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Icosahedral virus-like particles were isolated from Petunia hybrida cuttings with interveinal chlorotic mottling. The virus was transmitted by mechanical inoculation from infected to healthy P. hybrida, and was found to contain a bipartite RNA genome of 7.6 and 3.8 kilobases. Full genomic sequence was obtained by high-throughput sequencing combined with RACE amplification of the 5′-termini of RNAs 1 and 2, and reverse-transcription PCR amplification of the 3′-termini with oligo-dT and sequence specific primers. Based on particle morphology, genome organization, and phylogenetic analyses, it was concluded that the new virus is a member of the genus Nepovirus, subgroup A. This new virus causing a leaf mottling disease of petunia was provisionally named Petunia chlorotic mottle virus (PCMoV).

The mechanisms underlying host plant symptom development upon infection by viruses of the genus

ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown to be required for the temperature-dependent symptom recovery of Nicotiana benthamiana plants infected with tomato ringspot virus (isolate ToRSV-Rasp1) at 27 °C. In this study, we show that symptom recovery from isolate ToRSV-GYV shares similar hallmarks of antiviral RNA silencing but occurs at a wider range of temperatures (21–27 °C). At 21 °C, an early spike in AGO2 mRNAs accumulation was observed in plants infected with either ToRSV-Rasp1 or ToRSV-GYV but the AGO2 protein was only consistently detected in ToRSV-GYV infected plants. Symptom recovery from ToRSV-GYV at 21 °C was not prevented in an ago2 mutant or by silencing of AGO1 or AGO2. We conclude that other factors (possibly other AGOs) contribute to symptom recovery under these conditions. The results also highlight distinct expression patterns of AGO2 in response to ToRSV isolates and environmental conditions.

Kundu J.K., Gadiou S., Schlesingerova G., Dziakova M., Cermak V. (2015): Emergence of quarantine Tobacco ringspot virus in Impatiens walleriana in the Czech Republic. Plant Protect. Sci., 51: 115–122. Tobacco ringspot virus (TRSV) is a quarantine pathogen in Europe. During an official inspection in November 2011, Impatiens walleriana plants showing symptoms were found in a nursery in the Czech Republic. The causal agent of the disease was detected as Nepovirus group A by RT-PCR using specific primers of the Nepovirus group. Sequence analysis of PCR fragments confirms that the detected virus is TRSV. TRSV detection in these plants was further con firmed by ELISA and one-step RT-PCR using specific primers. The coat protein (CP) gene of the Czech TRSV isolate was sequenced, and the sequence analysis showed high identity of both nucleotide (99.28%) and amino acid (99.96%) levels compared with other known TRSV isolates from GenBank. Two amino acid motifs characteristic of nepoviruses, FDDY (FDAY) and FWGR (FYGR), were found equally at positions 80 and 497 of the TRSV CP genes, respectively, including the sequences described in this study.

A reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) has been developed for detection of tomato black ring virus (TBRV) isolates collected from different hosts. One-step RT-LAMP was performed with a set of four primers, the design of which was based on the coat protein gene. Results of RT-LAMP were visualized by direct staining of products with fluorescent dyes, agarose gel electrophoresis, and analysis of amplification curves. The sensitivity of RT-LAMP was 100-fold greater than that of RT-PCR. The RT-LAMP assay developed here is a useful and practical method for diagnosis of TBRV.

To investigate the prevalence and genetic variation of grapevine fanleaf virus (GFLV) in China, 142 grapevine samples from 13 provinces and regions were tested using DAS-ELISA, RT-PCR, and nested RT-PCR. Of the samples, 38% tested positive for GFLV by DAS-ELISA, and 26.8% tested positive by RT-PCR and nested RT-PCR. Movement protein (MP) and coat protein (CP) gene PCR products were cloned and sequenced. The MP or CP nucleotide and protein sequences shared identities that ranged from 94.9% to 100%. Phylogenetic analysis revealed that Chinese GFLV isolates obtained in this study were distinct from the isolates reported in GenBank.

Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing, and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary interaction RNA devices exhibit improved performance in terms of gene silencing, activation ratio, and ligand sensitivity as compared to optimized RNA devices that rely on secondary structure changes. We apply our method to building biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate understanding of the underlying sequence-structure-function relationships that empower rational design of complex biomolecules.

Little is known about the natural occurrence and genetic variability of nepovirus large satellite RNA (satRNA). This study screened 71 Grapevine fanleaf virus (GFLV) isolates mainly from Slovenia, but also from other countries in Europe and the USA, for the presence of satRNA, using a newly developed RT-PCR assay. GFLV satRNA (satGFLV) was detected in 72% of naturally GFLV-infected grapevines analysed, which is the highest frequency of occurrence of satGFLV reported to date. From 39 naturally GFLV-infected grapevines, 122 satGFLV clones were sequenced and compared to publicly available sequences of satGFLVs and the closely related satRNAs from Arabis mosaic virus (satArMVs). Phylogenetic analyses of these satRNAs revealed that their evolution was driven by substitutions, insertions, deletions, recombinations and reassortments between closely related helper viruses. Phylogenetic relationships of the satGFLVs and satArMVs show their separate and subsequent common evolution. Furthermore, the satGFLVs varied in size and showed higher variability at the amino acid level than at the nucleotide level, just as the 2AHP gene of their helper virus. This study shows that satGFLVs are also similar to their helper virus with respect to their quasispecies nature and their transmission route through anthropogenic exchange of propagation material.

