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evidence for translation of apple stem grooving capillovirus genomic rna.apple stem grooving virus (asgv) rna was translated in a rabbit reticulocyte lysate system and shown to direct the synthesis of several polypeptides of mr ranging from 200k to 43k. a polypeptide of 200k was a major product, but no polypeptide with electrophoretic mobility the same as that of the asgv coat protein was synthesized. immunoprecipitation experiments showed that a polypeptide of 200k was selectively precipitated by antiserum against purified asgv. these results indicate that asgv coat ...19921588329
cherry virus a: cdna cloning of dsrna, nucleotide sequence analysis and serology reveal a new plant capillovirus in sweet cherry.the nucleotide sequence (7383 nucleotides) of a newly identified member of the genus capillovirus, cherry virus a (cva), was obtained from cdna clones. the cdna was generated from dsrna extracted from plant tissue infected with little cherry virus (lcv). small amounts of lcv dsrna served as template nucleic acid and enabled the construction of a library of which, unexpectedly, 7.5% of the recombinant plasmids were specific for cva. the genome organization of cva resembles that of apple stem groo ...19957636482
striking similarities between the nucleotide sequence and genome organization of citrus tatter leaf and apple stem grooving capilloviruses.the sequence of the 3'-terminal 2956 nucleotides, excluding the poly(a) tail, of the citrus tatter leaf virus (ctlv) genome was determined and compared with that of the apple stem grooving virus (asgv) genome. the sequence of the 3'-terminal region of ctlv contains two overlapping open reading frames (orfs) and a 3'-terminal non-coding region of 142 nucleotides. the long, incomplete orf1 ends at uag (position 2812) and encodes a protein with at least 938 amino acids (m(r) > 108,703). this protei ...19938277280
a simple and reliable protocol for the detection of apple stem grooving virus by rt-pcr and in a multiplex pcr assay.primers were identified which amplify specifically a 499 bp fragment in the coat protein coding region of apple stem grooving virus (asgv) genome. these primers were used in various rt-polymerase chain reaction (pcr) analyses for the detection of asgv in chenopodium quinoa, nicotiana occidentalis, and in species of malus and pyrus. isolates of asgv in malus and pyrus from locations in canada, china, israel, japan, nepal, pakistan, south africa, and the u.s.a. were reliably detected in leaf and b ...199910598077
simultaneous detection of six citrus viroids and apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction.we developed a multiplex reverse transcription polymerase chain reaction (rt-pcr) to detect six citrus viroids: citrus exocortis viroid (cevd), citrus bent leaf viroid (cblvd), hop stunt viroid (hsvd), citrus viroid iii (cvd-iii), citrus viroid iv (cvd-iv) and citrus viroid os (cvd-os) and apple stem grooving virus (asgv, synonym: citrus tatter leaf virus (ctlv)) from citrus plants. the multiplex rt-pcr was also designed to distinguish cvd-i-lss (a distinct variant of cblvd) from cblvd. by the m ...200212393154
nucleotide sequences of a korean isolate of apple stem grooving virus associated with black necrotic leaf spot disease on pear (pyrus pyrifolia).pear black necrotic leaf spot (pbnls) is a disease of pears caused by capillovirus-like particles, which can be observed under the electron microscope. the disease was analyzed by western blot analysis with antisera raised against apple stem grooving virus (asgv) coat protein. cdnas covering the entire genome were synthesized by rt-pcr and race using rna isolated from chenopodium quinoa infected with sap extracted from pear leaves carrying black necrotic spot disease. the complete genome sequenc ...200415528995
determination of major viral and sub viral pathogens incidence in apple orchards in himachal pradesh.apple is the major commercial horticulture crop in himachal pradesh and other hill states of jammu & kashmir, uttarakhand and some parts of northeastern states of india. in order to gather data on health status and incidence of virus and virus-like pathogens in apple orchards, survey was conducted in the month of june and september, 2010 in hatkoti, rohru, kuthara, jubbal and khadapathar areas of major apple producing shimla district of himachal pradesh. a total of 250 samples were collected and ...201123730008
characterization of apple stem grooving virus and apple chlorotic leaf spot virus identified in a crab apple tree.apple stem grooving virus (asgv), apple chlorotic leaf spot virus (aclsv), and prunus necrotic ringspot virus (pnrsv) were identified in a crab apple tree by small rna deep sequencing. the complete genome sequence of aclsv isolate bj (aclsv-bj) was 7554 nucleotides and shared 67.0%-83.0% nucleotide sequence identity with other aclsv isolates. a phylogenetic tree based on the complete genome sequence of all available aclsv isolates showed that aclsv-bj clustered with the isolates sy01 from hawtho ...201727990565
