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In 2012, dormant canes of a proprietary wine grape (Vitis vinifera L.) accession were included in the collection of the University of California–Davis Foundation Plant Services. No virus-like symptoms were elicited when bud chips from propagated own-rooted canes of the accession were graft-inoculated onto a panel of biological indicators. However, chlorotic ringspot symptoms were observed on sap-inoculated Chenopodium amaranticolor Coste & A. Rein and C. quinoa Willd. plants, indicating the presence of a mechanically transmissible virus. Transmission electron microscopy of virus preparations from symptomatic C. quinoa revealed spherical, nonenveloped virions about 27 nm in diameter. Nepovirus-like haplotypes of sequence contigs were detected in both the source grape accession and symptomatic C. quinoa plants via high-throughput sequencing. A novel bipartite nepovirus-like genome was assembled from these contigs, and the termini of each RNA segment were verified by rapid amplification of complementary DNA ends assays. The RNA1 (7,186-nt) of the virus encodes a large polyprotein 1 of 231.1 kDa, and the RNA2 (4,460-nt) encodes a large polyprotein 2 of 148.9 kDa. Each of the polyadenylated RNA segments is flanked by 5′- (RNA1 = 156-nt; RNA2 = 170-nt) and 3′- (RNA1 = 834-nt; RNA2 = 261-nt) untranslated region sequences with >90% identities. Maximum likelihood phylogenetic analyses of the conserved Pro-Pol amino acid sequences revealed the clustering of the new virus within the genus Nepovirus of the family Secoviridae. Considering its biological and molecular characteristics, and based on current taxonomic criteria, we propose that the novel virus, named grapevine nepovirus A, be assigned to the genus Nepovirus.

Data mining and metagenomic analysis of 277 open reading frame sequences of bipartite RNA viruses of the genus Nepovirus, family Secoviridae, were performed, documenting how challenging it can be to unequivocally assign a virus to a particular species, especially those in subgroups A and C, based on some of the currently adopted taxonomic demarcation criteria. This work suggests a possible need for their amendment to accommodate pangenome information. In addition, we revealed a host-dependent structure of arabis mosaic virus (ArMV) populations at a cladistic level and confirmed a phylogeographic structure of grapevine fanleaf virus (GFLV) populations. We also identified new putative recombination events in members of subgroups A, B and C. The evolutionary specificity of some capsid regions of ArMV and GFLV that were described previously and biologically validated as determinants of nematode transmission was circumscribed in silico. Furthermore, a C-terminal segment of the RNA-dependent RNA polymerase of members of subgroup A was predicted to be a putative host range determinant based on statistically supported higher π (substitutions per site) values for GFLV and ArMV isolates infecting Vitis spp. compared with non-Vitis-infecting ArMV isolates. This study illustrates how sequence information obtained via high-throughput sequencing can increase our understanding of mechanisms that modulate virus diversity and evolution and create new opportunities for advancing studies on the biology of economically important plant viruses.

Complete genome sequences of two cycas necrotic stunt virus (CNSV) isolates from Paeonia suffruticosa and Daphne odora were determined. Phylogenetic trees and pairwise comparisons using complete RNA1- and RNA2-encoded polyproteins showed that the two CNSV isolates are divergent (83.19%-89.42% in polyprotein 1 and 73.61%-85.78% in polyprotein 2). A comparative analysis based on taxonomic criteria for the species demarcation of nepoviruses confirmed that they are not new species but distinct variants. This is the first report of the complete genome sequences of CNSV detected in P. suffruticosa and D. odora, and the first report of CNSV infecting P. suffruticosa.