Members of the family Secoviridae, referred to as secovirids, are plant viruses that share features with animal and human viruses of the family Picornaviridae and other insect or marine viruses of the order Picornavirales. These common features include a conserved structure of the icosahedral virus particles, the expression of viral proteins by proteolytic cleavage of large polyproteins and viral replication proteins with conserved sequence motifs, including the viral RNA-dependent RNA polymerase and protease. Secovirids also share the distinguishing feature of encoding specialised proteins that enable their movement in plant and counteract plant defence mechanisms, such as RNA silencing. The family Secoviridae includes eight genera. Members of the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus and Torradovirus have a bipartite positive-strand RNA genome, whereas members of the genera Sequivirus and Waikavirus have a monopartite genome. Key Concepts Many plant viruses are related to animal and human viruses of the family Picornaviridae and to other picorna-like viruses infecting algae and arthropods. A recent update in the taxonomy of plant picorna-like viruses has led to the creation of the family Secoviridae which amalgamates the previous families Comoviridae and Sequiviridae as well as the genera Cheravirus, Sequivirus and Torradovirus. Secovirids share common characteristics including having both similar virus particle structures and genomic organizations and requiring specialised proteins to facilitate their movement within the host plant or counteract plant defence mechanism. Secovirids produce their proteins in the form of large polyproteins that are cleaved at specific sites by a viral protease. Replication of the viral RNA occurs in large protein complexes in association with intracellular membranes from the host. Plant cells infected with secovirids generally display tubular structures that are composed of the viral movement protein, contain virus-like particles and traverse the cell wall. These tubular structures are involved in the movement of the virus from cell to cell. Secovirids can be transmitted through seeds and pollen or with the help of nematode or arthropod vectors and their spread in the field is largely dependent on their mode of transmission. Some secovirids have been successfully exploited as plant vectors, allowing epitope presentation for vaccine production, expression of proteins in plants and silencing of endogenous plant genes. Keywords: picornavirales; protease; virus taxonomy; plant–virus interactions; cell-to-cell movement; virus replication

A producing strain of an anti-tumor and antiviral enzyme L-lysine-α-oxidase from Trichoderma was cultured using a technological device of G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino). L-lysine-α-oxidase activity in the obtained metabolite concentrate was 5.4 U/ml. We studied the effects of the concentrate of active L-lysine-α-oxidase producer on the highly infectious Tobacco ringspot virus and revealed anti-viral activity of it when enzyme concentration was at least 1.0 U/ml.

The complete RNA1 and RNA2 sequences of a new grapevine fanleaf virus isolate (GFLV-SDHN) from northeastern China were determined. The two RNAs are 7,367 and 3,788 nucleotides (nt) in length, respectively, excluding the poly(A) tails. Compared to other GFLV isolates, GFLV-SDHN has a 22- to 24-nt insertion in the RNA1 5′ untranslated region, and there was 19.1-20.1 % and 11.7 %–13.0 % sequence divergence in RNA1, and 15.5 %–20.5 % and 8.5-13.5 % in RNA2, at the nt and amino acid level, respectively. Phylogenetic analysis revealed that the origins of GFLV-SDHN are distinct from those of other GFLV isolates. One recombination event was identified in the 2AHP region of RNA2 in GFLV-SDHN.

The soil-borne nepovirus Grapevine fanleaf virus (GFLV), causal agent of grapevine fanleaf degeneration disease, spreads mainly via the nematode vector Xiphinema index. Since the arbuscular mycorrhizal (AM) fungus Rhizophagus intraradices can induce local and systemic protection against X. index (Hao et al., 2012), therefore, the objective of the present study was to investigate if AM may represent a means for limiting GFLV infection by reducing nematode vector attack. Three-week-old mycorrhizal or nonmycorrhizal grapevine rootstock SO4 (Vitis berlandieri × V. riparia) plants were inoculated with 10 or 100 viruliferous nematodes per plant under glasshouse conditions. Plants were harvested 35 and 90 days after inoculation with X. index (DAIN), prior to and after the appearance of fanleaf degeneration symptoms, respectively. Both levels of viruliferous X. index inhibited root growth of nonmycorrhizal plants at 90 DAIN, while the presence of arbuscular mycorrhizal colonization significantly decreased gall formation on roots at both time points and reduced nematode reproduction in soil at 90 DAIN. GFLV was not detected by one-step reverse transcription PCR in the roots of any plant at 35 DAIN after viruliferous X. index inoculation. At 90 DAIN with 10 viruliferous nematodes per plant, the virus was present in nonmycorrhizal roots but absent from mycorrhizal grapevine. The virus was detected in both mycorrhizal and nonmycorrhizal plants 90 DAIN. It is suggested that inhibition of X. index proliferation and penetration by AM may protect grapevine against GFLV at a low abundance of the viruliferous nematode in soils and so contribute to virus control under these conditions.