pseudo-polyprotein translated from the full-length orf1 of capillovirus is important for pathogenicity, but a truncated orf1 protein without variable and cp regions is sufficient for replication.the first open-reading frame (orf) of the genus capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (rep) and coat protein (cp), while other viruses in the family flexiviridae have separate orfs encoding these proteins. to investigate the role of the full-length orf1 polyprotein of capillovirus, we generated truncation mutants of orf1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative ...201020381551
multiplex rt-pcr-elisa compared with bioassay for the detection of four apple viruses.a sensitive and reliable multiplex rt-pcr-elisa technique for the detection of apple chlorotic leaf spot virus, apple stem pitting virus, apple mosaic virus and apple stem grooving virus was developed. this technique is compared with the method used commonly for indexing by woody indicators, which is time consuming and expensive. for the rt-pcr-elisa technique, the amplified products were labeled with digoxigenin during the rt-pcr by incorporation of a digoxigenin labeled primer. after hybridiza ...200312798242
deep sequencing reveals a novel closterovirus associated with wild rose leaf rosette disease.a bizarre virus-like symptom of a leaf rosette formed by dense small leaves on branches of wild roses (rosa multiflora thunb.), designated as 'wild rose leaf rosette disease' (wrlrd), was observed in china. to investigate the presumed causal virus, a wild rose sample affected by wrlrd was subjected to deep sequencing of small interfering rnas (sirnas) for a complete survey of the infecting viruses and viroids. the assembly of sirnas led to the reconstruction of the complete genomes of three know ...201525187347
high-throughput sequencing reveals small rnas involved in asgv infection.plant small rnas (srnas) associated with virulent virus infections have been reported by previous studies, while the involvement of srnas in latent virus infection remains largely uncharacterised. apple trees show a high degree of resistance and tolerance to viral infections. we analysed two srna deep sequencing datasets, prepared from different rna size fractions, to identify srnas involved in apple stem grooving virus (asgv) infection.201424998458
infection of apple by apple stem grooving virus leads to extensive alterations in gene expression patterns but no disease symptoms.to understand the molecular basis of viral diseases, transcriptome profiling has been widely used to correlate host gene expression change patterns with disease symptoms during viral infection in many plant hosts. we used infection of apple by apple stem grooving virus (asgv), which produces no disease symptoms, to assess the significance of host gene expression changes in disease development. we specifically asked the question of whether such asymptomatic infection is attributed to limited chan ...201424736405
detection of four apple viruses by multiplex rt-pcr assays with coamplification of plant mrna as internal control.two multiplex rt-pcr assays with specific coamplification of plant mrna as an internal control from total nucleic acids are described for the parallel detection of apple chlorotic leaf spot virus, apple stem pitting virus, apple mosaic virus and apple stem grooving virus. all are important economically and common pathogens in commercial apple and pear cultivars, except for apple mosaic virus. four virus specific primer pairs and one primer pair which allows the specific amplification of mrna of ...200211684306
comparison of bioassays and laboratory assays for apple stem grooving virus.the standard field double-budding assay with the indicator virginia crab and the glasshouse test with the indicator malus micromalus, were compared with elisa and immunocapture pcr for the detection of apple stem grooving virus (asgv) in 102 apple trees and three oriental pear. twenty-two trees were positive for asgv by both elisa and ic-pcr but three of these trees were negative by virginia crab, three were negative by m. micromalus and one was negative by both these bioassays. the infected tre ...200111311355
integrated analyses using rna-seq data reveal viral genomes, single nucleotide variations, the phylogenetic relationship, and recombination for apple stem grooving virus.next-generation sequencing (ngs) provides many possibilities for plant virology research. in this study, we performed integrated analyses using plant transcriptome data for plant virus identification using apple stem grooving virus (asgv) as an exemplar virus. we used 15 publicly available transcriptome libraries from three different studies, two mrna-seq studies and a small rna-seq study.201627507588
