Two Novel Geminiviruses Identified in Bees (Apis mellifera and Nomia sp.).

Members of the Geminviridae family are circular single-stranded DNA plant-infecting viruses, some of which impact global food production. Geminiviruses are vectored by sap-feeding insects such as leafhoppers, treehoppers, aphids, and whiteflies. Additionally, geminivirus sequences have also been identified in other insects such as dragonflies, mosquitoes, and stingless bees. As part of a viral metagenomics study on honeybees and solitary bees (Nomia sp.), two geminivirus genomes were identified. These represent a novel citlodavirus (from honeybees collected from Westmoreland, Jamaica) and a mastrevirus-like genome (from a solitary bee collected from Tempe, Arizona, USA). The novel honeybee-derived citlodavirus genome shares ~61 to 69% genome-wide nucleotide pairwise identity with other citlodavirus genome sequences and is most closely related to the passion fruit chlorotic mottle virus identified in Brazil. Whereas the novel solitary bee-derived mastrevirus-like genome shares ~55 to 61% genome-wide nucleotide identity with other mastreviruses and is most closely related to tobacco yellow dwarf virus identified in Australia, based on pairwise identity scores of the full genome, replication-associated protein, and capsid protein sequences. Previously, two geminiviruses in the Begomovirus genus were identified in samples of stingless bee (Trigona spp.) samples. Here, we identify viruses that represent two new species of geminiviruses from a honeybee and a solitary bee, which continues to demonstrate that plant pollinators can be utilized for the identification of plant-infecting DNA viruses in ecosystems.

Molecular Characterization and Pathogenicity of a Novel Soybean-Infecting Monopartite Geminivirus in China.

Soybean is a major legume crop that plays an important role in food production, industrial production, and animal husbandry. Here, we characterize a novel soybean-infecting monopartite geminivirus identified in China. Analysis of the contigs de novo assembled from sequenced small interfering RNAs, followed by PCR, cloning, and sequencing, the complete viral genome was determined to be 2782 nucleotides. The genome contains the conserved nonanucleotide sequence, TAATATTAC and other sequence features typical of the family Geminiviridae, and encodes two and four open reading frames in the virion-sense and the complementary-sense strands, respectively. Genome-wide pairwise identity analysis revealed that the novel virus shares less than 65.6% identity with previously characterized geminiviruses. Phylogenetic and recombination analysis indicated that this virus was placed in a unique taxon within the family Geminiviridae and potentially arose from recombination. An infectious clone of this virus was further constructed and its infectivity was tested in different species of plants. Successful infection and characteristic symptoms were observed in Glycine max, Nicotiana benthamiana, N. tabacum, N. glutinosa, and N. tabacum cv. Samsun plants. Taken together, this virus represents a member of an unclassified genus of the family Geminiviridae, for which the name soybean yellow leaf curl virus is proposed.

Identification of a Novel Geminivirus in Fraxinus rhynchophylla in Korea.

Fraxinus rhynchophylla, common name ash, belongs to the family Oleaceae and is found in China, Korea, North America, the Indian subcontinent, and eastern Russia. It has been used as a traditional herbal medicine in Korea and various parts of the world due to its chemical constituents. During a field survey in March 2019, mild vein thickening (almost negligible) was observed in a few ash trees. High-throughput sequencing of libraries of total DNA from ash trees, rolling-circle amplification (RCA), and polymerase chain reaction (PCR) allowed the identification of a Fraxinus symptomless virus. This virus has five confirmed open reading frames along with a possible sixth open reading frame that encodes the movement protein and is almost 2.7 kb in size, with a nonanucleotide and stem loop structure identical to begomoviruses. In terms of its size and structure, this virus strongly resembles begomoviruses, but does not show any significant sequence identity with them. To confirm movement of the virus within the trees, different parts of infected trees were examined, and viral movement was successfully observed. No satellite molecules or DNA B were identified. Two-step PCR confirmed the virion and complementary strands during replication in both freshly collected infected samples of ash tree and Nicotiana benthamiana samples agro-inoculated with infectious clones. This taxon is so distantly grouped from other known geminiviruses that it likely represents a new geminivirus genus.

Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties.

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

Novel circular DNA virus identified in Opuntia discolor (Cactaceae) that codes for proteins with similarity to those of geminiviruses.

