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The family Convolvulaceae comprises approximately 50-60 genera with approximately 1600-1700 species, exhibiting a rich diversity of morphological characteristics and occupying a broad range of ecological habitats. High-throughput sequencing identified a tentative new virus in the family Geminiviridae infecting Calystegia sepium var. japonica in South Korea. The 2,706 nt long genome comprises six open reading frames (ORFs). The analysis of the nucleotide sequence of the genome and the putative amino acid sequences of ORFs indicated that the virus was closely related to the members of the family Geminiviridae. The genome organization of the virus was similar to that of members of the genus Topilevirus in terms of the number of ORFs and splicing signal. However, the virus, tentatively named bindweed mottle virus (BWMV), may not be assigned to current genera into the family Geminiviridae., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Using a high-throughput sequencing (HTS) approach, we report the discovery of a new alphasatellite identified in a winter barley plant collected in France in 2022 that was also infected by wheat dwarf virus (WDV). The presence of the satellite and of WDV was confirmed by several independent PCR assays, and the complete genome sequence was determined. The circular satellite genome is 1424 nt long and shows typical hallmarks of members of the subfamily Geminialphasatellitinae, including a replication-associated hairpin with a CAGTATTAC sequence and a Rep-encoding open reading frame (ORF). It also possesses a second ORF, embedded in a different frame within the Rep ORF, which is also observed in clecrusatellites and a few other members of the family Alphasatellitidae. Pairwise sequence comparisons and phylogenetic analysis showed that this satellite represents a novel species. Its closest relatives are in the genus Colecusatellite, but it likely represents a new genus given its divergence from other genera of the subfamily Geminialphasatellitinae. Given that WDV was the only virus observed in coinfection with the satellite, the name "wheat dwarf virus-associated alphasatellite" is proposed for this novel agent., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The diversity of Geminiviridae and Alphasatellitidae species in tomatoes was assessed via high-throughput sequencing of 154 symptomatic foliar samples collected from 2002 to 2017 across seven Brazilian biomes. The first pool (BP1) comprised 73 samples from the North (13), Northeast (36), and South (24) regions. Sixteen begomoviruses and one Topilevirus were detected in BP1. Four begomovirus-like contigs were identified as putative novel species (NS). NS#1 was reported in the semi-arid (Northeast) region and NS#2 and NS#4 in mild subtropical climates (South region), whereas NS#3 was detected in the warm and humid (North) region. The second pool (BP2) comprised 81 samples from Southeast (39) and Central-West (42) regions. Fourteen viruses and subviral agents were detected in BP2, including two topileviruses, a putative novel begomovirus (NS#5), and two alphasatellites occurring in continental highland areas. The five putative novel begomoviruses displayed strict endemic distributions. Conversely, tomato mottle leaf curl virus (a monopartite species) displayed the most widespread distribution occurring across the seven sampled biomes. The overall diversity and frequency of mixed infections were higher in susceptible (16 viruses + alphasatellites) in comparison to tolerant (carrying the Ty -1 or Ty -3 introgressions) samples, which displayed 9 viruses. This complex panorama reinforces the notion that the tomato-associated Geminiviridae diversity is yet underestimated in Neotropical regions.

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.

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.

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.

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.

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.

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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.

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.

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.

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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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.

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 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.

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.

A viral metagenomics study of the sugarcane virome in Florida was carried out in 2013 to 2014 to analyze occurrence of known and potentially new viruses. In total, 214 sugarcane leaf samples were collected from different commercial sugarcane (Saccharum interspecific hybrids) fields in Florida and from other Saccharum and related species taken from two local germplasm collections. Virion-associated nucleic acids (VANA) metagenomics was used for detection and identification of viruses present within the collected leaf samples. VANA sequence reads were obtained for 204 leaf samples and all four previously reported sugarcane viruses occurring in Florida were detected: Sugarcane yellow leaf virus (SCYLV, 150 infected samples out of 204), Sugarcane mosaic virus (1 of 204), Sugarcane mild mosaic virus (13 of 204), and Sugarcane bacilliform virus (54 of 204). High prevalence of SCYLV in Florida commercial fields and germplasm collections was confirmed by reverse-transcription polymerase chain reaction. Sequence analyses revealed the presence of SCYLV isolates belonging to two different phylogenetic clades (I and II), including a new genotype of this virus. This viral metagenomics approach also resulted in the detection of a new sugarcane-infecting mastrevirus (recently described and named Sugarcane striate virus), and two potential new viruses in the genera Chrysovirus and Umbravirus.