characterization of virus-derived small interfering rnas in apple stem grooving virus-infected in vitro-cultured pyrus pyrifolia shoot tips in response to high temperature treatment.heat treatment (known as thermotherapy) together with in vitro culture of shoot meristem tips is a commonly used technology to obtain virus-free germplasm for the effective control of virus diseases in fruit trees. rna silencing as an antiviral defense mechanism has been implicated in this process. to understand if high temperature-mediated acceleration of the host antiviral gene silencing system in the meristem tip facilitates virus-derived small interfering rnas (vsirna) accumulation to reduce ...201627716257
a real-time rt-qpcr assay for the detection of citrus tatter leaf virus.citrus tatter leaf virus (cltv) is globally distributed wherever citrus is grown, and, given the extensive use of ctlv sensitive rootstock, has the potential to be a significant threat to the citrus industry. in order to facilitate fast and reliable detection of this virus, we have developed a ctlv-specific real-time rt-qpcr assay. the optimized assay was found to be more reliable and sensitive compared to elisa and end-point rt-pcr, detecting ctlv in up to 70% more plants. the real-time rt-qpcr ...201728274745
phylogenetic analysis and recombination events in full genome sequences of apple stem grooving virus.apple stem grooving virus (asgv) is one of the most important viral pathogens infecting pome and stone fruit trees worldwide. in this study, with the complete nucleotide sequence of isolate asgv-t47, which we generated, molecular variation and recombination in asgv full genomic sequences worldwide were analyzed. asgv-t47 shared 79.7-97.6% nucleotide identity with the other isolates worldwide and had the highest identity with an isolate from japan. phylogenetic analysis based on whole genome clus ...201425518711
molecular variability of the genomes of capilloviruses from apple, japanese pear, european pear, and citrus trees.abstract the 3'-terminal regions of the genomes of apple stem grooving virus (asgv), isolated from apple, japanese pear, and european pear plants, and citrus tatter leaf virus (ctlv), isolated from citrus plants, were amplified by reverse transcription-polymerase chain reaction. the dna products were cloned and sequenced. the results indicated that the asgv isolates from apple, japanese pear, and european pear comprise at least two to four "sequence variants" that differ considerably from each o ...199718945117
the 50-kda protein of apple chlorotic leaf spot virus interferes with intracellular and intercellular targeting and tubule-inducing activity of the 39-kda protein of grapevine berry inner necrosis virus.to understand why transgenic nicotiana occidentalis plants expressing a functional movement protein (mp) of apple chlorotic leaf spot virus (aclsv) show specific resistance to grapevine berry inner necrosis virus (ginv), the mps of aclsv (50kp) and ginv (39kp) were fused to green, yellow, or cyan fluorescent proteins (gfp, yfp, or cfp). these fusion proteins were transiently expressed in leaf cells of both transgenic (50kp) and nontransgenic (nt) plants, and the intracellular and intercellular t ...200312650450
the amino acid sequence determination of a granulin and polyhedrin from two baculoviruses infecting agrotis segetum.the amino acid sequence of agrotis segetum granulosis virus (asgv) granulin and a. segetum nuclear polyhedrosis virus (asnpv) polyhedrin was determined by sequencing tryptic and chymotryptic peptides from reduced and carboxymethylated proteins and tryptic fragments of oxidized and maleylated granulin. the comparison of the established peptide structures with the primary structures of other occlusion body proteins from related baculoviruses was also used for the polypeptide chains' reconstruction ...19921604817
the nucleotide sequence of apple stem grooving capillovirus genome.the complete nucleotide sequence of apple stem grooving virus (asgv) genome has been determined. the genome is 6496 nucleotides in length excluding a 3'-terminal poly(a) tail and contains two overlapping open reading frames (orfs). orf1 begins at nucleotide position 37 and is terminated at position 6341, encoding a protein with a molecular weight of 241 kda. orf2, which is in a different reading frame within orf1, begins at position 4788 and can encode a 36-kda protein. the 241-kda protein conta ...19921413530
infection of capilloviruses requires subgenomic rnas whose transcription is controlled by promoter-like sequences conserved among flexiviruses.the first open-reading frame (orf) of apple stem grooving virus (asgv), of the genus capillovirus, encodes an apparently chimeric polyprotein containing conserved regions for replicase (rep) and coat protein (cp). however, our previous study revealed that asgv mutants with distinct and discontinuous rep- and cp-coding regions successfully infect plants, indicating that cp expressed via a subgenomic rna (sgrna) is sufficient for viability of the virus. here we identified a transcription start sit ...201222401846
