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1.
Viruses ; 13(10)2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34696511

RESUMO

The cottony grape scale Pulvinaria vitis is a scale insect colonizing grapevine; however, its capacity as a vector of grapevine viruses is poorly known in comparison to other scale species that are vectors of viral species in the genera Ampelovirus and Vitivirus. The ability of P. vitis to transmit the ampeloviruses Grapevine leafroll-associated viruses [GLRaV]-1, -3, and -4, and the vitivirus Grapevine virus A (GVA), to healthy vine cuttings was assessed. The scale insects used originated from commercial vine plots located in Alsace, Eastern France. When nymphs sampled from leafroll-infected vineyard plants were transferred onto healthy cuttings, only one event of transmission was obtained. However, when laboratory-reared, non-viruliferous nymphs were allowed to acquire viruses under controlled conditions, both first and second instar nymphs derived from two vineyards were able to transmit GLRaV-1 and GVA. This is the first report of GLRaV-1 and GVA transmission from grapevine to grapevine by this species.


Assuntos
Closteroviridae/patogenicidade , Flexiviridae/patogenicidade , Hemípteros/patogenicidade , Animais , Closteroviridae/classificação , Closteroviridae/genética , Flexiviridae/metabolismo , Hemípteros/metabolismo , Insetos Vetores/virologia , Doenças das Plantas/virologia , Vitis/parasitologia
3.
Viruses ; 13(6)2021 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072594

RESUMO

Cassava is one of the most important staple crops in Africa and its production is seriously damaged by viral diseases. In this study, we identify for the first time and characterize the genome organization of novel ampeloviruses infecting cassava plants in diverse geographical locations using three high-throughput sequencing protocols [Virion-Associated Nucleotide Acid (VANA), dsRNA and total RNA], and we provide a first analysis of the diversity of these agents and of the evolutionary forces acting on them. Thirteen new Closteroviridae isolates were characterized in field-grown cassava plants from the Democratic Republic of Congo (DR Congo), Madagascar, Mayotte, and Reunion islands. The analysis of the sequences of the corresponding contigs (ranging between 10,417 and 13,752 nucleotides in length) revealed seven open reading frames. The replication-associated polyproteins have three expected functional domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). Additional open reading frames code for a small transmembrane protein, a heat-shock protein 70 homolog (HSP70h), a heat shock protein 90 homolog (HSP90h), and a major and a minor coat protein (CP and CPd respectively). Defective genomic variants were also identified in some cassava accessions originating from Madagascar and Reunion. The isolates were found to belong to two species tentatively named Manihot esculenta-associated virus 1 and 2 (MEaV-1 and MEaV-2). Phylogenetic analyses showed that MEaV-1 and MEaV-2 belong to the genus Ampelovirus, in particular to its subgroup II. MEaV-1 was found in all of the countries of study, while MEaV-2 was only detected in Madagascar and Mayotte. Recombination analysis provided evidence of intraspecies recombination occurring between the isolates from Madagascar and Mayotte. No clear association with visual symptoms in the cassava host could be identified.


Assuntos
Closteroviridae/classificação , Closteroviridae/genética , Manihot/virologia , Doenças das Plantas/virologia , África Central , Closteroviridae/isolamento & purificação , Closteroviridae/patogenicidade , Variação Genética , Genoma Viral , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Ilhas do Oceano Índico , Fases de Leitura Aberta , Filogenia , RNA Viral/genética
4.
Viruses ; 13(4)2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807294

RESUMO

Grapevine leafroll is one of the most widespread and highly destructive grapevine diseases that is responsible for great economic losses to the grape and wine industries throughout the world. Six distinct viruses have been implicated in this disease complex. They belong to three genera, all in the family Closteroviridae. For the sake of convenience, these viruses are named as grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, and -13). However, their etiological role in the disease has yet to be established. Furthermore, how infections with each GLRaV induce the characteristic disease symptoms remains unresolved. Here, we first provide a brief overview on each of these GLRaVs with a focus on genome structure, expression strategies and gene functions, where available. We then provide a review on the effects of GLRaV infection on the physiology, fruit quality, fruit chemical composition, and gene expression of grapevine based on the limited information so far reported in the literature. We outline key methodologies that have been used to study how GLRaV infections alter gene expression in the grapevine host at the transcriptomic level. Finally, we present a working model as an initial attempt to explain how infections with GLRaVs lead to the characteristic symptoms of grapevine leafroll disease: leaf discoloration and downward rolling. It is our hope that this review will serve as a starting point for grapevine virology and the related research community to tackle this vastly important and yet virtually uncharted territory in virus-host interactions involving woody and perennial fruit crops.