Tomato black ring virus (TBRV) is the only member of the Nepovirus genus that is known to form defective RNA particles (D RNAs) during replication. Here, de novo generation of D RNAs was observed during prolonged passages of TBRV isolates originated from Solanum lycopersicum and Lactuca sativa in Chenopodium quinoa plants. D RNAs of about 500 nt derived by a single deletion in the RNA1 molecule and contained a portion of the 5' untranslated region and viral replicase, and almost the entire 3' non-coding region. Short regions of sequence complementarity were found at the 5' and 3' junction borders, which can facilitate formation of the D RNAs. Moreover, in this study we analyzed the effects of D RNAs on TBRV replication and symptoms development of infected plants. C. quinoa, S. lycopersicum, Nicotiana tabacum, and L. sativa were infected with the original TBRV isolates (TBRV-D RNA) and those containing additional D RNA particles (TBRV + D RNA). The viral accumulation in particular hosts was measured up to 28 days post inoculation by RT-qPCR. Statistical analyses revealed that D RNAs interfere with TBRV replication and thus should be referred to as defective interfering particles. The magnitude of the interference effect depends on the interplay between TBRV isolate and host species., This work was supported by the National Science Centre, Poland (grant number 2017/25/B/NZ9/01715); Ministry of Science and Higher Education, Poland (grant number IP2014 014973) (to B.H.-J.) and Spain Agencia Estatal de Investigación-FEDER (grant number BFU2015-65037P) (to S.F.E.).

An isometric virus was identified in mulberry leaves showing symptoms of mulberry mosaic leaf roll (MMLR) disease. Its genome consists of two (+)ssRNAs. RNA1 and RNA2 have 7183 and 3742 nucleotides, excluding the 3'-terminal poly(A) tail. Based on phylogenetic analysis of the RNA1-encoded polyprotein and CP amino acid sequences, the properties of the the 3'-UTR of RNA1 and RNA2, and

Ribozyme (Rz) is a very attractive RNA molecule in metabolic engineering and synthetic biology fields where RNA processing is required as a control unit or ON/OFF signal for its cleavage reaction. In order to use Rz for such RNA processing, Rz must have highly active and specific catalytic activity. However, current methods for assessing the intracellular activity of Rz have limitations such as difficulty in handling and inaccuracies in the evaluation of correct cleavage activity. In this paper, we proposed a simple method to accurately measure the "intracellular cleavage efficiency" of Rz. This method deactivates unwanted activity of Rz which may consistently occur after cell lysis using DNA quenching method, and calculates the cleavage efficiency by analyzing the cleaved fraction of mRNA by Rz from the total amount of mRNA containing Rz via quantitative real-time PCR (qPCR). The proposed method was applied to measure "intracellular cleavage efficiency" of sTRSV, a representative Rz, and its mutant, and their intracellular cleavage efficiencies were calculated as 89% and 93%, respectively.

The Panel on Plant Health performed a pest categorisation of Longidorus diadecturus (Nematoda: Longidoridae) for the EU. The nematode is a well‐defined taxon and was described from Ontario, Canada and later reported from some states in the USA. The nematode is not present in the EU. It is regulated by Council Directive 2000/29/EC, listed in Annex I A I as L. diadecturus Eveleigh and Allen. It is a migratory ectoparasitic nematode species puncturing cells of plant roots thereby able to transmit the nepovirus Peach rosette mosaic virus (PRMV). The pest is found in soil associated with plant species belonging to different families. L. diadecturus is able to cause direct damage to plants, but its main damage is caused by vectoring PRMV. Soil is a potential pathway for this nematode for entry into the EU. The nematode is able to survive adverse conditions, but the virus may not persist inside the nematode for extended periods. Climatic conditions in the EU are similar to those found in the countries where the pest is currently present. Hosts of the nematode (and the associated virus) are, e.g. peaches and grapes; those crops are also widely cultivated in the EU. The nematode only moves short distances (around 1 m) but may be spread with soil moving activities. Measures are available to inhibit entry via soil as such. Entry of the nematode with soil attached to plants for planting that are not regulated is possible. L. diadecturus does satisfy all the criteria that are within the remit of EFSA to assess to be regarded as a potential Union quarantine pest.