Viral metagenomic studies have enabled the discovery of many unknown viruses and revealed that viral communities are much more diverse and ubiquitous than previously thought. Some viruses have multiple genome components that are encapsidated either in separate virions (multipartite viruses) or in the same virion (segmented viruses). In this study, we identify what is possibly a novel bipartite plant-associated circular single-stranded DNA virus in a wild prickly pear cactus, Opuntia discolor, that is endemic to the Chaco ecoregion in South America. Two ~1.8 kb virus-like circular DNA components were recovered, one encoding a replication-associated protein (Rep) and the other a capsid protein (CP). Both of the inferred protein sequences of the Rep and CP are homologous to those encoded by members of the family Geminiviridae. These two putatively cognate components each have a nonanucleotide sequence within a likely hairpin structure that is homologous to the origins of rolling-circle replication (RCR), found in diverse circular single-stranded DNA viruses. In addition, the two components share similar putative replication-associated iterative sequences (iterons), which in circular single-stranded DNA viruses are important for Rep binding during the initiation of RCR. Such molecular features provide support for the possible bipartite nature of this virus, which we named utkilio virus (common name of the Opuntia discolor in South America) components A and B. In the infectivity assays conducted in Nicotiana benthamiana plants, only the A component of utkilio virus, which encodes the Rep protein, was found to move and replicate systemically in N. benthamiana. This was not true for component B, for which we did not detect replication, which may have been due to this being a defective molecule or because of the model plants (N. benthamiana) used for the infection assays. Future experiments need to be conducted with other plants, including O. discolor, to understand more about the biology of these viral components.

Genome characterization and diversity of trifolium virus 1: identification of a novel legume-infecting capulavirus.

A novel geminivirus was identified in France and Spain in asymptomatic plants of white clover (Trifolium repens) and shrub medick (Medicago arborea). Its genome has the hallmarks of a capulavirus, and its relationship to other capulaviruses was confirmed by phylogenetic analysis. White clover isolates formed a tight cluster in the phylogenetic tree, while shrub medick isolates formed two distinct, more divergent groups with sequence identity values close to the species cutoff. These three groups have likely participated in recombination events involving alfalfa leaf curl virus and French bean severe leaf curl virus. The name "trifolium virus 1" (TrV1) is proposed for this new Capulavirus. Three TrV1 genotypes (TrV1-A, TrV1-B, and TrV1-C) were clearly distinguished.

Identification of the Viral Determinant of Hypovirulence and Host Range in Sclerotiniaceae of a Genomovirus Reconstructed from the Plant Metagenome.

Uncharacterized viral genomes that encode circular replication-associated proteins of single-stranded DNA viruses have been discovered by metagenomics/metatranscriptomics approaches. Some of these novel viruses are classified in the newly formed family Genomoviridae. Here, we determined the host range of a novel genomovirus, SlaGemV-1, through the transfection of Sclerotinia sclerotiorum with infectious clones. Inoculating with the rescued virions, we further transfected Botrytis cinerea and Monilinia fructicola, two economically important members of the family Sclerotiniaceae, and Fusarium oxysporum. SlaGemV-1 causes hypovirulence in S. sclerotiorum, B. cinerea, and M. fructicola. SlaGemV-1 also replicates in Spodoptera frugiperda insect cells but not in Caenorhabditis elegans or plants. By expressing viral genes separately through site-specific integration, the replication protein alone was sufficient to cause debilitation. Our study is the first to demonstrate the reconstruction of a metagenomically discovered genomovirus without known hosts with the potential of inducing hypovirulence, and the infectious clone allows for studying mechanisms of genomovirus-host interactions that are conserved across genera. IMPORTANCE Little is known about the exact host range of widespread genomoviruses. The genome of soybean leaf-associated gemygorvirus-1 (SlaGemV-1) was originally assembled from a metagenomic/metatranscriptomic study without known hosts. Here, we rescued SlaGemV-1 and found that it could infect three important plant-pathogenic fungi and fall armyworm (S. frugiperda Sf9) insect cells but not a model nematode, C. elegans, or model plant species. Most importantly, SlaGemV-1 shows promise for inducing hypovirulence of the tested fungal species in the family Sclerotiniaceae, including Sclerotinia sclerotiorum, Botrytis cinerea, and Monilinia fructicola. The viral determinant of hypovirulence was further identified as replication initiation protein. As a proof of concept, we demonstrate that viromes discovered in plant metagenomes can be a valuable genetic resource when novel viruses are rescued and characterized for their host range.

New World Cactaceae Plants Harbor Diverse Geminiviruses.

The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus Curtovirus, which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental Rhizobium-mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated.

Olea Europaea Geminivirus: A Novel Bipartite Geminivirid Infecting Olive Trees.

