| 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 ... | 2000 | 10675398 |
| partial sequence identification of grapevine-leafroll-associated virus-1 and development of a highly sensitive ic-rt-pcr detection method. | using immunocapture reverse transcription pcr (ic-rt-pcr) a specific pcr product from glrav-1 infected vine samples was amplified with the help of degenerate primers deduced from the conserved hsp70 region of closteroviruses. 511 basepairs of the 5'end of glrav-1 hsp70 gene were identified. within this region, putative glrav-1 specific primers were designed and an ic-rt-pcr detection procedure was developed which is about 125 times more sensitive than the established elisa method. no pcr product ... | 2000 | 10713381 |
| hypervariable genes in grapevine leafroll associated virus 1. | analysis of nucleotide sequences of 10 open reading frames from the grapevine leafroll associated virus 1 (glrav-1), a tentative member of the genus closterovirus, revealed the presence of an unusually high degree of sequence variation in orfs 3, 6 and 7 encoding a homologue of heat shock protein 70 and two diverged copies of the coat protein (cpd1 and cpd2), respectively. overall, 75 clones corresponding to orfs 3, 6 and 7 were sequenced and 1916 nucleotide changes were recorded relative to the ... | 2001 | 11597756 |
| a spot multiplex nested rt-pcr for the simultaneous and generic detection of viruses involved in the aetiology of grapevine leafroll and rugose wood of grapevine. | a spot nested rt-pcr assay using degenerate deoxyinosine-containing primers was developed, allowing rapid and simultaneous detection of closterovirus sequences. nested pcr amplification increased the specificity and sensitivity of detection. the sensitivity was also increased by a factor of 10 by using in addition to the deoxyinosine (di)-containing primers, respective homologous primers in which di was substituted by dg in the region of sequence homology. these homologous primers are shorter, h ... | 2003 | 12711066 |
| 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 ... | 2005 | 15929397 |
| analysis of the molecular variability of grapevine leafroll-associated virus 1 reveals the presence of two distinct virus groups and their mixed occurrence in grapevines. | genetic diversity of eight grapevine leafroll-associated virus 1 (glrav-1) isolates recovered from grapevines in three distinct locations in the czech republic and slovakia was characterised by restriction fragment length polymorphism (rflp) analysis and by sequencing of cloned 540 bp fragment of the heat shock protein 70 (hsp70) gene. comparison and phylogenetic analysis of obtained and previously available sequence data revealed the existence of two groups of glrav-l isolates, tentatively name ... | 2005 | 16175330 |
| improved detection of grapevine leafroll-associated virus 1 by magnetic capture hybridisation rt-pcr on a conserved region of viral rna. | we report the development of a sensitive diagnostic method for the detection of the grapevine leafroll-associated virus 1 (glrav-1). we have considered the current shortcoming in detection of glrav-1 to be linked to two factors, sequence variation in the viral rna and low template concentration. sequence information available allowed the selection of optimal target sequences for detection by rt-pcr, having high copy number and low levels of sequence variation. this was combined with the use of m ... | 2006 | 16292599 |
| 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 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 |
| a sensitive one-step real-time rt-pcr method for detecting grapevine leafroll-associated virus 2 variants in grapevine. | grapevine leafroll syndrome is caused by a complex of up to nine different grapevine leafroll-associated viruses (glrav-1-9) with glrav-2 being reported as one of the most variable species of this group. many methods, including indexing, serological and molecular procedures, have been developed for the detection of glrav-2. however, due to the low concentration of the virus in plants and the high variability of glrav-2, a method with improved sensitivity and with the capacity to detect of all kn ... | 2007 | 17223202 |
| 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 ... | 2001 | 18943345 |
| 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 ... | 2001 | 18943347 |
| 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 |
| 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 ... | 2009 | 19082687 |
| 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 ... | 2009 | 19740031 |
| 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 |
| 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 |
| 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 ... | 2010 | 21167205 |
| grapevine leafroll-associated virus 1 occurs as genetically diverse populations. | the genetic diversity of thirty-four isolates of grapevine leafroll-associated virus 1 (glrav-1) from different wine, table and ornamental grape cultivars in california, new york and washington states in the u.s.a. was investigated. segments of the heat-shock protein 70 homolog (hsp70h) gene, coat protein (cp) gene, coat protein duplicate 2 (cpd2) gene and orf9 (p24) were amplified by rt-pcr, cloned and sequenced. a pairwise comparison of nucleotide sequences revealed intra- and inter-isolate se ... | 2011 | 21830956 |