Background Recent studies have begun to reveal the complex evolutionary and biogeographic histories of mainland anoles in Central America, but the origins and relationships of many taxa remain poorly understood. One such group is the Anolis (Norops) crassulus species subgroup, which contains ten morphologically similar highland taxa, the majority of which have restricted distributions. The nominal taxon A. crassulus has a disjunct distribution from Chiapas, Mexico, through Guatemala, in the highlands of El Salvador, and in the Chortís Highlands of Honduras. We test the relationships of these species using multiple mitochondrial and nuclear loci in concatenated and multispecies coalescent frameworks, in an effort to both resolve long-standing taxonomic confusion and present new insights into the evolution and biogeography of these taxa. Results Sequences of multiple mitochondrial and nuclear loci were generated for eight of the ten species of the Anolis crassulus species subgroup. We analyzed phylogenetic relationships and estimated divergence times and ancestral ranges of the subgroup, recovering a monophyletic subgroup within Anolis. Within the nominal taxon Anolis crassulus, we recovered multiple genetically distinct lineages corresponding to allopatric populations, and show that the Chortís Highland lineage split from the others over 13 MYA. Additionally, distinct mitochondrial lineages are present within the taxa A. heteropholidotus and A. morazani, and importantly, samples of A. crassulus and A. sminthus previously used in major anole phylogenetic analyses are not recovered as conspecific with those taxa. We infer a Chortís Highland origin for the ancestor of this subgroup, and estimate cladogenesis of this subgroup began approximately 22 MYA. Conclusions Our results provide new insights into the evolution, biogeography, and timing of diversification of the Anolis crassulus species subgroup. The disjunctly distributed Anolis crassulus sensu lato represents several morphologically conserved, molecularly distinct anoles, and several other species in the subgroup contain multiple isolated lineages. Electronic supplementary material The online version of this article (doi: 10.1186/s12862-017-1115-8) contains supplementary material, which is available to authorized users.

To determine the genetic diversity and population structure of grapevine fanleaf virus (GFLV), the complete nucleotide sequence of the coat protein gene of 41 isolates from different regions in Iran was determined. Phylogenetic analyses of these isolates together with those available in the GenBank revealed two evolutionary divergent lineages, designated GFLV-G and GFLV-Ir that reflect origin of the isolates. Analysis of the genetic variability in the coat protein of these isolates revealed 37 genotype groups in GFLV population. Analyses indicate that GFLV-G and GFLV-Ir clades are significantly differentiated populations of GFLV. Also, geographical subpopulations of the virus in Iran were completely distinct from each other. Examination of nonsynonymous/synonymous nucleotide diversity showed that the CP gene has been under purifying selection. The neutrality tests indicate balancing selection operating within isolates of the northwest of Iran and purifying selection within the other populations.

International audience; One of the greatest hindrances to the study of grapevine fanleaf virus (GFLV) is the dearth of robust protocols for reliable, scalable, and cost-effective inoculation of host plants, especially methods which allow for rapid and targeted manipulation of the virus genome. Agroinoculation fulfills these requirements: it is a relatively rapid, inexpensive, and reliable method for establishing infections, and enables genetic manipulation of viral sequences by modifying plasmids. We designed a system of binary plasmids based on the two genomic RNAs [RNA1 (1) and RNA2 (2)] of GFLV strains F13 (F) and GHu (G) and optimized parameters to maximize systemic infection frequency in Nicotiana benthamiana via agroinoculation. The genomic make-up of the inoculum (G1–G2 and reassortant F1–G2), the identity of the co-infiltrated silencing suppressor (grapevine leafroll associated virus 2 p24), and temperature at which plants were maintained (25 °C) significantly increased systemic infection, while high optical densities of infiltration cultures (OD600nm of 1.0 or 2.0) increased the consistency of systemic infection frequency in N. benthamiana. In contrast, acetosyringone in the bacterial culture media, regardless of concentration, had no effect. Plasmids in this system are amenable to rapid and reliable manipulation by one-step site-directed mutagenesis, as shown by the creation of infectious RNA1 chimeras of the GFLV-F13 and GHu strains. The GFLV agroinoculation plasmids described here, together with the optimized protocol for bacterial culturing and plant maintenance, provide a robust system for the establishment of systemic GFLV infection in N. benthamiana and the rapid generation of GFLV mutants, granting a much-needed tool for investigations into GFLV-host interactions.

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time.

International audience; Avirulence factors are critical for the arm's race between a virus and its host in determining incompatible reactions. The response of plants to viruses from the genus Nepovirus in the family Secoviridae, including Grapevine fanleaf virus (GFLV), is well characterized, although the nature and characteristics of the viral avirulence factor remain elusive. By using infectious clones of GFLV strains F13 and GHu in a reverse genetics approach with wild-type, assortant and chimeric viruses, the determinant of necrotic lesions caused by GFLV-F13 on inoculated leaves of Nicotiana occidentalis was mapped to the RNA2-encoded protein 2A(HP), particularly to its 50 C-terminal amino acids. The necrotic response showed hallmark characteristics of a genuine hypersensitive reaction, such as the accumulation of phytoalexins, reactive oxygen species, pathogenesis-related protein 1c and hypersensitivity-related (hsr) 203J transcripts. Transient expression of the GFLV-F13 protein 2A(HP) fused to an enhanced green fluorescent protein (EGFP) tag in N.occidentalis by agroinfiltration was sufficient to elicit a hypersensitive reaction. In addition, the GFLV-F13 avirulence factor, when introduced in GFLV-GHu, which causes a compatible reaction on N.occidentalis, elicited necrosis and partially restricted the virus. This is the first identification of a nepovirus avirulence factor that is responsible for a hypersensitive reaction in both the context of virus infection and transient expression.