The distribution and diversity of grapevine red blotch virus (GRBV) and wild Vitis virus 1 (WVV1) (genus Grablovirus; family Geminiviridae) were determined in free-living Vitis spp. in northern California and New York from 2013 to 2017. Grabloviruses were detected by polymerase chain reaction in 28% (57 of 203) of samples from California but in none of the 163 samples from New York. The incidence of GRBV in free-living vines was significantly higher in samples from California counties with high compared with low grape production (χ 2 = 83.09; P < 0.001), and in samples near (<5 km) to compared with far (>5 km) from vineyards (χ 2 = 57.58; P < 0.001). These results suggested a directional spread of GRBV inoculum predominantly from vineyards to free-living Vitis spp. WVV1 incidence was also significantly higher in areas with higher grape production acreage (χ 2 = 16.02; P < 0.001). However, in contrast to GRBV, no differential distribution of WVV1 incidence was observed with regard to distance from vineyards (χ 2 = 0.88; P = 0.3513). Two distinct phylogenetic clades were identified for both GRBV and WVV1 isolates from free-living Vitis spp., although the nucleotide sequence variability of the genomic diversity fragment was higher for WWV1 (94.3 to 99.8% sequence identity within clade 1 isolates and 90.1 to 100% within clade 2 isolates) than GRBV (98.3% between clade 1 isolates and 96.9 to 100% within clade 2 isolates). Additionally, evidence for intraspecific recombination events was found in WVV1 isolates and confirmed in GRBV isolates. The prevalence of grabloviruses in California free-living vines highlights the need for vigilance regarding potential grablovirus inoculum sources in order to protect new vineyard plantings and foundation stock vineyards in California.

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.

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.

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.

Plant viruses encode virulence factors or RNA silencing suppressors to reprogram plant cellular processes or to fine-tune host RNA silencing-mediated defense responses. In a previous study, Mulberry mosaic dwarf-associated virus (MMDaV), a novel, highly divergent geminivirus, has been identified from a Chinese mulberry tree showing mosaic and dwarfing symptoms, but the functions of its encoded proteins are unknown. In this study, all seven proteins encoded by MMDaV were screened for potential virulence and RNA silencing suppressor activities. We found that V2, RepA, and Rep affect the pathogenicity of a heterologous potato virus X. We showed that V2 could inhibit local RNA silencing and long-distance movement of the RNA silencing signal, but not short-range spread of the green fluorescent protein (GFP) silencing signal in Nicotiana benthamiana 16c plants. In addition, V2 localized to both subnuclear foci and the cytoplasm. Deletion mutagenesis of V2 showed that the basic motif from amino acids 61 to 76 was crucial for V2 to form subnuclear foci and for suppression of RNA silencing. Although the V2 protein encoded by begomoviruses or a curtovirus has been shown to have silencing suppressor activity, this is the first identification of an RNA silencing suppressor from a woody plant-infecting geminivirus.

