Partial sequence analysis of a Grapevine leafroll-associated virus 3 isolate from Slovakia.
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Genetic Variability
Slovak
【Objective】The objective of the study is to develop a sensitive detection technique on grapevine viruses and understand the phylogenetic relationships of Sichuan isolates with other geographic origins.【Method】Comparison with three different RNA extraction protocols,the Reverse Transcription-Polymerase Chain Reaction(RT-PCR) were optimized to detect three different grapevine viruses.Partial genomes of three different viruses isolated from Sichuan were cloned and the phylogenetic studies of each isolate were conducted,respectively.【Result】The results indicated high quality RNA was obtained quickly from the mature phloem of shoots by the modified silica particles extraction only.RT-PCR was successfully used to amplify partial fragments of the GLRaV-2,GVB and GVA with 18S RNA of grapevine as internal control,respectively.In addition,phylogenetic analysis and nucleotide identities studies clearly clustered the 16 CP genes of GLRaV-2 isolates collected from different geographic origins into five phylogenetic groups.The nucleotide identities of GVB CP genes between 7 isolates of distinct geographic origins were calculated to 79.1%-98.7%.Five isolates of GVA from Sichuan were clustered into 4 clades based on the phylogenetic structure,so it was suggested that the virus populations of GVA from Sichuan consisted of genetic diversity of variants.【Conclusion】The studies suggested that GLRaV-2,GVA and GVB had genetic variability at level of nucleotide acids.Furthermore,GVA populations in Sichuan had significant genetic diversity and included 4 different strains.
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The complete genome sequence of a highly divergent strain of Grapevine leafroll-associated virus 4 (GLRaV-4) was determined using 454 pyrosequencing technology. This virus, designated GLRaV-4 Ob, was detected in Vitis vinifera ‘Otcha bala’ from our grapevine virus collection at Agroscope. The GLRaV-4 Ob genome length and organization share similarities with members of subgroup II in the genus Ampelovirus (family Closteroviridae). Otcha bala was graft-inoculated onto indicator plants of cultivar Gamay to evaluate the biological properties of this new strain, and typical leafroll symptoms were induced. A monoclonal antibody for the rapid detection of GLRaV-4 Ob by enzyme-linked immunosorbent assay is available, thus facilitating large-scale diagnostics of this virus. Based on the relatively small size of the coat protein, the reduced amino acid identity and the distinct serological properties, our study clearly shows that GLRaV-4 Ob is a divergent strain of GLRaV-4. Furthermore, molecular and serological data revealed that the AA42 accession from which GLRaV-7 was originally reported is in fact co-infected with GLRaV-4 Ob and GLRaV-7. This finding challenges the idea that GLRaV-7 is a leafroll-causing agent.
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Nepovirus
Vineyard
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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 confi rmed by DAS-ELISA in 2010.Th e 3'fi nal 1769 nucleotides of the Slovenian GLRaV-4 isolate were assembled from amplicons obtained by IC RT-PCR.Th e complete coat protein (CP) and p23 gene sequences were compared with other GLRaV-4 sequences from GenBank.Results showed that CP and p23 amino acid sequences of Slovenian variant (055-SI) are 88% and 85%, respectively, identical to corresponding genes of reference sequence GLRaV-4 LR106 (GenBank Acc.No. FJ467503).Phylogenetic analyses show that Slovenian variant clusters together with other corresponding strains of GLRaV-4.Th e sequencing results show great variability of the N-terminal part of the CP sequence indicating that this part of the genome is not suitable for molecular detection of the virus.To our knowledge this is also the fi rst report of GLRaV-4 in Slovenian vineyards.