We present here the first complete genomic sequence of Arracacha virus A from a Peruvian arracacha sample collected in 1975 and compare it with the genomes of other nepoviruses. Its RNA1 and RNA2 both had greatest amino acid identities with those of the subgroup A nepovirus Melon mild mottle virus .

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.

The RNA2 of seven grapevine fanleaf virus (GFLV) isolates from vines with yellow mosaic (YM) symptoms from different origin were sequenced. These sequences showed a high variability in the homing protein (2A(HP)) and, in five of them, a putative recombination with arabis mosaic virus (ArMV) was detected. To investigate recombination frequency, the partial sequences of the 2A(HP) of 28 additional GFLV isolates from nine different countries, showing either YM or infectious malformations (MF) symptoms, were obtained and compared with those of GFLV isolates from GenBank. The analysis confirmed the high level of sequence variability (up to 41 % at the nucleotide level) among isolates. In phylogenetic trees constructed using different approaches, the sequenced isolates always clustered in four conserved groups, three of which comprised YM strains (groups 1, 2 and 3), and one (group 4) the MF strains. Potential interspecific recombination sites between GFLV and ArMV were predicted in the 2A(HP) gene of several isolates, all of which were associated with YM symptoms.

The N-terminal domains of the RNA 2-encoded 2A(HP) proteins of the arabis mosaic (ArMV) and grapevine fanleaf (GFLV) nepoviruses were shown to be highly variable and a hotspot for intra- and inter-species recombination events. Chimeric ArMV-NW clones in which the N-terminal domain of 2A(HP) or the entire 2A(HP) of GFLV isolates replaced the corresponding domains of ArMV retained their infectivity, showing that the 2A(HP) proteins of ArMV-NW and GFLV are exchangeable. ArMN-NW clones with deletions of the N-terminal, core, or C-terminal domains of the ArMV-NW 2A(HP) were infectious in Chenopodium quinoa although viral RNA (especially RNA 2) accumulated at reduced levels. In contrast, deletion of the entire 2A(HP) protein or of the C-terminal two thirds of the protein abolished infectivity of the ArMV-NW clones. These results suggest that multiple functional domains are distributed throughout the 2A(HP) protein and are essential for the accumulation of viral RNA 2.

Grapevine fanleaf virus (GFLV), a member of the genus Nepovirus in the family Secoviridae, is the most widely distributed grapevine virus around the world. In the present research, recombinant antibody to the GFLV coat protein (CP) was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for the detection of GFLV by the use of a pair of primers corresponding to the full length CP gene. In addition, double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was done by the use of recombinant antibody to detect the GFLV isolates. Eventually, an expected band with around 1512 base pair (bp) in length was revealed for three isolates by IC-RT-PCR. Also, DAS-ELISA revealed that the recombinant antibody was able to detect the virus in leaf sample. These results showed that application of recombinant antibody in these detection methods can be useful for rapid detection of viruses in plant tissue because by applying IC-RT-PCR along with recombinant antibody, it i...

Factors involved in symptom expression of viruses from the genus Nepovirus in the family Secoviridae such as grapevine fanleaf virus (GFLV) are poorly characterized. To identify symptom determinants encoded by GFLV, infectious cDNA clones of RNA1 and RNA2 of strain GHu were developed and used alongside existing infectious cDNA clones of strain F13 in a reverse genetics approach. In vitro transcripts of homologous combinations of RNA1 and RNA2 induced systemic infection in Nicotiana benthamiana and Nicotiana clevelandii with identical phenotypes to WT virus strains, i.e. vein clearing and chlorotic spots on N. benthamiana and N. clevelandii for GHu, respectively, and lack of symptoms on both hosts for F13. The use of assorted transcripts mapped symptom determinants on RNA1 of GFLV strain GHu, in particular within the distal 408 nt of the RNA-dependent RNA polymerase (1EPol), as shown by RNA1 transcripts for which coding regions or fragments derived thereof were swapped. Semi-quantitative analyses indicated no significant differences in virus titre between symptomatic and asymptomatic plants infected with various recombinants. Also, unlike the nepovirus tomato ringspot virus, no apparent proteolytic cleavage of GFLV protein 1EPol was detected upon virus infection or transient expression in N. benthamiana. In addition, GFLV protein 1EPol failed to suppress silencing of EGFP in transgenic N. benthamiana expressing EGFP or to enhance GFP expression in patch assays in WT N. benthamiana. Together, our results suggest the existence of strain-specific functional domains, including a symptom determinant module, on the RNA-dependent RNA polymerase of GFLV.