Spinach is a common vegetable crop and very little data is available about its virus infection. Symptomatic leaves of spinach were collected during field survey. Circular DNA molecules were amplified from symptomatic samples using rolling circle amplification (RCA). After restriction analysis, presumed bands of virus and satellites were cloned, sequenced and analyzed. Analysis of sequenced RCA product revealed the presence of chickpea chlorotic dwarf virus (CpCDV; Mastrevirus). Further analyses of the cloned virus showed that strain "C" of CpCDV was present in symptomatic samples of spinach collected from field associated with vein darkening, curling and enations on leaves. Amplification of alpha- and betasatellites with universal primers was performed. CpCDV showed association with cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellites (CLCuMA). Infectivity analysis of CpCDV and CpCDV/CLCuMB were done in N. benthamiana using particle bombardment method and the results showed that CpCDV was able to transreplicates CLCuMB in this host. To our knowledge, this is the first report of a dicot infecting mastrevirus (CpCDV) along with CLCuMB and CLCuMA associated with leaf curl disease of spinach in Pakistan. The significance of the results is discussed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nanoviruses and geminiviruses are circular, single stranded DNA viruses that infect many plant species around the world. Nanoviruses and certain geminiviruses that belong to the Begomovirus and Mastrevirus genera are associated with additional circular, single stranded DNA molecules (~ 1-1.4 kb) that encode a replication-associated protein (Rep). These Rep-encoding satellite molecules are commonly referred to as alphasatellites and here we communicate the establishment of the family Alphasatellitidae to which these have been assigned. Within the Alphasatellitidae family two subfamilies, Geminialphasatellitinae and Nanoalphasatellitinae, have been established to respectively accommodate the geminivirus- and nanovirus-associated alphasatellites. Whereas the pairwise nucleotide sequence identity distribution of all the known geminialphasatellites (n = 628) displayed a troughs at ~ 70% and 88% pairwise identity, that of the known nanoalphasatellites (n = 54) had a troughs at ~ 67% and ~ 80% pairwise identity. We use these pairwise identity values as thresholds together with phylogenetic analyses to establish four genera and 43 species of geminialphasatellites and seven genera and 19 species of nanoalphasatellites. Furthermore, a divergent alphasatellite associated with coconut foliar decay disease is assigned to a species but not a subfamily as it likely represents a new alphasatellite subfamily that could be established once other closely related molecules are discovered.

Next-generation sequencing of small RNAs and PCR amplification with two pairs of back-to-back primers, followed by cloning and sequencing, allowed identification of a novel geminivirus isolate provisionally named camellia chlorotic dwarf-associated virus (CaCDaV). The complete genome sequence of CaCDaV comprises 3,687 nucleotides (nts) and six open reading frames (ORFs). The genome and putative proteins of CaCDaV were most closely related to those of two unclassified geminiviruses: citrus chlorotic dwarf-associated virus (CCDaV, 3,640 nt) and mulberry mosaic dwarf-associated virus (MMDaV, 2,952 nt). Phylogenetic analysis revealed a distinct clade for CaCDaV, CCDaV and MMDaV, supporting their inclusion in a new genus within the family Geminiviridae. Because CaCDaV and CCDaV shared a high genome nucleotide sequence identity (54.8%), we propose that the CaCDaV should be included as a member in this new genus.

Beet mild curly top virus (BMCTV) is associated with an outbreak of curly top in chili pepper, tomato and other Solanaceae species, which can cause severe crop losses. The aim of this work was to obtain the 1 H NMR metabolomic profiling of both healthy chili peppers (cv. mirasol) and infected chili peppers with BMCTV in order to find chemical markers associated to the infection process. Significant differences were found between the two groups, according to principal component analysis and orthogonal projections to latent structure discriminant analysis. Compared to the asymptomatic peppers, the symptomatic fruits had higher relative abundance of fructose, isoleucine, histidine, phenylalanine and tryptophan. Contrarily, the asymptomatic samples showed greater amounts of malonate and isobutyrate. These results suggest that in diseased chili peppers there are metabolic changes related to the viral acquisition of energy for replication and capsid assembly. This is the first study describing the chemical profiling of a polar extract obtained from Capsicum annuum infected by BMCTV under open field conditions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Wheat dwarf disease caused by wheat dwarf virus (WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein (CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines (i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies (MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840 (w/v, g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120 (w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.

We investigated the molecular characteristics of an Argentinean isolate of alfalfa leaf curl virus (ALCV-Arg), a virus of the genus Capulavirus in the family Geminiviridae that was isolated from alfalfa plants showing dwarfism. The genome was found to be 2,750 nucleotides in length. In pairwise comparisons, this ALCV isolate shared 83.2% to 92.6% sequence identity with European ALCV isolates. Sequence comparisons and phylogenetic analysis showed that this isolate combines features of strains A and B of ALCV. Recombination analysis showed that ALCV-Arg is a recombinant isolate that was generated by intraspecific recombination between ALCV strains A and B. The results of this study not only show that ALCV-Arg is unique because it combines features of strains A and B but also show that ALCV naturally infects this forage crop on the American continent.

