Detection Of Grapevine Leafroll-associated Virus Research Articles

An Assessment of the Virome of Nigerian Vineyards

The virome of Nigerian vineyards was assessed via diagnostic surveys for viruses and viroids in 40 sites across six states in 2016. Leaf tissues from 360 grapevines were preserved dried under CaCl2. High-throughput sequencing of total RNA extracts from two composite dry leaf samples resulted in the detection of grapevine leafroll-associated virus 1 (GLRaV-1), hop stunt viroid (HSVd), grapevine yellow speckle viroid 1 (GYSVd-1), and GYSVd-2. The incidences and distribution of these agents were assessed via RT-PCR with specific primers and two additional primer pairs targeting grapevine rupestris stem pitting-associated virus (GRSPaV) and grapevine red blotch virus (GRBV). Approximately 47% (169/360) of the samples had mixed infections of two to five agents, ∼22% (78/360) had single infections, and ∼31% (113/360) tested negative. HSVd was the most common, with its occurrence in ∼53% (192/360; 39 sites) of samples, followed by GYSVd-2 (∼43%; 155/360; 34 sites), GYSVd-1 (∼33%; 120/360; 26 sites), GLRaV-1 (∼24%; 85/360; 26 sites), and GRSPaV (∼3%; 10/360; 7 sites). All 360 samples were negative for GRBV. In pairwise comparisons, GLRaV-1 isolates from Nigeria shared 97 to 100% nucleotide (nt) and amino acid identities among themselves based on partial HSP70h and complete p24 sequences. The nearly complete genome (18,689 nt) of one GLRaV-1 isolate was determined and it shared 76.5 to 94.5% identity with other fully sequenced GLRaV-1 isolates. The results revealed a simpler virome of Nigerian vineyards and underscore the need to strengthen phytosanitary measures and promote propagation of clean plant materials for vineyard establishment in Nigeria to forestall the introduction of additional viruses and viroids. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

A Novel and Highly Inclusive Quantitative Real-Time RT-PCR Method for the Broad and Efficient Detection of Grapevine Leafroll-Associated Virus 1.

Grapevine (Vitis vinifera L.) is one of the most important crops in the world due to its economic and social impact. Like many other crops, grapevine is susceptible to different types of diseases caused by pathogenic microorganisms. Grapevine leafroll-associated virus 1 (GLRaV-1) is a virus associated with grapevine leafroll disease and it is considered at the national and European level as a pathogen that must be absent in propagative plant material. For this reason, the availability of specific, sensitive and reliable detection techniques to ascertain the sanitary status of the plants is of great importance. The objective of this research was the development of a new GLRaV-1 detection method based on a TaqMan quantitative real-time RT-PCR targeted to the coat protein genomic region and including a host internal control in a duplex reaction. To this end, three new GLRaV-1 full genomes were recovered by HTS and aligned with all sequences available in the databases. The method has been validated following EPPO standards and applied for the diagnosis of field plant material and transmission vectors. The new protocol designed has turned out to be highly sensitive as well as much more specific than the current available methods for the detection and absolute quantitation of GLRaV-1 viral titer.

Development of a one-step RT-qPCR assay for the detection of Grapevine leafroll-associated virus 7.

Grapevine leafroll disease (GLD) is one of the most economically important viral diseases of grapevines. GLD is caused by a complex of several ssRNA (+) viruses referred to as Grapevine leafroll-associated viruses (GLRaVs). To date, five different GLRaV species have been identified. One of those species, GLRaV-7, was first reported from a symptomless white-fruited wine grape cultivar from Albania. Since its discovery, GLRaV-7 has been reported from 14 countries. Although serological assays have been developed to detect GLRaV-7, commercially available antibodies produce high background signals making them unsuitable for regulatory testing. Furthermore, while molecular detection assays have been shown to be more sensitive when compared to the serological assays, published molecular assays, except the one Reverse Transcription-quantitaive Polymerase Chain Reaction (RT-qPCR) assay based on heat shock protein 70 homologue (HSP70h) gene, have been reported to be inadequate in detecting all reported isolates of GLRaV-7. Availability of multiple assays provides flexibility to diagnostic laboratories in cases where the chosen assay fails to detect a strain or an isolate of a pathogen due to variation in its targeted region or where additional confirmation of the results is required. In this study, we developed a sensitive and specific RT-qPCR assay, based on a region of p61 gene of GLRaV-7, which detected all available isolates.

