Variants Of Grapevine Leafroll-associated Virus Research Articles

Real-time RT-PCR high-resolution melting curve analysis to detect and differentiate Brazilian variants of grapevine viruses

Detecting and identifying viral infections in perennial plants, such as grapevines, can be challenging. Therefore, the aim of this study was to perform a real-time RT-PCR (RT-qPCR) high-resolution melting (HRM) curve analysis to detect and differentiate Brazilian variants of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine fanleaf virus (GFLV) in 74 and 10 infected plants, respectively, maintained in a collection block of grapevines. A single amplification curve was generated for each sample by RT-qPCR. Considering the amplified region of genomes of these two viruses, it was possible to identify and distinguish different variants of GLRaV-3 and of GFLV, which showed significantly different melting temperature (Tm) values between themselves, reflecting differences in the nucleotide sequences of the respective amplicons, and allowing discriminating variants and assess the viral diversity in grapevine accessions. The HRM analysis was validated by sequencing and nucleotide comparisons among Brazilian isolates of GLRaV-3 and GFLV.

Characterisation of different GLRaV-3 variant infections by determining virus concentration ratios and miRNA expression profiles

Grapevine leafroll disease (GLD) is present in all grape-growing regions of the world and is considered the most significant grapevine viral disease. Grapevine leafroll-associated virus 3 (GLRaV-3) is considered the primary cause of GLD and in South African vineyards five genetic variant groups (I, II, III, VI and VII) have been confirmed. Biological distinctions between GLRaV-3 variants have not been fully validated. By characterising virus concentration and stress-responsive microRNA expression in GLRaV-3 infected plants, this study aimed to glean a better understanding of the possible biological distinctions between GLRaV-3 variants. Quantitative reverse transcription PCR was utilised for virus concentration ratio (VCR) determination and miRNA quantitation in GLRaV-3 positive and negative grapevines grown under greenhouse and field conditions. This study found statistically significant differences in VCRs in plants singly infected with different GLRaV-3 variants. Interestingly, no difference in mean VCRs were observed between data sets, despite notable differences in plant age, duration of GLRaV-3 infection, scion/rootstock combination and growing conditions. Several miRNAs showed statistically significant expression modulation between infected and healthy samples. miRNA expression between data sets varied substantially and a greater overall miRNA response was observed in plants with more established GLRaV-3 infections. The lack of significant differences in mean VCRs between data sets, coupled with the consistent modulation of certain miRNAs in plants that have likely been infected for longer is a promising result. This finding could indicate that successful inhibition of further virus replication by plant defence mechanisms occurred, and that these miRNAs are implicated in this response.

Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3’ untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.

Virus preparations from the mixed-infected P70 Pinot Noir accession exhibit GLRaV-1/GVA 'end-to-end' particles.

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.

Genetic variants of Grapevine leafroll-associated virus 2 infecting Portuguese grapevine cultivars

Genetic variability of 19 isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) from Portuguese grapevine cultivars was characterized by sequencing the entire capsid protein (CP) gene of the virus. Global phylogenetic analysis of the CP gene, which included nucleotide sequences obtained in this study and complete homologous sequences from GenBank, showed segregation of GLRaV-2 variants from Portuguese isolates into three major phylogroups (PN, 93/955 and H4). The novelty of these phylogenetic results is the evidence of well-supported subdivision within H4 as well as within PN, with subgroup PN3 composed exclusively of variants from a Portuguese isolate. These findings and the genetic analysis of global phylogroups indicate demographic expansion, mainly within PN and 93/955. Because the existence of a mixture of variants from different phylogroups was detected in some of the isolates, a typification assay based on reverse transcription reaction followed by polymerase chain reaction and restriction fragment length polymorphism analysis, was developed to complement molecular detection assay of the virus. This protocol discriminates variants from the phylogroups identified in this study, and is appropriate for routine testing for GLRaV-2.

Differential distribution and titre of selected grapevine leafroll-associated virus 3 genetic variants within grapevine rootstocks.

In this study of three grapevine leafroll-associated virus 3 (GLRaV-3) genetic variants in two grapevine rootstock hosts, GLRaV-3 detection was shown to be affected by the virus distribution, titre, and the genetic variant. Group VI and NZ2 GLRaV-3 variants had reduced detectability compared with the group I variant. Differences in the genomic and subgenomic RNA (sgRNA) expression levels, and differences in the level of expression between the genetic variants were also observed. The observed differences in virus titre and sgRNA expression levels suggest differences in plant-virus interactions by the various GLRaV-3 genetic variants.