The number of plant viruses that are known likely remains only a vanishingly small fraction of all extant plant virus species. Consequently, the distribution and population dynamics of plant viruses within even the best-studied ecosystems have only ever been studied for small groups of virus species. Even for the best studied of these groups very little is known about virus diversity at spatial scales ranging from an individual host, through individual local host populations to global host populations. To date, metagenomics studies that have assessed the collective or metagenomes of viruses at the ecosystem scale have revealed many previously unrecognized viral species. More recently, novel georeferenced metagenomics approaches have been devised that can precisely link individual sequence reads to both the plant hosts from which they were obtained, and the spatial arrangements of these hosts. Besides illuminating the diversity and the distribution of plant viruses at the ecosystem scale, application of these "geometagenomics" approaches has enabled the direct testing of hypotheses relating to the impacts of host diversity, host spatial variations, and environmental conditions on plant virus diversity and prevalence. To exemplify how such top-down approaches can provide a far deeper understanding of host-virus associations, we provide a case-study focusing on geminiviruses within two complex ecosystems containing both cultivated and uncultivated areas. Geminiviruses are a highly relevant model for studying the evolutionary and ecological aspects of viral emergence because the family Geminiviridae includes many of the most important crop pathogens that have emerged over the past century. In addition to revealing unprecedented degrees of geminivirus diversity within the analyzed ecosystems, the geometagenomics-based approach enabled the focused in-depth analysis of the complex evolutionary dynamics of some of the highly divergent geminivirus species that were discovered., (© 2018 Elsevier Inc. All rights reserved.)

Limited information is available on the spread of Grapevine red blotch virus (GRBV, genus Grablovirus, family Geminiviridae) in vineyards. To investigate ecological aspects of red blotch disease spread, sticky cards to catch flying insects were placed in 2015 (April to November) and 2016 (March to November) in a vineyard study site in California where disease incidence increased by nearly 20% between 2014 and 2016. Subsets of insect species or taxa were removed from sticky card traps and individual specimens were tested for the presence of GRBV by multiplex polymerase chain reaction. GRBV was consistently detected in Spissistilus festinus (Membracidae), Colladonus reductus (Cicadellidae), Osbornellus borealis (Cicadellidae), and a Melanoliarus sp. (Cixiidae). Populations of these four candidate vectors peaked from June to September, with viruliferous S. festinus peaking from late June to early July in both years. An assessment of co-occurrence and covariation between the spatial distribution of GRBV-infected vines and viruliferous insects identified a significant association only with viruliferous S. festinus. These findings revealed the epidemiological relevance of S. festinus as a vector of GRBV in a vineyard ecosystem. Sequencing coat protein and replicase-associated protein gene fragments of GRBV isolates from newly infected vines and viruliferous vector candidates further suggested secondary spread primarily from local sources and occasionally from background sources.

In fall 2014, 5 to 75% percent of chili and bell pepper (Capsicum annuum L.) in commercial fields located in the Mexican states of Durango, Zacatecas, and Michoacán had symptoms of deformed, small, mosaic, curled, and chlorotic leaves; shortened internodes; plant dwarfing; or phyllody and rosetting leaf tips. At the same time, leafhoppers and psyllids were observed in the fields, and more than 50 beet leafhoppers (Circulifer tenellus) and nearly 300 potato psyllids (Bactericera cockerelli) were collected from the pepper plants and adjacent weeds. Based on the insect pressure and observed symptoms, nearly 400 pepper samples were collected across this region of Mexico and tested for the presence of leafhopper- and psyllid-associated pathogens. In all, 76% of the pepper samples were found to be infected with 'Candidatus Liberibacter solanacearum', beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma, a strain of a curtovirus, or a combination of any two or three of these pathogens. Additionally, 77% of the collected leafhoppers and 40% of the psyllids were infected with one or more of these pathogens, in addition to Spiroplasma citri. Specifically, the leafhoppers were infected with BLTVA phytoplasma, S. citri, or a strain of curtovirus. Of particular interest, potato psyllids were not only infected with 'Ca. L. solanacearum' but also with phytoplasmas that belong to the groups 16SrVI subgroup A and 16SrI subgroup A. The presence of mixed infections in pepper plants and the insect vectors highlights the need for growers to effectively control both leafhoppers and potato psyllids from solanaceous crops in this region of Mexico in order to prevent the spread of these bacterial and viral pathogens.

