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High-throughput sequencing (HTS) was used to construct the virome profile of an old grapevine-leafroll-diseased grapevine (Vitis vinifera). De novo assembly of HTS data showed a complex infection, including a virus sequence with similarity to viruses of the genus Badnavirus, family Caulimoviridae. The complete genome sequence of this virus consists of 7090 nucleotides and has four open reading frames (ORFs). Genome organisation and phylogenetic analysis identify this virus as a divergent variant of grapevine Roditis leaf discoloration-associated virus (GRLDaV) with 90% nucleotide sequence identity to isolate w4 (NC_027131). This is the first genome sequence of a South African variant of GRLDaV. [ABSTRACT FROM AUTHOR]

To characterize their virome, double stranded RNAs extracted from scrapings of two Iranian grapevine varieties held in the Vassal-Montpellier Grapevine Biological Resources Center were analysed by high-throughput sequencing. In addition to several well-known grapevine viruses, divergent isolates of the newly described grapevine Kizil Sapak virus were identified in both accessions. Four complete genome sequences were determined, as well as two additional partial sequences (1,580 and 3,849 nucleotides long). These genomic sequences highlight the molecular diversity of this poorly known virus. In view of the absence of amplification of the GKSV isolates characterized here using the published primer pair, novel degenerate, polyvalent primers were designed, providing a more robust diagnosis. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically significant virus of grapevines, with secondary spread mediated by several species of mealybug and soft scale insects. To better understand virus-vector interactions, sensitive virus detection in these insects is a key tool. In this research, two new hydrolysis-probe-based real-time assays for GLRaV-3 detection were developed and compared to three existing assays. Of the five assays compared, the one-step RT-qPCR probe-based assay was the most sensitive and reliable, with as few as 10 virus RNA copies detected. This is the first description of a real-time molecular assay for virus detection in mealybugs with such sensitivity. [ABSTRACT FROM AUTHOR]

Grapevine red blotch virus (GRBV) is an emerging virus of significant viticultural importance throughout North America. Here, we report the development of a simple protocol for point-of-use detection of GRBV. Extraction of nucleic acids is not required; instead, the whole intact plant can simply be pricked with a sterile pipette tip, which is then incubated in sterile distilled water to provide the sample template in a loop-mediated isothermal amplification (LAMP) reaction. This method is 10,000 times more sensitive than conventional PCR, costs under a dollar per sample, and can be completed from sampling to readout in just over half an hour. [ABSTRACT FROM AUTHOR]

P70 is a Pinot Noir grapevine accession that displays strong leafroll disease symptoms. A high-throughput sequencing (HTS)-based analysis established that P70 was mixed-infected by two variants of grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus) and one of grapevine virus A (GVA, genus Vitivirus) as well as by two viroids (hop stunt viroid [HSVd] and grapevine yellow speckle viroid 1 [GYSVd1]) and four variants of grapevine rupestris stem pitting-associated virus (GRSPaV). Immunogold labelling using gold particles of two different diameters revealed the existence of ‘hybrid’ particles labelled at one end as GLRaV-1, with the rest labelled as GVA. In this work, we suggest that immunogold labelling can provide information about the biology of the viruses, going deeper than just genomic information provided by HTS, from which no recombinant or ‘chimeric’ GLRaV-1/GVA sequences had been identified in the dataset. Our observations suggest an unknown interaction between members of two different viral species that are often encountered together in a single grapevine, highlighting potential consequences in the vector biology and epidemiology of leafroll and rugose-wood diseases. [ABSTRACT FROM AUTHOR]

The impact of recombination on variant classification and the use of different genomic regions to identify virus variants were investigated using a diversity study performed on grapevine rupestris stem pitting-associated virus (GRSPaV). Three surveys were conducted to investigate the genetic diversity of GRSPaV and to compare the ability of the GRSPaV coat protein and replicase domains to classify virus variants. GRSPaV variants identified in the surveys clustered into five of the six currently recognised lineages, and a seventh, previously unclassified lineage was detected. A correlation was observed between the detection of recombinant GRSPaV sequences and inconsistencies in classification when using different genome regions for analysis. [ABSTRACT FROM AUTHOR]