Identification and distribution of multiple virus infections in Grapevine leafroll diseased vineyards

A survey of viruses affecting grapevine in the wine regions of the Western Cape Province in South Africa was conducted. The survey determined the relative abundance of five different Grapevine leafroll-associated virus 3 (GLRaV-3) variants. Virus profiles were also determined for individual vines. A total of 315 plants were sampled and analysed over two growing seasons. Five GLRaV-3 variants were detected as either single or mixed infections, with GLRaV-3 variant groups II and VI being the most prominent as single infections and in combinations with each other and other variants. An analysis of the distribution of variants per region showed that single infections of variant groups II and VI occurred predominantly in certain regions, and were equally distributed in the red and white cultivars studied. The distribution of a recently identified, unclassified variant of GLRaV-3 (represented by isolates GH24 and GTG10) was included in the study. The overall analysis showed that infection with variant groups II and VI were the most abundant among the samples with 49.8 and 47.6 %, respectively, followed by variant group I, variants similar to isolate GH24 and variant group III with 16.2, 13.3 and 2.5 % infection, respectively. Mixed infections, representing 36 virus combinations, were found in 251 plants. The most abundant virus combination was GLRaV-3 with Grapevine virus E (GVE), found in 28 % of the plants. GLRaV-3 was the predominant virus detected in the samples with a frequency of 80 % detection, followed by GVE (57.4 %), Grapevine rupestris stem pitting-associated virus (GRSPaV) (36.8 %), Grapevine virus A (GVA) (19.3 %), Grapevine virus F (GVF) (16.25 %), Grapevine leafroll-associated virus 2 (GLRaV-2) (8.25 %), Grapevine leafroll-associated virus 1 (GLRaV-1) (1.58 % infection) and Grapevine leafroll-associated virus 4 (GLRaV-4 like) (0.6 %). Most of the plants tested were infected with multiple viruses. The complexity of virus populations detected in this study, highlights the need for detection methods able to identify all viruses and their variants in vineyards. The information generated in this study will assist in the development of reliable detection assays that will benefit the monitoring of disease spread and aid in the efficient management of Grapevine leafroll disease (GLD).

Brief Report of a New Highly Divergent Variant of Grapevine leafroll-associated virus 3 (GLRaV-3)

The alignment of the complete genomes of genetic variants of Grapevine leafroll-associated virus 3 (GLRaV-3) representing phylogenetic groups I, II, III and VI revealed numerous regions with exceptionally high divergence between group I to III and group VI variants. Oligonucleotide primers universal for all the above groups of the virus were designed in conserved short stretches of sequences flanking the divergent regions in the helicase (Hel) and RNA-dependent RNA polymerase (RdRP) domains of the replicase gene and the divergent copy of the capsid protein (dCP) gene. Cloning and sequencing of the 549-bp RT-PCR amplicon of the helicase domain from grapevine cv. Shiraz lead to the detection of a variant of GLRaV-3, which shared only 69.6–74.1% nt similarity with other variants, including the recently reported, new, highly divergent variant, isolate 139. This was confirmed by the results of the analysis of 517-bp amplicon of the HSP70 gene of GLRaV-3 generated in RT-nested PCR based on degenerate primers for the simultaneous amplification of members of the Closteroviridae family designed by Dovas and Katis (J Virol Methods, 109, 2003, 217). In this genomic region, the variant shares 72.3–78.7% nt similarity with other variants of GLRaV-3. This previously unreported, new, highly divergent variant was provisionally named GTG10. From the alignment of the HSP70 sequences primers for the specific RT-nested PCR amplification of the variant GTG10 and members of group VI, and specific simultaneous amplification of variants of groups I, II and III, were designed. The results obtained from brief testing of various grapevines using all these primers suggest a relatively limited presence of GTG10 variant in vineyards.

Molecular characterisation of two divergent variants of grapevine leafroll-associated virus 3 in New Zealand

Partial genomic sequences of two divergent grapevine leafroll-associated virus 3 (GLRaV-3) variants, NZ1-B and NZ2, from New Zealand were determined and analysed (11,827 nt and 7,612 nt, respectively). At the nucleotide level, both variants are more than 20% different from the previously published GLRaV-3 sequences, from phylogenetic groups 1 to 5. Phylogenetic analysis indicated that NZ1-B is a variant of the previously identified divergent NZ-1, while NZ2 is a novel sequence with only 76% nucleotide sequence identity to GLRaV-3 variants NZ-1, GH11, and GH30. Therefore, NZ2 is a new variant of GLRaV-3. Amino acid sequence analysis of the NZ1-B and NZ2 coat proteins indicated significant substitutions that are predicted to alter the coat protein structure, which potentially leads to the observed reduced immunological reactivity of both variants to the Bioreba anti-GLRaV-3 conjugated monoclonal antibody.