Wheat dwarf virus (WDV), barley yellow striate mosaic virus (BYSMV), rice black-streaked dwarf virus (RBSDV) and northern cereal mosaic virus (NCMV) are four viruses infecting wheat and causing similar symptoms. In this paper, a multiplex reverse transcription polymerase chain reaction (m-RT-PCR) method has been developed for the simultaneous detection and discrimination of these viruses. The protocol uses specific primer set for each virus and produces four distinct fragments (273, 565, 783 and 1296bp), detecting the presence of RBSDV, BYSMV, WDV and NCMV, respectively. Annealing temperature, concentrations of dNTP, Taq polymerase and Mg 2+ were optimized for the m-RT-PCR. The detection limit of the assay was up to 10 -2 dilution. The amplification specificity of these primers was tested against a range of field samples from different regions of China, where RBSDV, BYSMV, WDV have been detected. This study fulfills the need for a rapid and specific wheat virus detection that also has the potential for investigating the epidemiology of these new viral diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

RNA silencing has an important role in defending against virus infection in plants. Plants with the deficiency of RNA silencing components often show enhanced susceptibility to viral infections. RNA-dependent RNA polymerase (RDRs) mediated-antiviral defense has a pivotal role in resistance to many plant viruses. In RDR6-mediated defense against viral infection, a plant-specific RNA binding protein, Suppressor of Gene Silencing 3 (SGS3), was also found to fight against some viruses in Arabidopsis . In this study, we showed that SGS3 from Nicotiana benthamiana ( NbSGS3 ) is required for sense-RNA induced post-transcriptional gene silencing (S-PTGS) and initiating sense-RNA-triggered systemic silencing. Further, the deficiency of NbSGS3 inhibited geminivirus-induced endogenous gene silencing (GIEGS) and promoted geminivirus infection. During TRV-mediated NbSGS3 or N. benthamiana RDR6 (NbRDR6) silencing process, we found that their expression can be effectively fine-tuned. Plants with the knock-down of both NbSGS3 and NbRDR6 almost totally blocked GIEGS, and were more susceptible to geminivirus infection. These data suggest that NbSGS3 cooperates with NbRDR6 against GIEGS and geminivirus infection in N. benthamiana , which provides valuable information for breeding geminivirus-resistant plants., Competing Interests: The authors declare no conflict of interest.

Viruses in the family Geminiviridae have single-stranded DNA genomes encapsulated in geminate icosahedral particles. High throughput sequencing (HTS) for metagenomic approaches are being extensively used for the identification of known and novel viruses. Using a HTS approach, we identified a novel geminivirus in a tomato (Solanum lycopersicum) sample and a Cleome sp. sample collected in the midwest region of Brazil. The genomes from the two samples share 99.96% identity and ∼61-63% to genomes in the genus Capulavirus. The novel virus has been tentatively named tomato associated geminivirus 1 (TaGV1). No visual symptoms were observed in the field tomato plant or in the inoculated Nicotiana benthamiana where the virus established a systemic infection. The replication associated protein of TaGV1 is most similar to that encoded by capulaviruses (sharing 62-70% identity), whereas the CP is most similar to that of tomato pseudo curly top virus (sharing ∼31% identity). In the TaGV1 positive Cleome sp. sample, begomovirus DNA A and B components were also detected sharing 96% and 90% sequence identity to cleome leaf crumple virus DNA A and B components, respectively. Using a HTS approach, we identified TaGV1 in tomato and Cleome sp. samples and this is the first report of a geminivirus that is non-begomovirus in Brazil., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: In Africa and Asia, sugarcane is the host of at least seven different virus species in the genus Mastrevirus of the family Geminiviridae. However, with the exception of Sugarcane white streak virus in Barbados, no other sugarcane-infecting mastrevirus has been reported in the New World. Conservation and exchange of sugarcane germplasm using stalk cuttings facilitates the spread of sugarcane-infecting viruses., Methods: A virion-associated nucleic acids (VANA)-based metagenomics approach was used to detect mastrevirus sequences in 717 sugarcane samples from Florida (USA), Guadeloupe (French West Indies), and Réunion (Mascarene Islands). Contig assembly was performed using CAP3 and sequence searches using BLASTn and BLASTx. Mastrevirus full genomes were enriched from total DNA by rolling circle amplification, cloned and sequenced. Nucleotide and amino acid sequence identities were determined using SDT v1.2. Phylogenetic analyses were conducted using MEGA6 and PHYML3., Results: We identified a new sugarcane-infecting mastrevirus in six plants sampled from germplasm collections in Florida and Guadeloupe. Full genome sequences were determined and analyzed for three virus isolates from Florida, and three from Guadeloupe. These six genomes share >88% genome-wide pairwise identity with one another and between 89 and 97% identity with a recently identified mastrevirus (KR150789) from a sugarcane plant sampled in China. Sequences similar to these were also identified in sugarcane plants in Réunion., Conclusions: As these virus isolates share <64% genome-wide identity with all other known mastreviruses, we propose classifying them within a new mastrevirus species named Sugarcane striate virus. This is the first report of sugarcane striate virus (SCStV) in the Western Hemisphere, a virus that most likely originated in Asia. The distribution, vector, and impact of SCStV on sugarcane production remains to be determined.