Assuntos
Closteroviridae/genética , Frutas/fisiologia , Expressão Gênica , Doenças das Plantas/virologia , Folhas de Planta/virologia , Vitis/fisiologia , Vitis/virologia , Closteroviridae/patogenicidade , Frutas/virologia , Genoma Viral , RNA Viral/genética , Análise de Sequência de DNA
5.
Virol J ; 17(1): 181, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208142

RESUMO

BACKGROUND: Virus disease is one of the main diseases in grapevine, and there has been no report on Plum bark necrosis and stem pitting-associated virus infecting grapevine in China. OBJECTIVE: The leaf samples of grapevine cultivar 'Cabernet Gernischt' were collected from Shandong province, which the leaves suffered from viral-like symptoms with spotting and crinkle. METHODS: Small RNA-seq combined with reverse transcription PCR (RT-PCR) were performed to detect the potential viruses in these field samples. Phylogenetic tree was constructed using the neighbor joining method in MEGA 5.1 CONCLUSIONS: This is the first report of PBNSPaV infecting grapevine in China, contributing to a better understanding of the epidemiology and host range distribution of this pathogen.


Assuntos
Closteroviridae/genética , Especificidade de Hospedeiro , Doenças das Plantas/virologia , Folhas de Planta/virologia , Prunus domestica/virologia , Vitis/virologia , China , Closteroviridae/classificação , Closteroviridae/patogenicidade , Genoma Viral , Filogenia , Casca de Planta/virologia , RNA Viral/genética
6.
Viruses ; 12(10)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-33007975

RESUMO

Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122-139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.


Assuntos
Closteroviridae/genética , Necrose/metabolismo , Nicotiana/virologia , Interferência de RNA/fisiologia , Agrobacterium/genética , Closteroviridae/patogenicidade , Necrose/virologia , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Potexvirus/genética , RNA Interferente Pequeno/metabolismo
7.
Arch Virol ; 165(11): 2653-2657, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32852617

RESUMO

A novel virus infecting yams (Dioscorea spp.), tentatively named "yam asymptomatic virus 1" (YaV1), was characterized and sequenced from an asymptomatic D. alata plant from Vanuatu. Sequence comparisons and phylogenetic analysis showed that YaV1 is a novel ampelovirus and has the smallest genome among "subgroup 1" members. RT-PCR-based screening of a yam germplasm collection conserved in Guadeloupe showed that YaV1 is prevalent in D. alata, D. bulbifera, D. cayennensis subsp. rotundata, D. esculenta and D. trifida accessions but causes no apparent symptoms. Additional phylogenetic analysis revealed a low variability of YaV1 in Guadeloupe in a limited part of the genome, and suggested the occurrence of plant-to-plant transmission.


Assuntos
Closteroviridae/classificação , Dioscorea/virologia , Filogenia , Doenças das Plantas/virologia , Closteroviridae/isolamento & purificação , Closteroviridae/patogenicidade , Variação Genética , Genoma Viral , Guadalupe , Prevalência , Reação em Cadeia da Polimerase em Tempo Real
8.
Viruses ; 12(8)2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32731601

RESUMO

Grapevine viruses are found throughout the viticultural world and have detrimental effects on vine productivity and grape and wine quality. This report provides a comprehensive and up-to-date review on grapevine viruses in Australia with a focus on "Shiraz Disease" (SD) and its two major associated viruses, grapevine virus A (GVA) and grapevine leafroll-associated virus 3 (GLRaV-3). Sensitive grapevine cultivars like Shiraz infected with GVA alone or with a co-infection of a leafroll virus, primarily GLRaV-3, show symptoms of SD leading to significant yield and quality reductions in Australia and in South Africa. Symptom descriptors for SD will be outlined and a phylogenetic tree will be presented indicating the SD-associated isolates of GVA in both countries belong to the same clade. Virus transmission, which occurs through infected propagation material, grafting, and naturally vectored by mealybugs and scale insects, will be discussed. Laboratory and field-based indexing will also be discussed along with management strategies including rogueing and replanting certified stock that decrease the incidence and spread of SD. Finally, we present several cases of SD incidence in South Australian vineyards and their effects on vine productivity. We conclude by offering strategies for virus detection and management that can be adopted by viticulturists. Novel technologies such as high throughput sequencing and remote sensing for virus detection will be outlined.