A virus was isolated from potted plants of an unidentified species of Aeonium, a succulent ornamental very common in Southern Italy, showing chlorotic spots and rings on both leaf surfaces. It was successfully transmitted by sap inoculation to a limited range of hosts, including Nicotiana benthamiana which was used for ultrastructural observations and virus purification. Virus particles are isometric, ca. 30nm in diameter, have a single type of coat protein (CP) subunits 54kDa in size, that encapsidate single-stranded positive-sense RNA species of 7,549 (RNA1) and 4,010 (RNA2) nucleotides. A third RNA molecule 3,472 nts in size entirely derived from RNA2 was also found. The structural organization of both genomic RNAs and the cytopathological features were comparable to those of nepoviruses. In addition, amino acid sequence comparisons of CP and the Pro-Pol region (a sequence containing parts of the proteinase and polymerase) with those of other nepoviruses showed that the Aeonium virus belongs to the subgroup A of the genus Nepovirus and is phylogenetically close to, but serologically distinct from tobacco ringspot virus (TRSV). Based on the species demarcation criteria for the family Secoviridae, the virus under study appears to be a novel member of the genus Nepovirus for which the name of Aeonium ringspot virus (AeRSV) is proposed.

For quarantine purpose, we developed the RT- and nested PCR module of Tomato black ring virus (TBRV), Arabis mosaic virus (ArMV), Cherry leafroll virus (CLRV) and Grapevine fanleaf virus (GFLV). The PCR modules, developed in this study make diagnosis more convenient and speedy because of same PCR condition. And also, the methods are more accurate because it can check whether the result is contamination or not using the mutation-positive control. We discard or return the 27 cases of Nepovirus infection seed by employing the module past 3 years. This study provides a rapid and useful method for detection of four quarantine plant viruses.

International audience; Arabis mosaic virus (ArMV) and Grapevine fanleaf virus (GFLV) are two picorna-like viruses from the genus Nepovirus, consisting in a bipartite RNA genome encapsidated into a 30 nm icosahedral viral particle formed by 60 copies of a single capsid protein (CP). They are responsible for a severe degeneration of grapevines that occurs in most vineyards worldwide. Although sharing a high level of sequence identity between their CP, ArMV is transmitted exclusively by the ectoparasitic nematode Xiphinema diversicaudatum whereas GFLV is specifically transmitted by the nematode X. index. The structural determinants involved in the transmission specificity of both viruses map solely to their respective CP. Recently, reverse genetic and crystallographic studies on GFLV revealed that a positively charged pocket in the CP B domain located at the virus surface may be responsible for vector specificity. To go further into delineating the coat protein determinants involved in transmission specificity, we determined the 6.5 Å resolution cryo-electron microscopy structure of ArMV and used homology modeling and flexible fitting approaches to build its pseudo-atomic structure. This study allowed us to resolve ArMV CP architecture and delineate connections between ArMV capsid shell and its RNA. Comparison of ArMV and GFLV CPs reveals structural differences in the B domain pocket, thus strengthening the hypothesis of a key role of this region in the viral transmission specificity and identifies new potential functional domains of Nepovirus capsid.

Grapevine fanleaf virus (GFLV) is the major causal agent of the grapevine degeneration disease. To characterize the genomic RNA2 segment from Iranian isolates of GFLV, leaf samples were collected from infected vineyards in different locations with a long history of vine cultivation. Four isolates were selected for cloning and sequencing on the basis of the restriction profiles of RT-PCR products. The sequencing data revealed that the RNA2 of the Iranian GFLV isolates were the shortest compared with that of all previously described GFLV isolates. The sizes were 3730 nucleotides (nt) for Shir-Amin and Urmia isolates and 3749 nt for Takestan and Bonab isolates (excluding the poly (A) tail), due to deletion events in both 5′ and 3′ non-coding regions. In the phylogenetic tree based on the full-length nucleotide sequences of GFLV RNA2, all the GFLV isolates clustered into two groups with the exception of the Hungarian isolate (GHu). The Iranian isolates grouped as a distinct cluster. Recombination analyses showed that GFLV-NW (Germany), GFLV-F13 (reference isolate), GFLV isolate Shir-Amin (Iran) and Arabis mosaic virus isolate Lv were recombinant isolates and one of their parents belonged to the same lineage as the Iranian isolates. These findings suggest that these isolates originated from a common ancestor.