A novel virus, with characteristics of viruses classified within the genus Vitivirus, was identified from a sample of Vitis vinifera cv. Chardonnay in New Zealand. The virus was detected with high throughput sequencing (small RNA and total RNA) and its sequence was confirmed by Sanger sequencing. Its genome is 7507 nt long (excluding the polyA tail) with an organisation similar to that described for other classifiable members of the genus Vitivirus. The closest relative of the virus is grapevine virus E (GVE) with 65% aa identity in ORF1 (65% nt identity) and 63% aa identity in the coat protein (66% nt identity). The relationship with GVE was confirmed with phylogenetic analysis, showing the new virus branching with GVE, Agave tequilina leaf virus and grapevine virus G (GVG). A limited survey revealed the presence of this virus in multiple plants from the same location where the newly described GVG was discovered, and in most cases both viruses were detected as co-infections. The genetic characteristics of this virus suggest it represents an isolate of a new species within the genus Vitivirus and following the current nomenclature, we propose the name “Grapevine virus I”. [ABSTRACT FROM AUTHOR]

During screening of non-cultivated (wild) grapevine ( Vitis sp.) from Napa County, California for the grapevine red blotch virus (GRBV; genus Glabrovirus, family Geminiviridae), an atypical polymerase chain reaction product pattern was observed. Rolling circle amplification followed by cloning and sequencing revealed the presence of a circular DNA characteristic of geminiviruses. The complete genome of nine isolates of the virus ranged from 3204 to 3278 nt in size. The genome most closely resembled that of GRBV in both sequence (57 to 59% identity) and organization. With limited sequence identity to described geminiviruses, this virus warrants designation as a new species, and the name ' Wild Vitis virus 1' is proposed. [ABSTRACT FROM AUTHOR]

This work describes the first molecular characterization of grapevine virus B (GVB) in Portuguese grapevine cultivars. During a routine screening of 44 accessions in the National Collection of Grapevine Varieties (CAN PRT051), 17 were found infected with GVB in DAS-ELISA assays with commercial antibodies. However, only six of the corresponding isolates were successfully amplified using primer pairs described in the literature. The sequence variants (ORF4-3'UTR, 1147 nt) retrieved from these isolates segregated into two phylogenetic groups, which included sequences from complete genomes available in GenBank. The highly discrepant results obtained using serological and RT-PCR-based diagnostic tools led to the design of a primer pair for detection of GVB, which allowed the amplification of a 606-bp GVB-specific fragment from all DAS-ELISA-positive isolates and also revealed the existence of false negatives in the serological testing. [ABSTRACT FROM AUTHOR]

As part of a grapevine metagenome study, total RNA extracted from grapevine phloem scrapings was analyzed by Illumina sequencing. For one 420A rootstock sample, reads mapping against a reference database and BLAST annotation of contigs identified the presence of a divergent isolate of Botrytis virus F (BVF). The full genome sequence of this isolate (IVC-5-77) was determined (6,828 nucleotides [nt], excluding the poly(A) tail) and shown to be collinear with that of the BVF reference isolate, with the two open reading frames encoding a replication-associated protein (REP) and a coat protein (CP). The IVC-5-77 isolate, however, is very divergent, showing only 81.3-81.6% nucleotide sequence identity to the two other sequenced BVF isolates. The internal non-coding region was also found to be highly variable between BVF isolates. Analysis of the RNASeq reads demonstrated that close to 20% of them belong to Botrytis cinerea, the putative host of the IVC-5-77 isolate. These results extend our knowledge of the diversity and variability of BVF and demonstrate its detectability, together with that of its B. cinerea host, in total RNA RNASeq data from grapevine phloem scrapings. [ABSTRACT FROM AUTHOR]

This paper describes the nucleotide sequence and genome organization of a novel RNA virus detected in grapevine (Vitis vinifera) cultivar ‘Kizil Sapak’ by high-throughput sequencing (HTS) and tentatively named “grapevine virus J” (GVJ). The full genome of GVJ is 7,390 nucleotides in length, which comprises five open reading frames (ORFs), including a 20K ORF (ORF 2) between the replicase (ORF 1) and the movement protein (ORF 3) genes. According to the level of sequence homology and phylogenetics, GVJ is proposed as a new member of the genus Vitivirus (subfamily Trivirinae; family Betaflexiviridae), with the closest characterized virus being grapevine virus D (GVD). [ABSTRACT FROM AUTHOR]