Assuntos
Closteroviridae/genética , Flexiviridae/genética , Filogenia , Doenças das Plantas/virologia , Animais , Austrália , Closteroviridae/classificação , Closteroviridae/patogenicidade , Efeito Citopatogênico Viral , Flexiviridae/classificação , Flexiviridae/patogenicidade , Insetos/virologia , África do Sul , Viroses/transmissão , Vitis/virologia , Vinho
9.
Biosensors (Basel) ; 8(4)2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445781

RESUMO

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the main pathogens of grapes, causing a significant loss in yield and decrease in quality for this agricultural plant. For efficient widespread control of this infection, rapid and simple analytical techniques of on-site testing are requested as a complementary addition for the currently applied hybridization (PCR) and immunoenzyme (ELISA) approaches. The given paper presents development and approbation of the immunochromatographic assay (ICA) for rapid detection of GLRaV-3. The ICA realizes a sandwich immunoassay format with the obtaining complexes ((antibody immobilized on immunochromatographic membrane)⁻(virus in the sample)⁻(antibody immobilized on gold nanoparticles (GNP)) during sample flow along the membrane compounds of the test strip. Three preparations of GNPs were compared for detection of GLRaV-3 at different dilutions of virus-containing sample. The GNPs with maximal average diameters of 51.0 ± 7.9 nm provide GLRaV-3 detection for its maximal dilutions, being 4 times more than when using GNPs with a diameter of 28.3 ± 3.3 nm, and 8 times more than when using GNPs with a diameter of 18.5 ± 3.3 nm. Test strips have been manufactured using the largest GNPs conjugated with anti-GLRaV-3 antibodies at a ratio of 1070:1. When testing samples containing other grape wine viruses, the test strips have not demonstrated staining in the test zone, which confirms the ICA specificity. The approbation of the manufactured test strips indicated that when using ELISA as a reference method, the developed ICA is characterized by a sensitivity of 100% and a specificity of 92%. If PCR is considered as a reference method, then the sensitivity of ICA is 93% and the specificity is 92%. The proposed ICA can be implemented in one stage without the use of any additional reactants or devices. The testing results can be obtained in 10 min and detected visually. It provides significant improvement in GLRaV-3 detection, and the presented approach can be transferred for the development of test systems for other grape wine pathogens.


Assuntos
Closteroviridae/patogenicidade , Imunoensaio/métodos
10.
Virology ; 523: 89-99, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30103103

RESUMO

Infectious cDNA clones were developed for Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus, family Closteroviridae). In vitro RNA transcripts generated from cDNA clones showed replication via the production of 3'-coterminal subgenomic (sg) mRNAs in Nicotiana benthamiana protoplasts. The detection of sgRNAs and the recovery of progeny recombinant virions from N. benthamiana leaves agroinfiltrated with full-length cDNA clones confirmed RNA replication and virion formation. The 5' non-translated region (5' NTR) of GLRaV-3 was exchangeable between genetic variants and complement the corresponding cognate RNA functions in trans. Mutational analysis of the 5' NTR in minireplicon cDNA clones showed that the conserved 40 nucleotides at the 5'-terminus were indispensable for replication, compared to downstream variable portion of the 5' NTR. Some of the functional mutations in the 5' NTR were tolerated in full-length cDNA clones and produced sgRNAs and virions in N. benthamiana leaves, whereas other mutations affected replication and virion formation.