In 2014, high-throughput sequencing of libraries of total DNA from olive trees allowed the identification of two geminivirus-like contigs. After conventional resequencing of the two genomic DNAs, their analysis revealed they belonged to the same viral entity, for which the provisional name of Olea europaea geminivirus (OEGV) was proposed. Although DNA-A showed a genome organization similar to that of New World begomoviruses, DNA-B had a peculiar ORF arrangement, consisting of a movement protein (MP) in the virion sense and a protein with unknown function on the complementary sense. Phylogenetic analysis performed either on full-length genome or on coat protein, replication associated protein (Rep), and MP sequences did not endorse the inclusion of this virus in any of the established genera in the family Geminiviridae. A survey of 55 plants revealed that the virus is widespread in Apulia (Italy) with 91% of the samples testing positive, although no correlation of OEGV with a disease or specific symptoms was encountered. Southern blot assay suggested that the virus is not integrated in the olive genome. The study of OEGV-derived siRNA obtained from small RNA libraries of leaves and fruits of three different cultivars, showed that the accumulation of the two genomic components is influenced by the plant genotype while virus-derived-siRNA profile is in line with other geminivirids reported in literature. Single-nucleotide polymorphism (SNP) analysis unveiled a low intra-specific variability.

Isolation of single-chain variable fragment (scFv) antibodies for detection of Chickpea chlorotic dwarf virus (CpCDV) by phage display.

Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus), has a wide host range and geographic distribution in many parts of the world, and it is one of the most important legume-infecting viruses. Detection of CpCDV-infected plants in the field and evaluation of viral resistance of plant cultivars are possible by conducting serological assays. Here, development and characterization of a specific recombinant monoclonal antibody for CpCDV as a diagnostic tool are described. For this purpose, the coat protein of CpCDV was expressed in Escherichia coli strain Rosetta (DE3) and used to screen a Tomlinson phage display antibody library to select a specific single-chain variable fragment (scFv). In each round of biopanning, the affinity of the phage for CpCDV-CP was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that the specificity of the eluted phages increased after each round of panning. Testing of individual clones by ELISA showed that five clones of the monoclonal phage were more strongly reactive against CpCDV than the other clones. All selected positive clones contained the same sequence. The phage-displayed scFv antibody, which was named CpCDV-scFvB9, did not bind to other tested plant pathogens and showed high sensitivity in the detection of CpCDV. A Western blot assay demonstrated that CpCDV-scFvB9 reacted with the recombinant coat protein of CpCDV. Finally, the interaction CpCDV-scFvB9 and CpCDV-CP was analyzed in a molecular docking experiment. This is the first report on production of an scFv antibody against CpCDV, which could be useful for immunological detection of the virus.

Isolation and characterization of a novel geminivirus from parsley.

Fresh leaf vegetables are a significant part of the Persian food. Following a survey for identification of nanoviruses and geminivirus infecting leaf vegetables, a novel geminivirus was identified in a diseased parsley sample showing upward marginal leaf curling, marginal leaf yellowing, dwarfing and reduced leaf size in south-eastern Iran. The genome was identified through combination of rolling circle amplification (RCA) and high throughput sequencing (HTS) approaches. The full-length genome (2779 nts) of the cloned geminivirus, parsley yellow leaf curl virus (PYLCV), shares <66 % genome-wide pairwise identity with all other known geminiviruses. The PYLCV genome has six open reading frames (ORFs) and appears to be a hybrid with the virion sense encoded proteins being most similar to those of becurtoviruses and curtoviruses, whereas the complementary sense encoded proteins are most similar to those of begomoviruses. In comparison with other geminivirus encoded capsid proteins (CPs) and replication associated proteins (Reps), the CP of PYLCV shares <56 % amino acid pairwise identity whereas the Rep shares <73 % amino acid pairwise identity. To demonstrate the pathogenicity of the geminivirus, a partial dimer infectious clone was constructed and used to agro-infect parsley as well as Nicotiana benthamiana, turnip, radish and tomato. The agro-inoculation resulted in infection with symptoms in 83.7 % (82/98) of the tested plant. Based on the similarity of the CP encoded by PYLCV to those of becurtoviruses and curtoviruses, it is likely that leafhoppers may be the primary transmission vector.

Alfalfa leaf curl virus is transmitted by Aphis craccivora in a highly specific circulative manner.