A novel nepovirus was identified and characterised from caraway, and tentatively named caraway yellows virus (CawYV). Tubular structures with isomeric virus particles typical for nepoviruses were observed in infected tissues by electron microscopy. The whole genome of CawYV was identified by high throughput sequencing (HTS). It consists of two segments with 8026 nt for RNA1 and 6405 nt for RNA2, excluding the poly(A) tails. CawYV-RNA1 shared closest nt identity to peach rosette mosaic virus (PRMV) with 63%, while RNA2 shared 41.5% with blueberry latent spherical virus (BLSV). The amino acid sequences of the CawYV protease-polymerase (Pro-Pol) and capsid protein (CP) regions share the highest identities with those of the subgroup C nepoviruses. The Pro-Pol region shared highest aa identity with PRMV (80.1%), while the CP region shared 39.6% to soybean latent spherical virus. Phylogenetic analysis of the CawYV-Pro-Pol and -CP aa sequences provided additional evidence of their association with nepoviruses subgroup C. Based on particle morphology, genomic organization and phylogenetic analyses, we propose CawYV as a novel species within the genus Nepovirus subgroup C. Electronic supplementary material The online version of this article (10.1186/s12985-019-1181-1) contains supplementary material, which is available to authorized users.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5’-viral genome-linked protein (VPg) and have a 3’ polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3’ untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5’ regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5’ region and 3’ UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5’ region and 3’ UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5’ proximal coding region of the RNA2 polyprotein and in the RNA2 3’ UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3’ UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3’ UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5’ region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773–972) of the 1547 nt long 3’ UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

International audience; High throughput sequencing was performed on virion-associated nucleic acids (VANA) from a pool of fifty asymptomatic rough bluegrasses (Poa trivialis L.) collected in a Belgian grazed pasture. Bioinformatics analyses produced some contigs presenting similarities with secovirid genomes, in particular nepoviruses and waikaviruses. Three distinct positive-sense single-stranded RNAs including 5? and 3? UTR were reconstructed and they represented two novel viruses infecting rough bluegrass, for which the provisional names poaceae Liege nepovirus A (PoLNVA, 7298 nt for RNA1 and 4263 nt for RNA2) and poaceae Liege virus 1 (PoLV1, 11,623 nt) were proposed. Compared to other Secoviridae members, the highest amino acid identity reached 90.7 % and 66.7 % between PoLNVA and nepoviruses for the Pro-Pol and CP regions respectively, while PoLV1 presented the highest amino acid identity with waikaviruses but with lower identities, i.e. 41.2 % for Pro-Pol and 25.8 % for CP regions, far below the ICTV demarcation criteria for novel secovirid. Based on sequence identity and phylogenetic analyses, PoLNVA was proposed to belong to the genus Nepovirus and PoLV1 as an unclassified secovirids. Detection of the two novel viruses was confirmed in high prevalence in rough bluegrass and ten other wild Poaceae species (Agropyron repens, Agrostis capillaris, Apera spica-venti, Anthoxanthum odoratum, Cynosorus cristatus, Festuca rubra, Holcus lanatus, Lolium perenne, Phleum bertolini and Phleum pratense) by RT-PCR and Sanger sequencing, revealing a diverse host range within Poaceae for these novel secovirids. Seed transmission was evaluated and confirmed for PoLNVA.

The genome sequence of a new subgroup C nepovirus from Stenotaphrum secundatum in Australia is described. This virus, tentatively named Stenotaphrum nepovirus (SteNV), was present in separate plants as a mixed infection with either sugarcane mosaic virus or Panicum mosaic virus. The virus genome was divided between two RNA segments, 7,824 and 7,104 nucleotides (nt) in length, which each encode a single long polyprotein with putative 3C-like cysteine protease sites of the type H/G, H/S or L/S. The 3' untranslated region of RNA2, at 2,155 nt, is the longest observed for any subgroup C nepovirus. Phylogenetic analyses using protease-polymerase and coat protein amino acid alignments suggest that SteNV is most closely related to cherry leaf roll virus. Using a newly developed RT-PCR assay, this virus was detected at multiple localities in New South Wales, Queensland and Western Australia, and in a second host species, Digitaria didactyla. No consistent association between virus infection and symptoms could be established. The economic importance, pathogenicity and transmission of this novel virus species warrant further investigation.