Nepoviruses supplied seminal landmarks to the historical trail of plant virology. Among the first agriculturally relevant viruses recognized in the late 1920s and among the first plant viruses officially classified in the early 1970s, nepoviruses also comprise the first species for which a soil-borne ectoparasitic nematode vector was identified. Early research on nepoviruses shed light on the genome structure and expression, biological properties of the two genomic RNAs, and mode of transmission. In recent years, research on nepoviruses enjoyed an extraordinary renaissance. This resurgence provided new insights into the molecular interface between viruses and their plant hosts, and between viruses and dagger nematode vectors to advance our understanding of some of the major steps of the infectious cycle. Here we examine these recent findings, highlight ongoing work, and offer some perspectives for future research.

International audience; Nematodes of the genera Longidorus, Paralongidorus, and Xiphinema in the family Longidoridae can parasitize grapevine roots. These ectoparasitic soil-borne nematodes live in proximity to the rhizosphere and use a stylet to feed on root cells. During the feeding process, some nematodes are able to acquire and transmit nepoviruses, the causal agents of grapevine degeneration disease. The association between nematode vectors and the virus(es) they transmit is specific. This specificity is linked to the nature, site, and mechanism of virus retention. Here, we present and discuss major features of the interaction between nematodes and viruses with regard to the biology, ecology, and life cycle of the nematodes. In addition, we discuss how reverse genetics in combination with virus structural data have recently provided insights into the specificity of the transmission process through the identification of a cavity-like domain on the virion surface that could be recognized by a ligand within the nematode feeding apparatus. Finally, we offer some perspectives on future research to further advance nematode-virus interactions.

Eleven of the 15 Vitis-infecting nepoviruses are thought to have an Old World origin, either Eurasian, i.e., Grapevine fanleaf virus (GFLV); European, i.e., Arabis mosaic virus (ArMV), tomato black ring virus (TBRV), Grapevine chrome mosaic virus (GCMV), Grapevine Bulgarian latent virus (GBLV), raspberry ringspot virus (RpRSV), artichoke Italian latent virus (AILV), and cherry leaf roll virus (CLRV); north African (Tunisia), i.e., Grapevine Tunisian ringspot virus (GTRSV); and Asiatic (Turkey and Iran), i.e., Grapevine deformation virus (GDeV) and Grapevine Anatolian ringspot virus (GARSV). Only four of these viruses (GFLV, ArMV, RpRSV, and TBRV) have ectoparasitic longidorid nematodes belonging to the genera Xiphinema, Longidorus, and Paralongidorus as recognized vectors. Whereas mechanical transfer to herbaceous indicators is readily achieved with all these viruses, their transmission through pollen and seeds is rare and does not seem to occur in grapevines. Some of these viruses (GFLV, GBLV, GCMV, GTRSV, GDeV, and GARSV) are apparently restricted to Vitis, while AILV, CLRV, RpRSV, and TBRV have a host range that includes woody and herbaceous crops, as well as weed species. All these viruses cause systemic, symptomatic infections in grapevines. Depending on the strains involved, infection with these viruses induces either chlorotic mottling and deformation of leaves and canes (by the distorting strains) or bright yellow discolorations of the leaves (by the chromogenic strains). Like all known nepoviruses, the grapevine-infecting ones from the Old World have a bipartite genome and require both genomic RNAs for infection. Planting selected stocks that have undergone sanitation and certification procedures in soils free of the nematode vectors should guarantee the sanitary conditions of new plantings for the lifespan of the vineyards. This is not the case for plantings in nematode-infested soils because removal of the roots from the previous stand and prolonged fallow period do not prevent the resurgence of the infection.

Dagger and needle nematodes included in the family Longidoridae (viz. Longidorus, Paralongidorus, and Xiphinema) are highly polyphagous plant-parasitic nematodes in wild and cultivated plants and some of them are plant-virus vectors (nepovirus). The mitochondrial (mt) genomes of the dagger and needle nematodes, Xiphinema rivesi, Xiphinema pachtaicum, Longidorus vineacola and Paralongidorus litoralis were sequenced in this study. The four circular mt genomes have an estimated size of 12.6, 12.5, 13.5 and 12.7 kb, respectively. Up to date, the mt genome of X. pachtaicum is the smallest genome found in Nematoda. The four mt genomes contain 12 protein-coding genes (viz. cox1-3, nad1-6, nad4L, atp6 and cob) and two ribosomal RNA genes (rrnL and rrnS), but the atp8 gene was not detected. These mt genomes showed a gene arrangement very different within the Longidoridae species sequenced, with the exception of very closely related species (X. americanum and X. rivesi). The sizes of non-coding regions in the Longidoridae nematodes were very small and were present in a few places in the mt genome. Phylogenetic analysis of all coding genes showed a closer relationship between Longidorus and Paralongidorus and different phylogenetic possibilities for the three Xiphinema species., This research was supported by grants P12-AGR 1486 and AGR-136 from ‘Consejeria de Economia, Innvovacion y Ciencia’ from Junta de Andalucia, and Union Europea, Fondo Europeo de Desarrollo regional, ‘Una manera de hacer Europa’, and grant 219262 ArimNET_ERANET FP7 2012–2015 Project PESTOLIVE ‘Contribution of olive history for the management of soilborne parasites in the Mediterranean basin’ from Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA). The James Hutton Institute receives financial support from the Scottish Government, Rural and Environment Science and Analyical Services Division.