Early detection of grapevine leafroll disease in a red-berried wine grape cultivar using hyperspectral imaging

This research was conducted to examine the potential use of hyperspectral imaging for non-destructive detection of Grapevine leafroll-associated virus 3 (GLRaV-3) during asymptomatic and symptomatic stages of grapevine leafroll disease (GLD) in a red-berried wine grape (Vitis vinifera) cultivar. Cabernet Sauvignon vines tested positive and negative for GLRaV-3 were used in this study. Leaves from infected and non-infected vines were detached at five phenological stages and individual leaf images were acquired by a hyperspectral imager in 2017, 2018, and 2019 seasons. Those images were then preprocessed using spectra normalization and Monte-Carlo method for eliminating spectral sample outliers. Least absolute shrinkage and selection operator was used to select feature wavelengths for each phenological stage within three-season datasets. The sensitivity of selected feature wavelengths was evaluated based on analysis of variance and linear regression. Six salient wavelengths (690, 715, 731, 1409, 1425 and 1582 nm) were determined as sensitive wavebands for detecting virus symptoms in leaf samples. The detectability of GLD using those six salient wavelengths was evaluated using least squares-support vector machine. The classification accuracy was found between 66.67 and 89.93% for test datasets collected at first asymptomatic stage over three seasons. These results indicated that the hyperspectral imaging technique has the potential for nondestructively detecting virus-infected grapevines during asymptomatic stages.

Detection of Grapevine Leafroll-Associated Virus 1 and 3 in White and Red Grapevine Cultivars Using Hyperspectral Imaging

Grapevine leafroll disease (GLD) is considered one of the most widespread grapevine virus diseases, causing severe economic losses worldwide. To date, six grapevine leafroll-associated viruses (GLRaVs) are known as causal agents of the disease, of which GLRaV-1 and -3 induce the strongest symptoms. Due to the lack of efficient curative treatments in the vineyard, identification of infected plants and subsequent uprooting is crucial to reduce the spread of this disease. Ground-based hyperspectral imaging (400–2500 nm) was used in this study in order to identify white and red grapevine plants infected with GLRaV-1 or -3. Disease detection models have been successfully developed for greenhouse plants discriminating symptomatic, asymptomatic, and healthy plants. Furthermore, field tests conducted over three consecutive years showed high detection rates for symptomatic white and red cultivars, respectively. The most important detection wavelengths were used to simulate a multispectral system that achieved classification accuracies comparable to the hyperspectral approach. Although differentiation of asymptomatic and healthy field-grown grapevines showed promising results further investigations are needed to improve classification accuracy. Symptoms caused by GLRaV-1 and -3 could be differentiated.

Development of sensitive molecular assays for the detection of grapevine leafroll-associated virus 3 in an insect vector.

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.

Visible-near infrared spectroradiometry-based detection of grapevine leafroll-associated virus 3 in a red-fruited wine grape cultivar

Grapevine leafroll disease (GLD) is one of the major threats to wine grapes (Vitis vinifera) causing substantial economic losses to the growers. This study was undertaken to evaluate the applicability of visible and near infrared (VIS-NIR) spectroradiometery as a rapid, robust and non–destructive optical sensing method for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3) at different phenological stages in a red-berried wine grape cultivar. Using VIS-NIR spectroradiometer, data was collected from the healthy and GLRaV-3-infected leaf samples from cv. Cabernet Sauvignon for two seasons at specific intervals during asymptomatic and symptomatic stages of the disease. Fiber optic leaf clip was used to collect spectral responses from grapevine leaves under field conditions. Salient feature extraction using stepwise multilinear regression and partial least square regression methods showed significant differences between healthy and virus–infected leaves in the visible (351, 377, 501, 526, 626 and 676 nm) and near infrared (701, 726, 826, 901, 951, 976, 1001, 1027, 1052 and 1101 nm) regions. Spectral wavelengths from near infrared region (1001, 1027 and 1052 nm) were validated at different phenological stages spanning both asymptomatic and symptomatic stages of the disease. Selected spectral wavelengths demonstrated robustness in virus detection with overall classification accuracies in the range of 75–99% using quadratic discriminant analysis (QDA) classifier. QDA based classification accuracies for healthy, infected and overall classes were significantly higher compared to Naïve Bayes classifier. The accuracy for virus detection during asymptomatic stages was not significantly different from the symptomatic phase, indicating reliability of the selected features for early detection of GLRaV–3–infected grapevines.

Lateral Flow Immunoassay for Rapid Detection of Grapevine Leafroll-Associated Virus.

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.

Development of immunodiagnostics for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.

Serological detection of Grapevine leafroll-associated virus 4 in grapevine growing areas of India using polyclonal antiserum raised against the recombinant coat protein

Grapevine leafroll disease (GLD), caused by a complex of serologically and genetically distinct or related viral species termed as Grapevine leafroll associated viruses (GLRaVs), is a serious threat to the grape production worldwide. Recently, Grapevine leafroll-associated virus 4 (GLRaV-4) has been reported in the Indian vineyards, mostly as a mixed infection with GLRaV-3 and GLRaV-1. In order to screen the planting material, a polyclonal antiserum against the in vitro expressed coat protein (CP) of GLRaV-4 was produced. The antiserum, purified IgGs and conjugated IgGs detected GLRaV-4 in crude saps of infected grapevines and purified coat protein when used in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Western blotting and dot immunobinding assay (DIBA). GLRaV-4 could be detected in 54% (39 out of 72) of symptomatic grapevine samples from different grape growing regions of India using DAS-ELISA. The validation of DAS-ELISA results was done by RT-PCR using GLRaV-4 CP-specific primers. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in vitro expressed CP of GLRaV-4 and its successful application in diagnosis of the virus in grape vineyards of India.