A nepovirus was isolated from Begonia ricinifolia showing chlorotic ringspot and line pattern symptoms. The purified virus had spherical particles of ca. 30 nm and contained a single coat protein subunit of ca. 56 kDa. The complete nucleotide sequence of the bipartite viral genome was determined. RNA 1 is 7394 nucleotides long, flanked by 5' and 3' untranslated regions (UTR), and followed by a 3' poly-A tail. It contains a single 6810 nt long open reading frame (ORF), which is translated into a 255 kDa polyprotein composed of 2269 amino acids. The 4684 nt long RNA 2 has a 4053 nt long ORF which encodes a single polyprotein of 1350 amino acids with a molecular weight of 149 kDa. Sequence comparisons revealed that the virus isolated from B. ricinifolia has the highest sequence similarity to beet ringspot virus and should be considered as a strain of BRSV. This is the first report on the occurrence of BRSV in B. ricinifolia and the presence of this virus outside Scotland.

During high throughput sequencing (HTS) of leaves from a symptomatic red clover plant, a new RNA virus, tentatively named red clover nepovirus A (RCNVA), was discovered. The complete genomic sequence was determined and characterized. Particularly noteworthy was that RCNVA shares high sequence identities in RNA1 with a group of phylogenetically related nepoviruses while homologies in the RNA2 segments are markedly lower. Based on the genomic organization and phylogenetic attributes, RCNVA should be classified as a novel virus of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order Picornavirales).

Grapevine fanleaf virus (GFLV) and arabis mosaic virus (ArMV) are nepoviruses responsible for grapevine degeneration. They are specifically transmitted from grapevine to grapevine by two distinct ectoparasitic dagger nematodes of the genus Xiphinema. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two viruses, were previously defined and exchanged to test their involvement in virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in virus transmission, thus representing a new viral determinant of nematode transmission.

Grapevine fanleaf virus (GFLV) is responsible for a widespread disease in vineyards worldwide. Its genome is composed of two single-stranded positive-sense RNAs, which both show a high genetic diversity. The virus is transmitted from grapevine to grapevine by the ectoparasitic nematode Xiphinema index. Grapevines in diseased vineyards are often infected by multiple genetic variants of GFLV but no information is available on the molecular composition of virus variants retained in X. index following nematodes feeding on roots. In this work, aviruliferous X. index were fed on three naturally GFLV-infected grapevines for which the virome was characterized by RNAseq. Six RNA-1 and four RNA-2 molecules were assembled segregating into four and three distinct phylogenetic clades of RNA-1 and RNA-2, respectively. After 19 months of rearing, single and pools of 30 X. index tested positive for GFLV. Additionally, either pooled or single X. index carried multiple variants of the two GFLV genomic RNAs. However, the full viral genetic diversity found in the leaves of infected grapevines was not detected in viruliferous nematodes, indicating a genetic bottleneck. Our results provide new insights into the complexity of GFLV populations and the putative role of X. index as reservoirs of virus diversity.

Raspberry ringspot virus (RpRSV) is an important virus that infects horticultural crops including grapevine, cherry, berry fruit and rose. The genome sequences of RpRSV are highly diverse between isolates and this makes the design of a PCR-based detection method difficult. In this study, a TaqMan real-time RT-PCR assay was developed for the rapid and sensitive detection of RpRSV. Primers and probes targeting the most conserved region of the movement protein gene were designed to amplify a 229 bp fragment of RpRSV RNA-2. The assay was able to amplify all RpRSV isolates tested. The detection limit of the RpRSV target region was estimated to be 61–98 copies, depending on the RpRSV strain. The sensitivity was about 100 times greater than the conventional RT-PCR assay using the same primers as the real-time RT-PCR assay. A comparison with published conventional RT-PCR assays indicated that both published assays lacked reliability and sensitivity, as neither were able to amplify all RpRSV isolates tested, and both were at least 1000 times less sensitive than the novel TaqMan real-time RT-PCR assay. The assay can also be run as a duplex reaction with the nad5 plant internal control primers and probe to simultaneously verify the PCR competency of the samples. The amplicon obtained with the real-time RT-PCR assay is suitable for direct sequencing if it is necessary to further confirm the RpRSV identity or determine the RpRSV strain.

Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detection of virus at concentrations as low as 101 copies was possible, although 102 copies was more consistent. Furthermore, comparison against extant assays showed that in both pollen and plant samples, the newly developed RT-qPCR assays were more sensitive and could detect a greater range of isolates than extant endpoint RT-PCR and ELISA assays. Use of updated assays will improve biosecurity protocols as well as the study of viruses infecting pollen.

High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique’s success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.

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

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.

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.

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.

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.

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.

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.

The complete nucleotide sequence of a new soybean-infecting member of the genus Nepovirus (provisionally named “soybean latent spherical virus” [SLSV]) was identified by high-throughput sequencing of RNAs from soybean leaf samples from North Dakota, USA. The sequences of RNAs 1 (8,190 nt) and 2 (5,788 nt) were completed by rapid amplification of cDNA ends. Each contained a single long open reading frame and a 3’ nontranslated region of greater than 1,500 nt. The predicted amino acid sequences of the two ORFs were most closely related to nepoviruses in subgroup C. Full-length cDNAs of RNAs 1 and 2 were cloned and used to inoculate soybean plants, which did not display obvious symptoms. These results suggest that SLSV represents a new species in the genus Nepovirus.

Grapevine (Vitis spp.) can be infected by numerous viruses that are often widespread and of great economic importance. Reliable detection methods are necessary for sanitary selection which is the only way to partly control grapevine virus diseases. Biological indexing and ELISA are currently the standard methods for screening propagation material, and PCR-methods are becoming increasingly popular. Due to the diversity of virus isolates, it is essential to verify that the tests allow the detection of the largest possible virus populations. We developed three quadruplex TaqMan

The nucleotide sequence of RNA1 of grapevine Anatolian ringspot virus (GARSV), a nepovirus of subgroup B, was determined from cDNA clones. It is 7,288 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame (ORF), extending from nucleotides 272 to 7001, encoding a polypeptide of 2,243 amino acids with a predicted molecular mass of 250 kDa. The primary structure of the polyprotein, compared with that of other viral polyproteins, revealed the presence of all the characteristic domains of members of the order Picornavirales, i.e., the NTP-binding protein (1B(Hel)), the viral genome-linked protein (1C(VPg)), the proteinase (1D(Prot)), the RNA-dependent RNA polymerase (1E(Pol)), and of the protease cofactor (1A(Pro-cof)) shared by members of the subfamily Comovirinae within the family Secoviridae. The cleavage sites predicted within the polyprotein were found to be in agreement with those previously reported for nepoviruses of subgroup B, processing from 1A to 1E proteins of 67, 64, 3, 23 and 92 kDa, respectively. The RNA1-encoded polyprotein (p1) shared the highest amino acid sequence identity (66 %) with tomato black ring virus (TBRV) and beet ringspot virus (BRSV). The 5'- and 3'-noncoding regions (NCRs) of GARSV-RNA1 shared 89 % and 95 % nucleotide sequence identity respectively with the corresponding regions in RNA2. Phylogenetic analysis confirmed the close relationship of GARSV to members of subgroup B of the genus Nepovirus.

The complete sequences of RNA1 and RNA2 have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SAPCS3). The two RNAs are, respectively, 7,342 and 3,817 nucleotides in length, excluding the poly(A) tails. RNA1 has a large open reading frame (ORF) of 6,852 nucleotides and a 5'-UTR and a 3'-UTR of 243 and 244 nucleotides, respectively. RNA2 encodes for an ORF of 3,330 nucleotides and has the highest nucleotide identity (90.4 %) with GFLV-F13. The full length nucleotide sequence of GFLV-SAPCS3 RNA1 had the highest nucleotide identity (86.5 %) to the French isolate GFLV-F13. The 5'- and 3'-UTRs of GFLV-SAPCS3 RNA2 are 272 nucleotides and 212 nucleotides (nt) in length, respectively. The GFLV-SAPCS3 RNA2 5'-UTR is 32-53 nt longer compared to other GFLV isolates. The GFLV-SAPCS3 RNA2 5'-UTR is also more closely related to GFLV-GHu and Arabis mosaic virus (ArMV) isolates than to other GFLV isolates. Putative intra- and interspecies recombination events between GFLV and ArMV isolates involving GFLV-SAPCS3 RNA1 and RNA2 were investigated. Recombination analysis software has indicated that the GFLV-SAPCS3 5'-UTR might have evolved from a recombinational event between GFLV-F13-type and ArMV-Ta-type isolate.