Assuntos
Closteroviridae/genética , DNA Complementar/genética , Nicotiana/virologia , RNA Viral/genética , Vírion/genética , Vitis/virologia , Regiões 5' não Traduzidas , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Células Clonais , Closteroviridae/metabolismo , Closteroviridae/patogenicidade , DNA Complementar/metabolismo , Mutação , Doenças das Plantas/virologia , Folhas de Planta/virologia , Plantas Geneticamente Modificadas/virologia , Plasmídeos/química , Plasmídeos/metabolismo , Protoplastos/virologia , RNA Viral/metabolismo , Transformação Genética , Vírion/metabolismo , Vírion/patogenicidade , Replicação Viral
11.
Transgenic Res ; 27(4): 379-396, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29876789

RESUMO

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.


Assuntos
Quitinases/genética , Closteroviridae/genética , Plantas Geneticamente Modificadas/genética , Vitis/genética , Agrobacterium/genética , Botrytis/genética , Botrytis/patogenicidade , Closteroviridae/patogenicidade , DNA Bacteriano/genética , Resistência à Doença/genética , Epigênese Genética , Metarhizium/enzimologia , Metarhizium/genética , Metarhizium/virologia , Melhoramento Vegetal , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Solanum nigrum/genética , Vitis/crescimento & desenvolvimento , Vitis/virologia
12.
Physiol Plant ; 160(2): 171-184, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28044321

RESUMO

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 context of virus infection is an important topic that needs to be understood. In this work, we have focused our attention on determining the interaction of biotic and abiotic stresses on WUE and hydraulic conductance (Kh ) parameters in two white grapevine cultivars (Malvasia de Banyalbufar and Giró Ros). Under well-watered (WW) conditions, virus infection provokes a strong reduction (P < 0.001) in Kpetiole in both cultivars; however, Kleaf was only reduced in Malvasia de Banyalbufar. Moreover, the presence of virus also reduced whole-plant hydraulic conductance (Khplant ) in 2013 and 2014 for Malvasia de Banyalbufar and in 2014 for Giró Ros. Thus, the effect of virus infection on water flow might explain the imposed stomatal limitation. Under WS conditions, the virus effect on Kplant was negligible, because of the bigger effect of WS than virus infection. Whole-plant WUE (WUEWP ) was not affected by the presence of virus neither under WW nor under WS conditions, indicating that plants may adjust their physiology to counteract the virus infection by maintaining a tight stomatal control and by sustaining a balanced carbon change.


Assuntos
Vírus de Plantas/patogenicidade , Vitis/metabolismo , Vitis/virologia , Água/metabolismo , Closteroviridae/patogenicidade , Desidratação , Fotossíntese/fisiologia , Folhas de Planta/metabolismo , Folhas de Planta/virologia
13.
PLoS One ; 10(5): e0126819, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25992606

RESUMO

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 of all 13 available whole genomes of GLRaV-3 isolates and all other publicly available GLRaV-3 sequence data. Using multiple recombination detection methods we identified a clear signal for recombination in one whole genome sequence and further evidence for recombination in two more, including GH24. We inferred phylogenetic trees and networks and estimated the ages of common ancestors of GLRaV-3 clades by means of relaxed clock models calibrated with asynchronous sampling dates. Our results generally confirm previously identified variant groups as well as two new groups (VII and VIII). Higher order groups were defined as supergroups designated A to D. Supergroup A includes variant groups I-V and supergroup B group VI and its related unclassified isolates. Supergroups C and D are less well known, including the newly identified groups VII (including isolate GH24) and VIII respectively. The inferred node ages suggest that the origins of the major groups of GLRaV-3, including isolate GH24, may have occurred prior to worldwide cultivation of grapevines, whilst the current diversity represents closely related isolates that diverged from common ancestors within the last century.


Assuntos
Closteroviridae/genética , Doenças das Plantas/virologia , Vitis/virologia , Closteroviridae/classificação , Closteroviridae/patogenicidade , Evolução Molecular , Variação Genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Genéticos , Filogenia , Recombinação Genética
14.
Virus Genes ; 45(2): 333-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22714284

RESUMO

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 benthamiana. Differences in the suppressing activity were found among the variants assayed. Some constructs originated viral-like mosaic symptoms that evolved to necrosis. The intensity of these symptoms appeared to be related to the strength of the suppressor activity. A comparison of the protein sequences revealed a few amino acid substitutions that may be associated with the observed differences in the suppressing activity.