| 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 ... | 2011 | 22039446 |
| 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 |
| 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 |
| 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 ... | 2013 | 23219809 |
| 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 |
| 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 ... | 2013 | 24426260 |
| 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 ... | 2016 | 27329635 |
| 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- ... | 2016 | 27329628 |
| molecular characterization of a novel putative ampelovirus tentatively named grapevine leafroll-associated virus 13. | a novel putative ampelovirus was detected in grapevines that showed typical leafroll symptoms and was tentatively named grapevine leafroll-associated virus (glrav)-13 following the series of numbering of other glravs. the complete genome of glrav-13 comprised 17,608 nt and contained eleven putative open reading frames, showing genetic features similar to those of viruses belonging to subgroup i of genus ampelovirus. phylogenetic trees based on the rna-dependent rna polymerase, heat shock protein ... | 2016 | 27289224 |
| 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 ... | 2015 | 26223949 |
| genetic diversity and recombination analysis of grapevine leafroll-associated virus 1 from china. | grapevine leafroll-associated virus 1 (glrav-1) is one of the causal agents of grapevine leafroll disease (gld). to investigate the prevalence and genetic variation of glrav-1 in china, 132 grapevine samples from 14 chinese provinces and regions were tested using reverse transcription pcr (rt-pcr) and reverse transcription nested pcr (rt-npcr). the samples included symptomatic and asymptomatic cultivars, and 36.4% of them tested positive for glrav-1. 'beida' samples, previously identified as vir ... | 2015 | 25925705 |
| molecular data mining to improve antibody-based detection of grapevine leafroll-associated virus 1 (glrav-1). | testing for grapevine leafroll-associated virus 1 (glrav-1) is mandatory in certification schemes of propagation material within the eu. accurate and reliable diagnostic assays are necessary for implementation of this measure. during routine detection of glrav-1, using double antibody sandwich enzyme-linked immunosorbent assay (das-elisa) and reverse transcription (rt) followed by polymerase chain reaction (pcr), evidence was obtained that positive samples could be overlooked by either or both d ... | 2013 | 24056263 |
| analysis of a short genomic region of grapevine leafroll-associated virus 1 (glrav-1) reveals the presence of two different molecular groups of isolates in slovakia. | although grapevine leafroll-associated virus 1 (glrav-1) is one of the most important agents of the grapevine leafroll disease, the data about its molecular variability are scarce. in order to assess the glrav-1 diversity in slovakia, the sequence of a genome region encoding the central part of the capsid protein (cp) gene was determined from 18 glrav-1 isolates. despite the fact that the analysis targeted a relatively short portion of the genome, comparison of obtained sequences has revealed th ... | 2013 | 24020761 |
| 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 ... | 2013 | 23774971 |
| macroarray detection of grapevine leafroll-associated viruses. | grapevine leafroll-associated viruses (glravs) are an emerging group of viruses that represent a significant threat to the global productivity and sustainability of the grapevine industry. their control is achieved through the identification and elimination of infected vines, and the use of planting material derived from virus-tested, certified stocks. as such, much effort has been invested in developing reliable molecular diagnostic techniques. in this work, we report the development of a macro ... | 2012 | 22580028 |
| transmission of six ampeloviruses and two vitiviruses to grapevine by phenacoccus aceris. | grapevine leafroll disease is caused by grapevine leafroll-associated viruses (glravs). these viruses are common in vineyards worldwide and often associated with vitiviruses that are involved in the rugose wood complex of grapevine. ten mealybug species are known as vectors of one or several of these grapevine viruses, including the apple mealybug phenacoccus aceris which is widespread in holarctic regions and able to transmit grapevine leafroll-associated virus-1 and -3 (glrav-1 and -3). our ai ... | 2012 | 22439861 |
| an analysis of the complete genome sequence and subgenomic rnas reveals unique features of the ampelovirus, grapevine leafroll-associated virus 1. | despite being the first closterovirus documented in grapevines (vitis sp.), the molecular biology of grapevine leafroll-associated virus 1 (glrav-1, genus ampelovirus, family closteroviridae) is still in its infancy. in this study, the complete genome sequence of two glrav-1 isolates was determined to be 18,731 (isolate wa-ch) and 18,946 (isolate wa-pn) nucleotides (nt). the genome of wa-ch and wa-pn isolates encodes nine putative open reading frames (orfs) and the arrangement of these orfs in b ... | 2017 | 28686140 |