Development of an immunocapture–reverse transcription–polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3)

Grapevine leafroll-associated virus 3(GLRaV-3) is the most destructive virus causing leaf roll disease in grapevine. ELISA has been widely used to screen the propagating materials for indexing of this virus at nursery stage. But the uneven distribution of GLRaV-3 in vines, its confinement to phloem tissues and impact of seasonal influences on its concentration limit the scope of ELISA. RT-PCR (reverse transcription-polymerase chain reaction), is a more sensitive technique, but not feasible for large scale screening purpose because of the tedious process of RNA isolation. Furthermore, location of virus particles and the presence of inhibitory compounds in the woody tissues of grapevine make RNA isolation problematic. Immunocapture-RT-PCR (IC-RT-PCR), more sensitive than ELISA and RT-PCR alone, is a technique where the virus can be detected without isolating the RNA. In this study, IC-RT-PCR was performed using different combinations of three virus extraction buffers and two virus nucleic acid releasing buffers along with one virus RNA releasing condition for the detection of GLRaV-3. The modified extraction and RNA release protocol developed in this study was validated for specific detection of the virus in the vines of five infected grapevine cultivars. This protocol can help in complementing the GLRaV-3 specific certification program of the country.

Molecular data mining to improve antibody-based detection of Grapevine leafroll-associated virus 1 (GLRaV-1)

Testing for Grapevine leafroll-associated virus 1 (GLRaV-1) is mandatory in certification schemes of propagation material within the EU. Accurate and reliable diagnostic assays are necessary for implementation of this measure. During routine detection of GLRaV-1, using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT) followed by polymerase chain reaction (PCR), evidence was obtained that positive samples could be overlooked by either or both detection methods. With the aim of improving serological detection tools for GLRaV-1, a total of 20 isolates were analyzed and 83 new complete capsid protein (CP) gene sequences were obtained. This set, together with the CP sequences available at GenBank was used for a comprehensive in silico analysis. To obtain a specific antibody able to recognize all known CP variants, conserved regions with suitable antigenicity profile were identified along the deduced CP AA sequences and a 14 AA sequence was chosen for commercial peptide synthesis and immunization. Initially polyclonal antibodies were produced and tested, followed by purification of the respective monospecific antibody and conjugation with alkaline phosphatase or fluorescein isothiocyanate (FITC). These serological tools were tested successfully on all the available positive samples and proved adequate for in situ immunoassay (ISIA). Further testing showed that the monospecific antibody could also be used in tissue print immunoblotting (TPIB), a technique that allows rapid processing of large sample sets, which is highly suitable to implement protocols ensuring that, for each vine analyzed, enough random samples are taken and processed, before certification.

Detection of Grapevine leafroll-associated virus 7 using real time qRT-PCR and conventional RT-PCR

Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.

First detection of Grapevine leafroll-associated virus 9 in Italy.

First detection of Grapevine leafroll-associated virus 9 in Italy.

An assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody

Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevine. A previously described single-chain fragment variable (scFv) antibody (scFvLR3), directed against the coat protein (CP) of GLRaV-3, was expressed in Escherichia coli and used to develop a diagnostic ELISA kit. The antibody was fused to the light chain constant domain of human immunoglobulin to create the bivalent reagent C(L)-LR3, which was purified from the periplasmic fraction, giving a yield of ~5 mg/l. The sensitivity of the reagent against recombinant GLRaV-3 CP was 0.1 ng. The sensitivity, specificity and durability of the reagent was similar to a commercial kit. The C(L)-LR3 showed a weak cross-reaction in immune electron microscopy assays to GLRaV-1 and -7, but not with the phylogenetically more distant GLRaV-2. A fully recombinant kit was developed with the inclusion of a recombinant GLRaV-3 CP expressed in bacteria, thus avoiding problems associated with virus propagation and purification. This system represents a rapid, simple, sensitive and standardized diagnostic protocol for GLRaV-3 detection.

Improved detection of grapevine leafroll-associated virus 1 by magnetic capture hybridisation RT-PCR on a conserved region of viral RNA

We report the development of a sensitive diagnostic method for the detection of the grapevine leafroll-associated virus 1 (GLRaV-1). We have considered the current shortcoming in detection of GLRaV-1 to be linked to two factors, sequence variation in the viral RNA and low template concentration. Sequence information available allowed the selection of optimal target sequences for detection by RT-PCR, having high copy number and low levels of sequence variation. This was combined with the use of magnetic capture hybridisation to allow the removal of RT-PCR inhibitors and the addition of 100-fold excess template RNA to a single RT-PCR. The reproducibility of the technique was confirmed using field samples.