Genomic and biological analysis of Grapevine leafroll-associated virus 7 reveals a possible new genus within the family Closteroviridae.
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ABSTRACT:
SUBMITTER: Al Rwahnih M
PROVIDER: S-EPMC5898237 | biostudies-literature | 2012 Jan
REPOSITORIES: biostudies-literature
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Genomic and biological analysis of Grapevine leafroll-associated virus 7 reveals a possible new genus within the family Closteroviridae.
Al Rwahnih Maher M Dolja Valerian V VV Daubert Steve S Koonin Eugene V EV Rowhani Adib A
Virus research 20111025 1
Deep sequencing analysis of an asymptomatic grapevine revealed a virome containing five RNA viruses and a viroid. Of these, Grapevine leafroll-associated virus 7 (GLRaV-7), an unassigned closterovirus, was by far the most prominently represented sequence in the analysis. Graft-inoculation of the infection to another grape variety confirmed the lack of the leafroll disease symptoms, even though GLRaV-7 could be detected in the inoculated indicator plants. A 16,496 nucleotide-long genomic sequence ...[more]
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Project description: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 type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.
| S-EPMC3627144 | biostudies-literature
Project description:BackgroundThe 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.ResultsThe gene expression and cis-acting elements of Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus) was examined and compared to that of other members of the family Closteroviridae. Six putative 3'-coterminal subgenomic (sg) RNAs were abundantly present in grapevine (Vitis vinifera) infected with GLRaV-3. The sgRNAs for coat protein (CP), p21, p20A and p20B were confirmed using gene-specific riboprobes in Northern blot analysis. The 5'-termini of sgRNAs specific to CP, p21, p20A and p20B were mapped in the 18,498 nucleotide (nt) virus genome and their leader sequences determined to be 48, 23, 95 and 125 nt, respectively. No conserved motifs were found around the transcription start site or in the leader sequence of these sgRNAs. The predicted secondary structure analysis of sequences around the start site failed to reveal any conserved motifs among the four sgRNAs. The GLRaV-3 isolate from Washington had a 737 nt long 5' nontranslated region (NTR) with a tandem repeat of 65 nt sequence and differed in sequence and predicted secondary structure with a South Africa isolate. Comparison of the dissimilar sequences of the 5'NTRs did not reveal any common predicted structures. The 3'NTR was shorter and more conserved. The lack of similarity among the cis-acting elements of the diverse viruses in the family Closteroviridae is another measure of the complexity of their evolution.ConclusionsThe results indicate that transcription regulation of GLRaV-3 sgRNAs appears to be different from members of the genus Closterovirus. An analysis of the genome sequence confirmed that GLRaV-3 has an unusually long 5'NTR of 737 nt compared to other monopartite members of the family Closteroviridae, with distinct differences in the sequence and predicted secondary structure when compared to the corresponding region of the GLRaV-3 isolate from South Africa.
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Project description:A putative new ampelovirus was detected in Vitis vinifera cv. Carnelian showing mild leafroll symptoms and molecularly characterized. The complete genome consisted of 13,625 nt and had a structure similar to that of members of subgroup I in the genus Ampelovirus (fam. Closteroviridae). In-depth analyses showed that the virus from cv. Carnelian is the most distinct member of the "GLRaV-4 lineage" of ampeloviruses, which comprises GLRaV-4, -5, -6, -9, and the recently characterized GLRaV-Pr, and GLRaV-De. This virus appears to be a new member of the family Closteroviridae, for which the provisional name grapevine leafroll-associated Carnelian virus is proposed.
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Project description: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 & 3), Grapevine virus A (GVA), Grapevine fan leaf virus (GFLV), Grapevine fleck virus (GFkV) and successfully detected GLRaV-1 & 3 and GFkV. All these samples were complemented with RT- PCR along with GVb and phytoplasma (additional to ELISA) using specific primers. Specific amplification in RT-PCR for GLRaV-1 (~232 bp), GLRaV-3 (~300 bp), GFkV (~179 bp) and GVB (~440 bp) confirmed the presence of these pathogens. Overall, ELISA and RT-PCR results confirmed the presence GLRaV-3 (66.7 %), GLRaV-1& GFkV (50 %), and Grapevine virus B (GVB) (12.5 %) in symptomatic plants. None of the samples were found positive for GFLV, GLRaV-2 and phytoplasma. Mixed infection was common and none of the plants were found virus free. To the best of our knowledge this is the first report of detection of GFkV and GVB in India.
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Project description: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.
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Project description:Thioalkalivibrio is a genus of obligate chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria. Their habitat are soda lakes which are dual extreme environments with a pH range from 9.5 to 11 and salt concentrations up to saturation. More than 100 strains of this genus have been isolated from various soda lakes all over the world, but only ten species have been effectively described yet. Therefore, the assignment of the remaining strains to either existing or novel species is important and will further elucidate their genomic diversity as well as give a better general understanding of this genus. Recently, the genomes of 76 Thioalkalivibrio strains were sequenced. On these, we applied different methods including (i) 16S rRNA gene sequence analysis, (ii) Multilocus Sequence Analysis (MLSA) based on eight housekeeping genes, (iii) Average Nucleotide Identity based on BLAST (ANIb) and MUMmer (ANIm), (iv) Tetranucleotide frequency correlation coefficients (TETRA), (v) digital DNA:DNA hybridization (dDDH) as well as (vi) nucleotide- and amino acid-based Genome BLAST Distance Phylogeny (GBDP) analyses. We detected a high genomic diversity by revealing 15 new "genomic" species and 16 new "genomic" subspecies in addition to the ten already described species. Phylogenetic and phylogenomic analyses showed that the genus is not monophyletic, because four strains were clearly separated from the other Thioalkalivibrio by type strains from other genera. Therefore, it is recommended to classify the latter group as a novel genus. The biogeographic distribution of Thioalkalivibrio suggested that the different "genomic" species can be classified as candidate disjunct or candidate endemic species. This study is a detailed genome-based classification and identification of members within the genus Thioalkalivibrio. However, future phenotypical and chemotaxonomical studies will be needed for a full species description of this genus.
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