The discovery and full-genome sequences of two isolates of a fourth capulavirus species are reported. The viruses were discovered during a viral metagenomics survey of uncultivated Plantago lanceolata plants in the Åland archipelago of south western Finland. The newly discovered viruses apparently produce no symptoms in P. lanceolata. They have a genome organization that is very similar to that of the three known capulavirus species and additionally share between 62.9 and 67.1% genome-wide sequence identity with the isolates of these species. It is therefore proposed that these viruses be assigned to a new capulavirus species named "Plantago lanceolata latent virus".

Most known mastreviruses (family Geminiviridae) infect members of the grass family, Poaceae. Although the greatest number of grass-infecting mastrevirus species have been discovered in Africa, it is apparent that the ten grass-infecting mastrevirus species that have so far only been discovered in south-east Queensland have a degree of diversity that rivals that observed in Africa. In this study, we have used a deep sequencing approach to identify two new mastrevirus species, tentatively named rice latent virus 1 and 2 (RLV 1 and 2), from two, undescribed wild rice species (Oryza AA genome group) in Cape York Peninsula, Queensland. The sequences of these new viruses had less than 70% identity with any previously identified mastrevirus, and therefore their discovery vastly expands the known diversity of monocot-infecting mastreviruses in Australia. This study also highlights the potential risks of novel crop pathogens emerging from uncultivated grass species, as the wild rice hosts are very closely related to domesticated rice., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Maize streak virus (MSV), an important pathogen of maize in Africa, is the most extensively studied member of the Mastrevirus genus in the family Geminiviridae. Comparatively little is known about other monocot-infecting African mastreviruses, most of which infect uncultivated grasses. Here we determine the complete sequences of 134 full African mastrevirus genomes from predominantly uncultivated Poaceae species. Based on established taxonomic guidelines for the genus Mastrevirus, these genomes could be classified as belonging to the species Maize streak virus, Eragrostis minor streak virus, Maize streak Reunion virus, Panicum streak virus, Sugarcane streak Reunion virus and Sugarcane streak virus. Together with all other publicly available African monocot-infecting mastreviruses, the 134 new isolates extend the known geographical distributions of many of these species, including MSV which we found infecting Digitaria sp. on the island of Grand Canaria: the first definitive discovery of any African monocot-infecting mastreviruses north-west of the Saharan desert. These new isolates also extend the known host ranges of both African mastrevirus species and the strains within these. Most notable was the discovery of MSV-C isolates infecting maize which suggests that this MSV strain, which had previously only ever been found infecting uncultivated species, may be in the process of becoming adapted to this important staple crop., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Geminiviruses are plant-infecting single-stranded DNA viruses that occur in most parts of the world. Currently, there are seven genera within the family Geminiviridae (Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus). The rate of discovery of new geminiviruses has increased significantly over the last decade as a result of new molecular tools and approaches (rolling-circle amplification and deep sequencing) that allow for high-throughput workflows. Here, we report the establishment of two new genera: Capulavirus, with four new species (Alfalfa leaf curl virus, Euphorbia caput-medusae latent virus, French bean severe leaf curl virus and Plantago lanceolata latent virus), and Grablovirus, with one new species (Grapevine red blotch virus). The aphid species Aphis craccivora has been shown to be a vector for Alfalfa leaf curl virus, and the treehopper species Spissistilus festinus is the likely vector of Grapevine red blotch virus. In addition, two highly divergent groups of viruses found infecting citrus and mulberry plants have been assigned to the new species Citrus chlorotic dwarf associated virus and Mulberry mosaic dwarf associated virus, respectively. These species have been left unassigned to a genus by the ICTV because their particle morphology and insect vectors are unknown.

