthamiana revealed distinct a ...201728710957
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