Nepoviruses supplied seminal landmarks to the historical trail of plant virology. Among the first agriculturally relevant viruses recognized in the late 1920s and among the first plant viruses officially classified in the early 1970s, nepoviruses also comprise the first species for which a soil-borne ectoparasitic nematode vector was identified. Early research on nepoviruses shed light on the genome structure and expression, biological properties of the two genomic RNAs, and mode of transmission. In recent years, research on nepoviruses enjoyed an extraordinary renaissance. This resurgence provided new insights into the molecular interface between viruses and their plant hosts, and between viruses and dagger nematode vectors to advance our understanding of some of the major steps of the infectious cycle. Here we examine these recent findings, highlight ongoing work, and offer some perspectives for future research.

Grapevine fanleaf virus (GFLV) is one of the 15 viruses causing fanleaf degeneration, one of the most detrimental viral diseases of grapevines worldwide. GFLV belongs to the genus Nepovirus in the family Secoviridae. It was the first phytovirus for which transmission by an ectoparasitic dagger nematode vector was demonstrated and the first Nepovirus for which infectious clones were obtained, paving the way to studies on virus-vector-host interactions. Information on subcellular localization of GFLV-encoded proteins and the use of modified synthetic virus constructs resulted in a better understanding of virus movement and transmission. In recent years, advances on the identification of viral determinants involved in the specific transmission of GFLV by Xiphinema index were made, and the atomic structure of the virus was obtained at a 2.7 A resolution, revealing potential sites of interaction with the nematode vector at the surface of the particle. Host factors involved in the early steps of the virus cell-to-cell movement and viral determinants of symptom development in herbaceous hosts were identified. Here we review the current knowledge of GFLV with a special emphasis on some of its unique features compared to other nepoviruses. We also discuss the recent progress in regard to new antiviral strategies and suggest future research priorities.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is a tumorigenic rhadinovirus that is associated with all forms of Kaposi's sarcoma. Current serological detection of KSHV is based on enzyme-linked immunosorbent or immunofluorescence assays that suffer from a variety of problems, including the lack of defined standards for test comparison. While KSHV is the only known human rhadinovirus, two lineages of KSHV-like rhadinoviruses are found in Old World primates: the RV1 lineage includes KSHV and retroperitoneal fibromatosis herpesvirus (RFHV) in macaques, and the RV2 lineage includes RRV and MneRV2 from different macaque species. To develop animal models of KSHV-associated diseases, we developed quantitative multiplex bead-based serological assays to detect antibodies against rhadinovirus antigens. Proteins from KSHV (RV1) and MneRV2 (RV2) virions were coupled to spectrally distinct fluorescent beads and used in Luminex flow cytometry-based assays to detect immune responses in macaques. Both assays showed large dynamic ranges with high levels of seroreactivity to both KSHV and MneRV2 proteins. A large set of macaque serum samples from the Washington National Primate Research Center was screened, and most of the samples (82%) were positive in both assays, consistent with the high level of RV1-RV2 coinfection detected by PCR. The macaque sera showed broad, variable, and unique serological responses to the different viral antigens, allowing an initial seroprevalence to be determined for the macaque viruses. The Luminex assays offer a novel multiplexed approach to assess rhadinovirus infection patterns in both humans and nonhuman primates. This will help advance our understanding of rhadinovirus biology and associated host immunological responses.

The antioxidative response of grapevine leaves (Vitis vinifera cv. Trebbiano) affected by the presence of grapevine fanleaf virus was studied during the summer of 2010 at three different harvest times (July 1st and 26th, and August 30th). At the first and second harvest, infected leaves showed increases in the concentration of superoxide radical and hydrogen peroxide, the latter increasing for enhanced activity of superoxide dismutase. In contrast, at the last harvest time, increases in the ascorbate pool and ascorbate peroxidase activity maintained hydrogen peroxide to control levels. The glutathione pool was negatively affected as summer progressed, showing a decrease in its total and reduced form amounts. At the same time, increases in the ascorbate pool were observed, making antioxidant defenses of grapevine effective also at the last harvest time. Increases in phenolic acids, and in particular in p-hydroxybenzoic acid, at the first and second harvest might have enhanced the efficiency of the antioxidant system through an interrelation between a peroxidase/phenol/ascorbate system and the NADPH/glutathione/ascorbate cycle. The lack of increase in p-hydroxybenzoic acid at the third harvest could be due instead to the enhanced utilization of this acid for hydrogen peroxide detoxification. With time, grapevine plants lost their capacity to contrast the spread of grapevine fanleaf virus, but acquired a greater ability to counteract pathogen-induced oxidative stress, being endowed with more reduced antioxidant pools.