This is the first description of full genome sequences of chickpea chlorotic dwarf virus (CpCDV; genus Mastrevirus; family Geminiviridae) identified in papaya and tomato plants sampled in Burkina Faso. The CpCDV full genome sequences from papaya and tomato share the highest pairwise sequence identity (84% and 93.5%) with Sudanese isolates of the CpCDV-K and CpCDV-M strains, respectively. Based on the strain demarcation threshold (>94% identity) for mastreviruses, we propose two new strains, CpCDV-Q and CpCDV-R, identified in papaya and tomato, respectively. Phylogenetic analysis confirmed that the sequences belong to a distinct clade of the highly diverse population of CpCDVs. Evidence of inter-strain recombination provided more support for the important role of recombination in CpCDV evolution. The discovery of CpCDV on papaya, a previously unsuspected host, raises many questions about the natural and potential host range of this dicot-infecting mastrevirus species that is reported to be emerging worldwide.

A novel virus was detected in grapevines by Illumina sequencing during the screening of two table grape (Vitis vinifera) accessions, cultivars Black Beet and Nagano Purple, from South Korea. The monopartite circular ssDNA genome sequence was subsequently confirmed by rolling cycle amplification, cloning and Sanger sequencing. The complete viral genomic sequence from both accessions ranged from 2,903 to 2,907 nucleotides in length and contained the conserved nonanucleotide sequence TAATATT↓AC and other sequence features typical of the family Geminiviridae, including two predicted sense and four complementary-sense open reading frames. Phylogenetic analysis placed the novel virus in a unique taxon within the family Geminiviridae. A naturally occurring defective subviral DNA was also discovered. This defective DNA molecule carried a deletion of approximately 46% of the full-length genome. Both the genomic and defective DNA molecules were graft-transmissible although no disease is yet correlated with their occurrence in Vitis spp. The tentative names Grapevine geminivirus A (GGVA) and GGVA defective DNA (GGVA D-DNA) are proposed. PCR assays developed using primers designed in the coat protein gene led to the detection of GGVA in 1.74% of 1,262 vines derived from 15 grapevine cultivars from six countries across three continents.

Little is known about the prevalence, diversity, evolutionary processes, genomic structures and population dynamics of viruses in the divergent geminivirus lineage known as the capulaviruses. We determined and analyzed full genome sequences of 13 Euphorbia caput-medusae latent virus (EcmLV) and 26 Alfalfa leaf curl virus (ALCV) isolates, and partial genome sequences of 23 EcmLV and 37 ALCV isolates. While EcmLV was asymptomatic in uncultivated southern African Euphorbia caput-medusae, severe alfalfa disease symptoms were associated with ALCV in southern France. The prevalence of both viruses exceeded 10% in their respective hosts. Besides using patterns of detectable negative selection to identify ORFs that are probably functionally expressed, we show that ALCV and EcmLV both display evidence of inter-species recombination and biologically functional genomic secondary structures. Finally, we show that whereas the EcmLV populations likely experience restricted geographical dispersion, ALCV is probably freely moving across the French Mediterranean region., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Turnip curly top virus (TCTV) is the only member of the newly established genus Turncurtovirus (family Geminiviridae). As part of an ongoing study to identify additional plant hosts and the diversity of turncurtoviruses, between 2012 and 2014, we sampled symptomatic turnip plants and other crops in the provinces Fars and Khorasan Razavi (southern and northeastern Iran, respectively). Infection by turncurtoviruses was tested by PCR and/or rolling-circle amplification (RCA) coupled with restriction enzyme digests. Turncurtoviruses were identified in turnip as well as seven other field crops, including eggplant, basil, radish, lettuce, sugar beet, red beet and spinach. Full turncurtovirus genomes were recovered from 25 of these samples, leading to the identification of TCTV and a new putative turncurtovirus, turnip leaf roll virus (TLRV; 13 isolates), which shares <80% genome-wide pairwise identity with TCTV. Agroinoculation of plants with an infectious clone of TLRV demonstrated that this virus could infect several plant hosts under greenhouse conditions and could be transmitted by the leafhopper Circulifer haematoceps (Mulsant and Rey, 1855) from agroinoculated to healthy plants.