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At least 58 viruses have been reported to infect grapevines, causing economic damage globally. Our lab has reported previously the presence of more than 10 viral species in Chilean grapevines (2,3). Grapevine Syrah virus-1 (GSyV-1) is a novel marafivirus recently described in California vineyards (1). Grapevine virus Q (GVQ) was described shortly after GSyV-1 and both genomes share more than 99% nucleotide identity (4). Since GSyV-1 and GVQ correspond to the same viral species, the name GSyV-1 will be used in the current note to avoid confusion. Forty dormant cane samples from 12 different cultivars were collected from different regions of Chile and screened by reverse transcription-PCR. One of the 40 samples (cv. Syrah) collected from the VI region of Chile was found to be infected with GSyV-1 using two different pairs of GSyV-1-specific primers. The first pair of primers GSyV-1Det-F: 5'-CAAGCCATCCGTGCATCTGG -3' and GSyV-1Det-R: 5'-GCCGATTTGGAACCCGATGG -3' (1), was used to amplify a 297-bp fragment corresponding to a partial region of the putative methyltransferase gene. The sequence (GenBank Accession No. GU566025) shared 87% nucleotide and 100% amino acid identities with the corresponding fragment of a Californian GSyV-1 isolate (GenBank Accession No. FJ436028). Since there are no commercial antibodies available for GSyV-1 detection, a second pair of primers, GVQCP-F: 5'-TCCCAGCTTCAGGGTGAATT -3' and GVQCP-R: 5'-GCATTGCTGCGCATTGGAGG -3' (4), that amplified a 720-bp fragment of the putative coat protein gene was also used. The sequence of 720 bp from the Chilean sample (GenBank Accession No. GU566024) shared 92% nucleotide and 98% amino acid identities with the corresponding fragment of a Californian GSyV-1 isolate (GenBank Accession No. FJ436028). The GSyV-1-positive sample was also infected with Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus that have been reported previously in Chile. To our knowledge, this is the first report of GSyV-1 in Chile. Further studies will help to establish the incidence and effects of this virus in Chilean grapevines. References: (1) M. Al Rwahnih et al. Virology 387:395, 2009. (2) E. Engel et al. J. Virol. Methods. 163:445, 2010. (3) P. F. Escobar et al. Plant Dis. 92:1474, 2008. (4) S. Sabanadzovic et al. Virology 394:1, 2009.
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The sanitary status of grapevines has not yet been considered sufficiently in vineyards throughout Bosnia and Herzegovina (BiH). An extensive survey of five major grapevine viruses in the country was carried out in 2019. A total of 630 samples from the two dominant autochthonous cultivars, named Žilavka and Blatina, were tested by DAS-ELISA for the presence of grapevine leafroll-associated viruses (GLRaV-1 and 3), grapevine fleck virus (GFkV), grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV). Eighty-eight % of the samples were positive for at least one virus, and all five viruses were detected, thought with different incidence, i.e. GLRaV-3 (84%), GFLV (43%), GLRaV-1 (14%), GFkV (10%) and ArMV (0.2%). The majority of infected plants (about 75%) were asymptomatic. Specific virus symptoms were observed in the remaining infected plants, together with the reported GLRaV vectors, Planococcus ficus and Parthenolecanium corni , while nematodes of the Xiphinema genus were not found in the GFLV- or ArMV-infected vineyards. The GLRaV-3 CP phylogenetic analyses showed 75–100% nucleotide identity between the BiH and reference isolates, and the BiH isolates clustered into the major group. The dNS/dS ratio indicated a negative selection of the virus population, and the lack of geographical structuring within the population was observed. In addition, putative GLRaV-3 recombinants with breakpoints in the 5’ of the CP gene were detected, while no recombinant strains were identified for the other four viruses. The obtained results indicate a deteriorated sanitary status of the cultivated grapevines, the prevalence and intraspecies genetic diversity of GLRaV-3 throughout the country. The establishment of certified grapevine material and adequate virus vector control is therefore of primary importance to prevent further spread of these viruses. This study presents the results of the first molecular characterisation of grapevine viruses in Bosnia and Herzegovina.
Xiphinema
Nepovirus
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Abstract Background Grapevine leafroll disease is one of the most economically important viral diseases affecting grape production worldwide. Grapevine leafroll-associated virus 4 (GLRaV-4, genus Ampelovirus , family Closteroviridae ) is one of the six GLRaV species documented in grapevines ( Vitis spp.). GLRaV-4 is made up of several distinct strains that were previously considered as putative species. Currently known strains of GLRaV-4 stand apart from other GLRaV species in lacking the minor coat protein. Methods In this study, the complete genome sequence of three strains of GLRaV-4 from Washington State vineyards was determined using a combination of high-throughput sequencing, Sanger sequencing and RACE. The genome sequence of these three strains was compared with corresponding sequences of GLRaV-4 strains reported from other grapevine-growing regions. Phylogenetic analysis and SimPlot and Recombination Detection Program (RDP) were used to identify putative recombination events among GLRaV-4 strains. Results The genome size of GLRaV-4 strain 4 (isolate WAMR-4), strain 5 (isolate WASB-5) and strain 9 (isolate WALA-9) from Washington State vineyards was determined to be 13,824 nucleotides (nt), 13,820 nt, and 13,850 nt, respectively. Multiple sequence alignments showed that a 11-nt sequence (5′-GTAATCTTTTG-3′) towards 5′ terminus of the 5′ non-translated region (NTR) and a 10-nt sequence (5′-ATCCAGGACC-3′) towards 3′ end of the 3′ NTR are conserved among the currently known GLRaV-4 strains. LR-106 isolate of strain 4 and Estellat isolate of strain 6 were identified as recombinants due to putative recombination events involving divergent sequences in the ORF1a from strain 5 and strain Pr. Conclusion Genome-wide analyses showed for the first time that recombinantion can occur between distinct strains of GLRaV-4 resulting in the emergence of genetically stable and biologically successful chimeric viruses. Although the origin of recombinant strains of GLRaV-4 remains elusive, intra-species recombination could be playing an important role in shaping genetic diversity and evolution of the virus and modulating the biology and epidemiology of GLRaV-4 strains.