Two members of the genus Capulavirus (Geminiviridae) are transmitted by aphids including Alfalfa leaf curl virus (ALCV) transmitted by Aphis craccivora. The capulavirus Euphorbia caput-medusae latent virus was shown here to be transmitted also by A. craccivora, using the population EuphorbiaSA. ALCV was transmissible by several A. craccivora populations including Robinia, but not the EuphorbiaSA population, reflecting a high transmission specificity. Typical of the circulative-persistent mode of transmission, ALCV persists through insect molts. ALCV accumulation and localization were analyzed in whole insects, midguts, hemolymphs, and heads of aphids from vector and non-vector populations of A. craccivora and from the non-vector species Acyrthosiphon pisum. Vector and non-vector populations could be distinguished by contrasted virus accumulations and midgut intracellular localization consistent with a gut barrier to the transmission of ALCV in A. pisum and a primary salivary gland barrier in A. craccivora.

A Novel Divergent Geminivirus Identified in Asymptomatic New World Cactaceae Plants.

Cactaceae comprise a diverse and iconic group of flowering plants which are almost exclusively indigenous to the New World. The wide variety of growth forms found amongst the cacti have led to the trafficking of many species throughout the world as ornamentals. Despite the evolution and physiological properties of these plants having been extensively studied, little research has focused on cactus-associated viral communities. While only single-stranded RNA viruses had ever been reported in cacti, here we report the discovery of cactus-infecting single-stranded DNA viruses. These viruses all apparently belong to a single divergent species of the family Geminiviridae and have been tentatively named Opuntia virus 1 (OpV1). A total of 79 apparently complete OpV1 genomes were recovered from 31 different cactus plants (belonging to 20 different cactus species from both the Cactoideae and Opuntioideae clades) and from nine cactus-feeding cochineal insects (Dactylopius sp.) sampled in the USA and Mexico. These 79 OpV1 genomes all share > 78.4% nucleotide identity with one another and < 64.9% identity with previously characterized geminiviruses. Collectively, the OpV1 genomes display evidence of frequent recombination, with some genomes displaying up to five recombinant regions. In one case, recombinant regions span ~40% of the genome. We demonstrate that an infectious clone of an OpV1 genome can replicate in Nicotiana benthamiana and Opuntia microdasys. In addition to expanding the inventory of viruses that are known to infect cacti, the OpV1 group is so distantly related to other known geminiviruses that it likely represents a new geminivirus genus. It remains to be determined whether, like its cactus hosts, its geographical distribution spans the globe.

Naked-Eye Detection of Grapevine Red-Blotch Viral Infection Using a Plasmonic CRISPR Cas12a Assay.

Herein, we described a novel plasmonic CRISPR Cas12a assay for the visual, colorimetric detection of grapevine viral infections. Our assay generates rapid and specific colorimetric signals for nucleic acid amplicons by combining the unique target-induced incriminate single-stranded DNase activity of Cas12a with plasmon coupling of DNA functionalized gold nanoparticles. The practical applicability of our plasmonic assay was successfully demonstrated through the detection of emerging red-blotch viral infections in grapevine samples collected from commercial vineyards.

A rapid, sensitive and inexpensive method for detection of grapevine red blotch virus without tissue extraction using loop-mediated isothermal amplification.

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.

Chickpea chlorotic dwarf virus: An Emerging Monopartite Dicot Infecting Mastrevirus.

Chickpea stunt disease (CSD), caused by Chickpea chlorotic dwarf virus (CpCDV) is a threat to chickpea production leading to yield losses of 75⁻95%. Chickpea chlorotic dwarf virus is a monopartite, single-stranded circular DNA virus in the genus Mastrevirus and family Geminiviridae. It is transmitted by Orosius albicinctus in a circulative (persistent) and nonpropagative manner. Symptoms of CSD include very small leaves, intense discoloration (yellowing (kabuli type) and reddening (desi type)), and bushy stunted appearance of the plant. Presently, CpCDVs occurs in Africa, Asia, Australia, and the Middle East, causing extensive losses on economically important crops in in the families Fabaceae, Asteraceae, Amaranthaceae, Brassicaceae, Cucurbitaceae, Caricaceae, Chenopodiaceae, Leguminosae, Malvaceae, Pedaliaceae, and Solanaceae. High frequency of recombinations has played a significant role in the wide host range, diversification, and rapid evolution of CpCDVs. This review highlights the extensive research on the CpCDV genome diversity, host range, plant⁻virus⁻insect interactions, and RNA interference-based resistance of CpCDV, providing new insights into the host adaptation and virus evolution.

Identification of a new turncurtovirus in the leafhopper Circulifer haematoceps and the host plant species Sesamum indicum.