Geminiviruses are among the most serious pathogens of many economically important crop plants and RNA interference (RNAi) is an important strategy for their control. Although any fragment of a viral genome can be used to generate a double stranded (ds) RNA trigger, the precursor for generation of siRNAs, the exact sequence and size requirements for efficient gene silencing and virus resistance have so far not been investigated. Previous efforts to control geminiviruses by gene silencing mostly targeted AC1, the gene encoding replication-associated protein. In this study we made RNAi constructs for all the genes of both the genomic components (DNA-A and DNA-B) of African cassava mosaic virus (ACMV-CM), one of the most devastating geminiviruses causing cassava mosaic disease (CMD) in Africa. Using transient agro-infiltration studies, RNAi constructs were evaluated for their ability to trigger gene silencing against the invading virus and protection against it. The results show that the selection of the DNA target sequence is an important determinant for the amount of siRNA produced and the extent of resistance. The ACMV genes AC1, AC2, AC4 from DNA-A and BC1 from DNA-B were effective targets for RNAi-mediated resistance and their siRNA expression was higher compared to other RNAi constructs. The RNAi construct targeting AC2, the suppressor of gene silencing of ACMV-CM gave highest level of resistance in the transient studies. This is the first report of targeting DNA-B to confer resistance to a bipartite geminivirus infection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The RNAi concept was explored to silence the rep gene from the bean golden mosaic virus (BGMV) and a genetically modified (GM) bean immune to the virus was previously generated. We investigated if BGMV-viruliferous whiteflies would reduce viral amount after feeding on GM plants. BGMV DNA amount was significantly reduced in whiteflies feeding in GM-plants (compared with insects feeding on non-GM plants) for a period of 4 and 8 days in 52% and 84% respectively., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Maize streak Reunion virus (MSRV) is a member of the Mastrevirus genus in the family Geminiviridae. Of the diverse and increasing number of mastrevirus species found so far, only Wheat dwarf virus and Sweetpotato symptomless virus 1 have been discovered in China. Recently, a novel, unbiased approach based on deep sequencing of small interfering RNAs followed by de novo assembly of siRNA, has greatly offered opportunities for plant virus identification., Methods: Samples collected from maize leaves was deep sequencing for virus identification. Subsequently, the assay of PCR, rolling circle amplification and Southern blot were used to confirm the presence of a mastrevirus., Results: Maize streak Reunion virus Yunnan isolate (MSRV-[China:Yunnan 06:2014], abbreviated to MSRV-YN) was identified from maize collected from Yunnan Province, China, by small RNA deep sequencing. The complete genome of this virus was ascertained as 2,880 nucleotides long by conventional sequencing. A phylogenetic analysis showed it shared 96.3 % nucleotide sequence identity with the isolate of Maize streak Reunion virus from La Reunion Island. To our knowledge, this is the first identification of MSRV in China. Analyses of the viral derived small interfering RNAs (vsiRNAs) profile showed that the most abundant MSRV-YN vsiRNAs were 21, 22 and 24 nt long and biased for A and G at their 5' terminal residue. There was a slightly higher representation of MSRV-YN siRNAs derived from the virion-sense strand genome than the complementary-sense strand genome. Moreover, MSRV-YN vsiRNAs were not uniformly distributed along the genome, and hotspots were detected in the movement protein and coat protein-coding region., Conclusions: A mastrevirus MSRV-YN collected in Yunnan Province, China, was identified by small RNA deep sequencing. This vsiRNAs profile derived from MSRV-YN was characterized, which might contribute to get an insight into the host RNA silencing defense induced by MSRV-YN, and provide guidelines on designing antiviral strategies using RNAi against MSRV-YN.