simultaneous detection and differentiation of three viruses in pear plants by a multiplex rt-pcr.a multiplex rt-pcr (mrt-pcr) assay was developed for detection and differentiation of the apple stem pitting virus (aspv), apple stem grooving virus (asgv) and apple chlorotic leaf spot virus (aclsv), which are viruses frequently occurring in pear trees. different combinations of mixed primer pairs were tested for their specificity and sensitivity for the simultaneous detection of the three viruses. three primer pairs were used to amplify their fragments of 247bp, 358bp and 500bp, respectively. ...201424269332
multiplex rt-pcr detection and distribution of four apple viruses in china.apple trees are natural hosts of four economically important virus species in china. we used a simple, sensitive multiplex rt-pcr protocol with an internal control to simultaneously detect and differentiate four apple viruses: apple chlorotic leaf spot virus (aclsv), apple mosaic virus (apmv), apple stem pitting virus (aspv), and apple stem grooving virus (asgv). this multiplex rt-pcr could be used as an alternative to other routinely used detection methods. we used this protocol to evaluate the ...201324294957
simultaneous detection of major pome fruit viruses and a viroid.a rapid and sensitive two-step rt-pcr protocol for simultaneous detection of major apple viruses, namely apple mosaic virus (apmv), apple stem pitting virus (aspv), apple stem grooving virus (asgv), apple chlorotic leaf spot virus (aclsv) and apple scar skin viroid (assvd), was developed. five specific primer pairs were tested and confirmed for these viruses and viroid together in a single tube, giving amplicons of ~198, ~330, ~370, ~547 and ~645 bp corresponding to asgv, assvd, aspv, apmv and a ...201425320423
a simplified strategy for studying the etiology of viral diseases: apple stem grooving virus as a case study.a simple method to amplify infective, complete genomes of single stranded rna viruses by long distance pcr (ld pcr) from woody plant tissues is described in detail. the present protocol eliminates partial purification of viral particles and the amplification is achieved in three steps: (i) easy preparation of template rna by incorporating a pre processing step before loading onto the column (ii) reverse transcription by amv or superscript reverse transcriptase and (iii) amplification of cdna by ...201525486082
cryotherapy as a method for reducing the virus infection of apples (malus sp.).there is an urgent need in kazakhstan for virus-free nursery stock to reinvigorate the industry and preserve historic cultivars. an in vitro collection of apples could be used for virus testing and elimination and to provide virus-free elite stock plants to nurseries.201626964019
a multiple rt-pcr assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees.apple chlorotic leaf spot virus (aclsv), apple stem grooving virus (asgv), and apple stem pitting virus (aspv) are three latent viruses frequently occurring in apple trees worldwide. in field orchards, these viruses are frequently found in a mixed infection with viroids in the genus apscarviroid, including apple scar skin viroid, and apple dimple fruit viroid. together these viruses and viroids could cause serious damage to apple fruit production worldwide. rapid and efficient detection methods ...201627054889
deep sequencing analysis of apple infecting viruses in korea.deep sequencing has generated 52 contigs derived from five viruses; apple chlorotic leaf spot virus (aclsv), apple stem grooving virus (asgv), apple stem pitting virus (aspv), apple green crinkle associated virus (agcav), and apricot latent virus (aplv) were identified from eight apple samples showing small leaves and/or growth retardation. nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. sequ ...201627721694
evaluation of reference genes for the relative quantification of apple stem grooving virus and apple mosaic virus in apple trees.a sybr green(®)-based one step rt-qpcr assay was developed for the detection and quantification of apple stem grooving virus (asgv) and apple mosaic virus (apmv). the rt-qpcr assay employed seven plant-expressed genes-glyceraldehyde 3-phosphate dehydrogenase (gapdh), 18s ribosomal rna, ubiquitin, ribosomal protein s19, rubisco, rna polymerase subunit ii and β-actin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. idared, champion, fragra ...201223730001
molecular characterization of citrus tatter leaf virus historically associated with meyer lemon trees: complete genome sequence and development of biologically active in vitro transcripts.citrus tatter leaf virus isolated from meyer lemon trees (ctlv-ml) from california and florida induces bud union incompatibility of citrus trees grafted on the widely used trifoliate and trifoliate hybrid rootstocks. the complete genome sequence of ctlv-ml was determined to be 6,495 nucleotides (nts), with two overlapping open reading frames (orfs) and a poly (a) tail at the 3' end. the genome organization is similar to other capilloviruses, with orf1 (nts 37 to 6,354) encoding a putative 242-kd ...200919271984