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Molecular reagents have been developed for virus-specific and simultaneous (virus-non-specific) detection of Grapevine fleck virus (GFkV) and allied viruses, ie. Grapevine asteroid mosaic-associated virus (GAMaV) and Grapevine red globe virus (GRGV). Degenerate primers designed on nucleotide sequences of the RNA-dependent RNA polymerase (RD) and methyltransferase (MTR) domains of the GFkV genome, were able to give amplification products of the expected size from total nucleic acid extracts of: vines infected with GFkV, GAMaV, and GRGV; a Californian grapevine accession infected by a marafi-like virus; Greek grapevine accessions infected by an unidentified agent that induced symptoms reminiscent of those elicited by GAMaV in Vitis rupestris . Degenerate primers designed on the nucleotide sequence of the helicase (HEL) domain of the GFLV genome recognized all the above viruses except for GAMaV and the unidentified Greek viral agent. RD primer set worked well also with crude grapevine cortical scrapings, thus constituting a useful universal reagent for the non-specific molecular identification of GFkV-like viruses in Vitis . The marafi-like virus from California was amplified by all sets of primers, but was recognized only by the GRGV-specific probe, suggesting that it is a likely isolate of GRGV: Likewise, the unidentified virus from Greek vines shared sequence homology with GFkV and allied viruses (GAMaV and GRGV) but exhibited differences relevant enough that call for further investigations to establish its taxonomic position. While GRGV was identified, though with a very low incidence, in some 11 southern Italian grapevine cultivars, no evidence was obtained for infection by GAMaV in any of 50 cultivars analyzed.
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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 diverged copy of the GLRaV-1 coat protein gene or GLRaV-3 heat-shock protein 70-homologue gene. Virus-specific RT-PCR products were amplified from immature insect vectors and adult mealybugs. Single viral amplicons were obtained mostly from immature vectors (35%, 30 of 85) and dual viral amplicons from immature (16%, 10 of 61) and adult (100%, 14 of 14) mealybugs, including individuals. These observations suggested a simultaneous uptake of GLRaV-1 and GLRaV-3 by individual mealybugs. Furthermore, a comparative nucleotide sequence analysis of viral amplicons from soft scales, mealybugs, and grapevines from which vectors were collected showed identical or highly similar haplotypes, indicating that uptake of GLRaV-1 and GLRaV-3 likely occurred by direct feeding of vectors on their host plants.
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Co-infection of Grapevine leafroll associated virus-3(GLRaV-3)and Grapevine virus A(GVA)was a common phenomenon on some cultivars of Vitis vinifera L.× Vitis labrusca L.in Sichuan Province.Semi-quantitative RT-PCR and qRT-PCR were used to investigate the relative accumulations of the aforementioned two viruses in different tissues of infected grape trees(Vitis vinifera L.× V.labrusca L.‘Himrod’).The results suggested the relative accumulations of viruses in petiole and phloem were higher than that of others tissues.The multiple RT-PCR was developed to amplify the target genes of GLRaV-3 and GVA in phloem tissues of mature shoots(Vitis vinifera L.× V.labrusca L.‘Fujiminori’) simultaneously.The full length of coat protein gene(CP)of GLRaV-3 and partial genome region of GVA(including partial sequence of CP and full length of viral suppressor of RNAsilencing gene,VSR)were cloned from V.vinifera L.× V.labrusca L.‘Fujiminori’separately.The nucleotide homology analysis and phylogenetic studies on different isolates of each virus were possibly revealed that CP from different GLRaV-3 isolates was highly conserved,whereas GVA Sichuan isolate contained very divergent variants and thus it could be quasispecies.
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