Generic and sequence-variant specific molecular assays for the detection of the highly variable Grapevine leafroll-associated virus 3

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically important virus, which is found in all grapevine growing regions worldwide. Its accurate detection in nursery and field samples is of high importance for certification schemes and disease management programmes. To reduce false negatives that can be caused by sequence variability, a new universal primer pair was designed against a divergent sequence data set, targeting the open reading frame 4 (heat shock protein 70 homologue gene), and optimised for conventional one-step RT-PCR and one-step SYBR Green real-time RT-PCR assays. In addition, primer pairs for the simultaneous detection of specific GLRaV-3 variants from groups 1, 2, 6 (specifically NZ-1) and the outlier NZ2 variant, and the generic detection of variants from groups 1 to 5 were designed and optimised as a conventional one-step multiplex RT-PCR assay using the plant nad5 gene as an internal control (i.e. one-step hexaplex RT-PCR). Results showed that the generic and variant specific assays detected in vitro RNA transcripts from a range of 1×101–1×108 copies of amplicon per μl diluted in healthy total RNA from Vitis vinifera cv. Cabernet Sauvignon. Furthermore, the assays were employed effectively to screen 157 germplasm and 159 commercial field samples. Thus results demonstrate that the GLRaV-3 generic and variant-specific assays are prospective tools that will be beneficial for certification schemes and disease management programmes, as well as biological and epidemiological studies of the divergent GLRaV-3 populations.

A divergent variant of Grapevine leafroll-associated virus 3is present in California

BackgroundGrapevine leafroll-associated viruses are a problem for grape production globally. Symptoms are caused by a number of distinct viral species. During a survey of Napa Valley vineyards (California, USA), we found evidence of a new variant of Grapevine leafroll-associated virus 3 (GLRaV-3). We isolated its genome from a symptomatic greenhouse-raised plant and fully sequenced it.FindingsIn a maximum likelihood analysis of representative GLRaV-3 gene sequences, the isolate grouped most closely with a recently sequenced variant from South Africa and a partial sequence from New Zealand. These highly divergent GLRaV-3 variants have predicted proteins that are more than 10% divergent from other GLRaV-3 variants, and appear to be missing an open reading frame for the p6 protein.ConclusionsThis divergent GLRaV-3 phylogroup is already present in grape-growing regions worldwide and is capable of causing symptoms of leafroll disease without the p6 protein.

Novel variants of grapevine leafroll-associated virus 4 and 7 detected from a grapevine showing leafroll symptoms

Novel variants of grapevine leafroll-associated virus 4 and 7 detected from a grapevine showing leafroll symptoms

Complete nucleotide sequence of a new strain of grapevine leafroll-associated virus 3 in South Africa

The complete genome sequences of two newly identified genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) are described. Two isolates, GH11 (18671 nt) and GH30 (18576 nt) were sequenced and found to be less than 70 % similar to other South African GLRaV-3 variants. The genome organization of GH11 and GH30 is similar to that of previously described GLRaV-3 isolates. However, no corresponding open reading frame 2 (ORF2) could be identified. Phylogenetic analysis indicates that GH11 and GH30 cluster in a subgroup (group VI) that is separate from the previously described GLRaV-3 isolates and should be regarded as a different strain of GLRaV-3.

Distribution of grapevine leafroll associated virus-3 variants in South African vineyards

Three genetic variants of Grapevine leafroll-associated virus 3 (GLRaV-3) were identified from vineyards of the Western Cape, South Africa. In a previous study, three full genome sequences of isolates representing each of the variant groups were determined. The three variant groups were represented by accessions 621, 623 and PL-20, of variant groups I, II and III respectively. A specific single strand conformation polymorphism (SSCP) profile was assigned to each variant which was used as a quick, reliable detection and differentiation method. In this study we analysed the occurrence of these three GLRaV-3 variants in mother blocks in different cultivars and from different vine growing regions using SSCP. The majority of the plants studied, were infected with the group II variant, similar to isolates 623 and GP18. The distribution of three GLRaV-3 variants within a spatio-temporally recorded cluster of diseased plants was studied by means of SSCP profile analysis of ORF5 amplified PCR products. We showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection cluster. Results showed that, in some leafroll disease clusters, the variant that was present in the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row and across rows.

Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5′UTR

Three genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) were identified in vineyards of the Western Cape, South Africa. The GLRaV-3 variants were identified by single-strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. ORF5 sequence data confirmed the three genetic variant groups, and a specific SSCP profile was assigned to each variant group. The results of SSCP analysis of this region in ORF5 showed that this method gives a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5' ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5'UTRs indicated significant sequence and length variation in this region between the three South African variant groups. Alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three variants of GLRaV-3 in South Africa and the presence of two or three additional variant groups elsewhere in the world.