Assuntos
Closteroviridae/patogenicidade , Regulação Viral da Expressão Gênica , Interferência de RNA , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Closteroviridae/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Doenças das Plantas/virologia , Plantas Geneticamente Modificadas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Nicotiana/genética , Proteínas Virais/genética , Virulência , Fatores de Virulência/genética
15.
Phytopathology ; 98(10): 1093-8, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18943455

RESUMO

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 characteristics of GLRaV-3 transmission by the vine mealybug. Our results indicate that the vine mealybug transmits GLRaV-3 in a semipersistent manner. First instars were more efficient vectors than adult mealybugs. GLRaV-3 transmission lacked a latent period in the vector. Virus transmission occurred with a 1-h acquisition access period (AAP) and peaked with a 24-h AAP. Mealybugs inoculated GLRaV-3 with a 1-h inoculation access period (IAP), and transmission efficiency increased with longer plant access period up to 24 h, after which transmission rate remained constant. After an AAP of 24 h, mealybugs lost GLRaV-3 and infectivity 4 days after virus acquisition. In addition, GLRaV-3 was not transovarially transmitted from infected females to their progeny as detected by reverse transcription polymerase chain reaction. In summary, we systematically analyzed transmission parameters of GLRaV-3 by the vine mealybug and showed that transmission of this virus occurs in a semipersistent manner. This research fills in important gaps in knowledge of leafroll virus transmission, which is critical for development of leafroll disease management practices.


Assuntos
Closteroviridae/patogenicidade , Insetos/patogenicidade , Insetos/virologia , Doenças das Plantas/virologia , Vitis/parasitologia , Vitis/virologia , Animais , California , Transmissão de Doença Infecciosa/estatística & dados numéricos , Insetos/crescimento & desenvolvimento , Larva/patogenicidade , Fatores de Tempo
16.
J Virol Methods ; 141(1): 22-9, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17223203

RESUMO

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Grapevine Leafroll associated viruses 1-5 and 9 (GLRaV-1-5 and -9). The assays were evaluated against a wide range of geographically distributed isolates. Geographical locations included South Africa, Europe, Australia, Asia, Latin America and the United States. Sequences were piled up from the most conserved regions of these geographically diverse isolates and TaqMan primers and probes were designed, targeting the regions with 100% sequence identity. Improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control to validate the quality of the extracted RNA proved to generate better diagnostic assays. The real-time TaqMan RT-PCR assays were compared to the conventional RT-PCR assays for the detection of viruses using purified total RNA as well as crude extract. The data showed that when using total RNA extracted either by the Qiagen RNeasy method or by an ABI automated system more isolates were detected in comparison to crude extract. The optimum volume of crude extract prepared in GES for use in real-time TaqMan RT-PCR cocktail was determined to be 1 microl per reaction. In addition this report showed that TaqMan RT-PCR was more sensitive than conventional one-step RT-PCR for testing different isolates of these viruses either using RNA or crude tissue extract.


Assuntos
Closteroviridae/genética , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Taq Polimerase/metabolismo , Vitis/virologia , Closteroviridae/classificação , Closteroviridae/isolamento & purificação , Closteroviridae/patogenicidade , Genoma Viral , Doenças das Plantas/virologia , Plantas/virologia , RNA Viral/isolamento & purificação
17.
Virus Genes ; 31(3): 247-55, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16175330

RESUMO

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 named A and E (genetic divergence between A and E group reached 13.9%). An RT-PCR detection method followed by simple restriction analysis was developed, showing the potential to differentiate GLRaV-1 isolates of these groups. Interestingly, a mixed infection of two GLRaV-1 groups in the same plant was frequently detected together with a high intra-group variability in some isolates.


Assuntos
Closteroviridae/genética , Vitis/virologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Closteroviridae/isolamento & purificação , Closteroviridae/patogenicidade , República Tcheca , DNA Viral/genética , Genes Virais , Variação Genética , Proteínas de Choque Térmico HSP70/genética , Dados de Sequência Molecular , Filogenia , Doenças das Plantas/virologia , Polimorfismo de Fragmento de Restrição , Homologia de Sequência de Aminoácidos , Eslováquia , Proteínas Virais/genética
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