A new genetic variant of grapevine virus A (GVA) of phylogenetic group I was identified during comparative analysis of the viruses infecting two sibling grapevines cv. Shiraz. The grapevines were propagated from a single mother plant. One of them become infected with Shiraz disease (SD), which is highly destructive on noble grapevine cultivars Shiraz and Merlot in South Africa. The new variant was not associated with SD, as it was present in both SD-affected and SD-free plants. However, unlike in an earlier study of grapevines affected by this disease, this GVA variant of group I strongly dominated over a coinfecting variant of group II associated with SD and a variant of group III. The variant, named I327-5, was mechanically transmitted from SD-affected grapevine to Nicotiana benthamiana, and its genome was fully sequenced. The sequence data revealed that the most distinctive genomic feature of variant I327-5 is the deletion of three nucleotides in the region where the ORF2 and ORF3 genes overlap. These genes of GVA encode a 19.8-kDa protein, the function of which remains unknown, and a 31-kDa protein that is indispensable for the movement of the virus in plants. An alignment of the amino acid sequences of these proteins encoded by variant I327-5 with the corresponding proteins encoded by other members of group I suggested that, as the result of mutations, a neutral threonine or alanine and a negatively charged glutamic acid, respectively, were removed from the proteins of GVA variant I327-5. [ABSTRACT FROM AUTHOR]

A number of vitiviruses infect grapevine, arguably the most important fruit crop, and the host from which they derive their genus name. In contrast to most grapevine viruses, the etiological role of these viruses is unclear, albeit that they are associated with several well-known - and a number of emerging - diseases of grapevine. Here, we review the genus Vitivirus, with special reference to its most omnipresent member, grapevine virus A. We discuss the latest taxonomic status of the genus, as well as the genome and genomic organisation, replication mechanism, and genetic variability of GVA, and we also present the latest research progress with vitivirus-based vectors; the identification of a new vitivirus, GVE, the discovery of VIGG, a unique GVA-induced host protein, the molecular characterisation of hitherto unknown or puzzling genetic elements in the GVA genome, and the latest developments in vitivirus diagnostics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article offers information on the molecular characteristics of a newly discovered specie of grapevine viroid. According to the authors, there are five species of such virus, in which, infections on plants include cultivated grapevines. The authors focused on the Chinese grapevine viroid, which was a result of nucleotide sequence, phylogenetic analysis and molecular characterization.

New virus-like sequences, TvAQ7 and TvP15, were found in a Japanese grapevine accession of OKY-AQ7 (cv. Aki Queen) and of OKY-P15 (cv. Pione) by nested RT-PCR to simultaneously amplify a segment of the RNA-dependent RNA polymerase (RdRp) gene from members of the genera Vitivirus and Foveavirus. The TvAQ7 genome, except for an exact 5′ terminus, is composed of about 7.6 kb containing five potential open reading frames. The genomic organization resembles those of grapevine virus A and other known vitiviruses. The 199-amino-acid sequence deduced from the ORF4 of TvAQ7 has 38.5–54.0% identity with the coat protein (CP) of known grapevine vitiviruses and 86.9% identity with TvP15. Phylogenetic analysis based on amino acid sequences of the CP showed that TvAQ7 and TvP15 were closely related to the vitiviruses. In addition, we confirmed that TvAQ7 and TvP15 were transmitted from infected grapevine to healthy seedlings by the mealybug Pseudococcus comstocki Kuwanae. On the basis of our findings, TvAQ7 and TvP15 should be considered isolates of a new species of the genus Vitivirus, and both isolates are probably genetic variants of the new species. We propose to name this virus grapevine virus E (GVE). [ABSTRACT FROM AUTHOR]