Turncurtoviruses (family: Geminiviridae; genus: Turncurtovirus) appear to have a high degree of genetic variation in Iran. Leafhoppers of the species Circulifer haematoceps (Mulsant and Rey, 1855) (family: Cicadellidae) were collected in 2014 from three geographical regions in south-eastern Iran (Orzoeyeh, Jiroft and Sirjan; Kerman province) and screened for the presence of turncurtoviruses using a combination of PCR and rolling circle amplification (RCA) methods. Eleven genomes of turncurtovirus were recovered and sequenced. Leafhoppers were sampled off sesame (S. indicum L.) and turnip (Brassica rapa sub sp. rapa). Thus, we identified three symptomatic sesame plants (yellowing, boat-shaped leaf curling, vein swelling on the lower leaf surfaces) from sesame farms in Jiroft. In these samples, we identified the same turncurtovirus as in the leafhoppers and have named it sesame curly top virus (SeCTV). Collectively, these SeCTV share > 98% genome-wide pairwise identity and ~ 87.3% to a recently identified turncurtovirus (sesame yellow mosaic virus; SeYMV) from sesame in Pakistan (GenBank accession MF344550). The SeCTV and SeYMV sequences share < 70% genome-wide pairwise identity with isolates of Turnip curly top virus and Turnip leaf roll virus, the two species in the genus Turncurtovirus. Based on the pairwise identities and phylogenetic analysis, SeCTV (n = 12) and SeYMV (n = 1) represent two strains of a new species in the genus Turncurtovirus.

The Westward Journey of Alfalfa Leaf Curl Virus.

Alfalfa leaf curl virus (ALCV), which causes severe disease symptoms in alfalfa (Medicago sativa L.) and is transmitted by the widespread aphid species, Aphis craccivora Koch, has been found throughout the Mediterranean basin as well as in Iran and Argentina. Here we reconstruct the evolutionary history of ALCV and attempt to determine whether the recent discovery and widespread detection of ALCV is attributable either to past diagnostic biases or to the emergence and global spread of the virus over the past few years. One hundred and twenty ALCV complete genome sequences recovered from ten countries were analyzed and four ALCV genotypes (ALCV-A, ALCV-B, ALCV-C, and ALCV-D) were clearly distinguished. We further confirm that ALCV isolates are highly recombinogenic and that recombination has been a major determinant in the origins of the various genotypes. Collectively, the sequence data support the hypothesis that, of all the analyzed locations, ALCV likely emerged and diversified in the Middle East before spreading to the western Mediterranean basin and Argentina.

The Relative Occurrence of Grapevine leafroll-associated virus 3 and Grapevine red blotch virus in Washington State Vineyards.

Vineyard surveys were conducted for three consecutive seasons in eastern Washington State, the major grapevine-growing region in the state, to document the occurrence of Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine red blotch virus (GRBV). The majority of samples were collected from red-berried wine grape (Vitis vinifera) cultivars exhibiting symptoms of or suspected for grapevine leafroll (GLD) and red blotch (GRBD) diseases. A limited number of samples from white-berried cultivars were collected randomly due to the lack of visual symptoms. Samples were collected from a total of 2,063 grapevines from 18 red-berried cultivars and seven white-berried cultivars planted in eight American Viticultural Areas and tested for GLRaV-3 and GRBV using RT-PCR and PCR, respectively. The results showed 67.77% and 6.01% of total samples positive for GLRaV-3 and GRBV, respectively, and 9.06% of samples positive for both viruses. About 17% of samples tested negative for the two viruses, but some of these samples were positive for GLRaV-2 and GLRaV-4. Overall results indicated that GLRaV-3 was more common than GRBV, independent of cultivars and the geographic origin of samples. Due to variability in symptoms in red-berried cultivars, virus-specific diagnostic assays were deemed necessary for reliable identification of GLRaV-3 and GRBV and to differentiate GLD and GRBD symptoms from those induced by biotic and abiotic stresses in vineyards. A multiplex PCR protocol was developed for simultaneous detection of GLRaV-3 and GRBV in grapevine samples. A global phylogenetic analysis of GRBV genome sequences revealed segregation of virus isolates from Washington State vineyards into two distinct clades, with the majority of isolates belonging to clade II.

The Distribution and Detection of Grapevine red blotch virus in its Host Depend on Time of Sampling and Tissue Type.

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative polymerase chain reaction (qPCR) method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a pattern similar to that found for the greenhouse-grown plants but, most strikingly, close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.