transgenic nicotiana occidentalis plants expressing the 50-kda protein of apple chlorotic leaf spot virus display increased susceptibility to homologous virus, but strong resistance to grapevine berry inner necrosis virus.abstract the 50-kda protein (p50) encoded by the open reading frame 2 of apple chlorotic leaf spot virus (aclsv), a putative movement protein, was expressed in transgenic nicotiana occidentalis plants. p50 in transgenic plants was mainly detected in a modified form in the cell wall fraction, similar to that in infected leaves. the p50-expressing plants (p50 plants) complemented the systemic spread of the p50-defective mutants of an infectious cdna clone of aclsv (pclsf), indicating that p50 in t ...200018944625
a rapid and effective rna release procedure for virus detection in woody plants by reverse transcription-polymerase chain reaction.a rapid and effective rna release procedure (rna-rp) for detection of the apple stem pitting virus (aspv) and the apple stem grooving virus (asgv) in woody plants by reverse transcription-polymerase chain reaction (rt-pcr) was developed. rna-rp released rna into crude homogenate. rna-rp was compared with classical phenol/chlorophorm extraction of rna. the rt-pcr detection of aspv and asgv was shown to be similar by both the rna preparation procedures. rna-rp in contrast to the classical phenol/c ...200314658842
a single silent substitution in the genome of apple stem grooving virus causes symptom attenuation.among randomly mutagenized clones derived from an infectious cdna copy of genomic rna of apple stem grooving virus (asgv), we previously identified a clone, prm21, whose in vitro transcript (asgv-rm21) does not induce any symptoms characteristic of the original (wild-type) cdna clone (asgv-wt) in several host plants. interestingly, asgv-rm21 has only a single, translationally silent nucleotide substitution, u to c, at nucleotide 4646 of the viral genome within open reading frame (orf) 1. here, w ...200312917479
production of monoclonal antibodies specific for the recombinant viral coat protein of apple stem grooving virus-citrus isolate and their application for a simple, rapid diagnosis by an immunochromatographic assay.a simple and rapid immunochromatographic assay (ica) for the diagnosis of apple stem grooving virus (asgv) in citrus was developed. nine lines of monoclonal antibodies (mabs) were produced by immunizing with a recombinant viral coat protein of asgv as the antigen. according to the competitive-binding elisa results, the 9 mabs comprised 2 paratope groups, a and b. after screening for the most effective combination of mabs, the two lines from different paratope groups (4a12 from group a and 6n31 f ...201424121136
molecular evolution of the genomic rna of apple stem grooving capillovirus.the complete genome of the german isolate ac of apple stem grooving virus (asgv) was sequenced. it encodes two overlapping open reading frames (orfs), similarly to previously described asgv isolates. two regions of high variability were detected between the asgv isolates, variable region 1 (v1, from amino acids (aa) 532 to 570), and variable region 2 (v2, from aa 1,583 to 1,868). the phylogenetic analysis of the v1 and v2 regions suggested that the asgv diversity was structured by host plant spe ...201223149596
complete sequence of an apple stem grooving virus (asgv) isolate from china.the complete genome sequence of a chinese isolate of apple stem grooving virus (asgv) was determined to be 6,495 nucleotides long, single-stranded, plus-sense rna. the viral rna has two overlapping open reading frames (orfs): orf1 and orf2. compared with the genome sequences of asgv isolates available in genbank, the nucleotide identities ranged from 80.1 to 86.3 %. the amino acid identities of proteins encoded by orf1 and orf2 ranged from 79.5 to 86.1 % and 82.0 to 85.9 %, respectively.201222864549
identification and characterization of micrornas from in vitro-grown pear shoots infected with apple stem grooving virus in response to high temperature using small rna sequencing.micrornas (mirnas) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. thermotherapy is an effective approach for elimination of viruses from fruit trees. however, the role of mirnas in this process remains elusive. previously, we showed that high temperature treatment reduces the titers of apple stem grooving virus (asgv) from the tips of in vitro-grown pyrus pyrifolia plants. in this study, we identified high t ...201526573813
simultaneous detection and identification of four pome fruit viruses by one-tube pentaplex rt-pcr.a pentaplex reverse-transcription polymerase chain reaction (pentaplex rt-pcr) in a single tube was developed for the simultaneous detection of the pome fruit viruses: apple stem pitting virus (aspv), apple stem grooving virus (asgv), apple chlorotic leaf spot virus (aclsv) and apple mosaic virus (apmv). this is the first report of the simultaneous detection of all four viruses and host mrna as an internal specific control. pentaplex rt-pcr was applied successfully throughout the year, using dif ...200616337013
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