The naturally occurring Grapevine fanleaf virus (GFLV) recombinant isolate A17b was recovered from its grapevine host by sap inoculation and serial passages onto Gomphrena globosa, a pseudo local lesion herbaceous host, and Chenopodium quinoa, a systemic herbaceous host, to characterize some of its biological properties. Sequence analysis of the CP gene, in which a recombinational event was previously detected, demonstrated the genetic stability of recombinant isolate A17b over a 5-year period in its natural host as well as in C. quinoa. Also, recombinant isolate A17b was graft transmissible, as shown by an in vitro heterologous approach, and transmitted by the nematode Xiphinema index as readily as nonrecombinant GFLV isolates. Furthermore, despite a lower pathogenicity on Chenopodium amaranticolor, recombinant isolate A17b had a similar host range and induced similar symptoms in type and severity to nonrecombinant GFLV isolates. Interestingly, the use of infectious chimeric RNA2 transcripts in combination to RNA1 transcripts of GFLV strain F13 suggested no implication of the recombination event in the CP gene of isolate A17b in the reduced pathogenicity on C. amaranticolor. Altogether, recombinant isolate A17b had similar biological properties to GFLV nonrecombinant isolates. [ABSTRACT FROM AUTHOR]

Summary.Maculavirus is a new genus of plant viruses typified by Grapevine fleck virus (GFkV). A possible second member is Grapevine redglobe virus (GRGV). Maculaviruses are phloem-limited non-mechanically transmissible viruses with isometric particles c. 30 nm in diameter that have a rounded contour and prominent surface structure. Vectors, if any, are unknown. GFkV preparations contain two centrifugal components, T made up of empty protein shells and B, which contains 35% RNA. The coat protein (CP) has a molecular mass of 24 kDa. The genome is a single-stranded RNA that has c. 50% cytosine residues. It is 7564 nt in size, excluding the poly(A) tail and contains four putative open reading frames (ORF) that encode a 215.4 kDa polypeptide with the conserved motifs of replication-associated proteins of positive-strand RNA viruses (ORF1), the CP (ORF2), and one (GRGV) or two (GFkV) proline-rich polyproteins of 31.4 kDa (ORF3) and 15.9 kDa (ORF4), respectively, with unknown function. Replication-associated proteins and CP are phylogenetically related to those of members of the genera Tymovirus and Marafivirus. GFkV-infected grapevine cells contain vesiculated mitochondria, the possible site of RNA replication. In the natural host, GFkV particles accumulate in great quantity, sometimes in crystalline arrays in phloem cells. [ABSTRACT FROM AUTHOR]

Summary. Two sets of degenerate primers for the specific amplification of 572–575 nt and 386 nt segments of the methyltransferase and RNA-dependent RNA polymerase cistrons of members of the genera Tymovirus and Marafivirus and of the unassigned virus Grapevine fleck virus (GFkV) were designed on the basis of available sequences. These primers were used for amplifying and subsequent cloning and sequencing part of the open reading frame 1 of the genome of GFkV, Grapevine asteroid mosaic-associated virus (GAMaV) and of another previously unreported virus, for which the name Grapevine red globe virus (GRGV) is proposed. Computer-assisted analysis of the amplified genome portions showed that the three grapevine viruses are phylogenetically related with one another and with sequenced tymoviruses and marafiviruses. The relationships with tymoviruses was confirmed by the type of ultrastructural modifications induced in the host cells. RdRp-specific degenerate primers were successfully used for the aspecific detection of the three viruses in crude grapevine sap extracts. Specific virus identification was obtained with RT-PCR using antisense virus-specific primers. [ABSTRACT FROM AUTHOR]

The complete genome sequence of a South African isolate of grapevine virus F (GVF) is presented. It was first detected by metagenomic next-generation sequencing of field samples and validated through direct Sanger sequencing. The genome sequence of GVF isolate V5 consists of 7539 nucleotides and contains a poly(A) tail. It has a typical vitivirus genome arrangement that comprises five open reading frames (ORFs), which share only 88.96 % nucleotide sequence identity with the existing complete GVF genome sequence (JX105428). [ABSTRACT FROM AUTHOR]

The article focuses on the complete genome sequencing of the Arabis mosaic virus. It states that the virus possesses polyhedral virions and that its vector is Xiphinema diversicatum. It mentions that the virus's genome is similar to that of the grapevine fanleaf virus, and that two protein domains corresponded with domains in the tomato ringspot virus.