The complete nucleotide sequence of cherry leaf roll virus (CLRV, genus Nepovirus) from a naturally infected cherry tree (Prunus avium cv. Bing) in North America was determined. RNA1 and RNA2 consist of 7,893 and 6,492 nucleotides, respectively, plus a poly-(A) tail. Each RNA encodes a single potential open reading frame. The first 657 nucleotides of RNA1 and RNA2 are 99% identical and include the 5′-UTR and the first 214 deduced amino acids of the polyproteins following the first of two in-frame start codons. Phylogenetic analysis reveals close relationships between CLRV and members of subgroup C of the genus Nepovirus. Electronic supplementary material The online version of this article (doi:10.1007/s00705-011-1208-4) contains supplementary material, which is available to authorized users.

The complete genome sequence of grapevine Bulgarian latent virus (GBLV) has been determined. RNA-1 (7,452 nt in length) contains a single ORF of 6,285 nt, encoding a polyprotein with conserved motifs characteristic of the viral protease cofactor (Prot-cofact), the NTP-binding protein (NTP), the cysteine-like protease (Cyst-Prot) and the RNA-dependent RNA polymerase (RdRp) of members of the order Picornavirales and show high aa sequence identity with blackcurrant reversion virus (BRV, 64%). RNA-2 (5,821 nt) contains a single ORF of 4,500 nt, encoding a polyprotein in which the conserved motifs of the movement protein (MP) and coat protein (CP) have been identified. The GBLV CP aa sequence shows highest homology with that of blueberry leaf mottle virus (BLMoV, 68%). Both RNAs have a poly(A) tail and a NCR at the 3' and 5' termini, respectively. The results of this study confirm the classification of GBLV as a member of a distinct species in subgroup C of the genus Nepovirus.

Mekuria, T. A., Gutha, L. R., Martin, R. R., and Naidu, R. A. 2009. Genome diversity and intra- and interspecies recombination events in Grapevine fanleaf virus. Phytopathology 99:1394-1402. Grapevine fanleaf virus (GFLV) was documented in self-rooted vines of four grapevine (Vitis vinifera) cultivars in eastern Washington. GFLV was found as mixed infection in cvs. Pinot Noir, Chardonnay, and Cabernet Franc and as single infections in cv. Merlot. Fanleaf disease symptoms were only observed in the first two cultivars. The spatial distribution of GFLV-infected grapevines was random, suggesting primary spread through planting virus-infected cuttings rather than infield transmission. RNA1 sequences of Washington isolates showed 87 to 89% nucleotide sequence identity between them and with strain F13. RNA2 of Washington isolates was variable in size, showing 85 to 99% sequence identity between them and 81 to 92% with other isolates. As in other GFLV isolates, three conserved putative stem-loop structures were present in the 5′ noncoding regions of both RNAs of Washington isolates. Phylogenetic incongruence of GFLV isolates from Washington in 2A HP and 2BMP-based trees and identification of putative recombination events suggested that their genomic RNA2 originated from inter- and intraspecies recombination events between GFLV, Grapevine deformation virus, and Arabis mosaic virus. These results confirm interspecies recombination in RNA2 of grapevine-infecting nepoviruses as an important strategy for GFLV evolution.

International audience; Recombination was assessed in a vineyard site in which grapevines cross-protected with mild strains GHu of Grapevine fanleaf virus (GFLV) or Ta of Arabis mosaic virus (ArMV) were superinfected with GFLV field isolates following transmission by the nematode vector Xiphinema index. The genetic structure and variability within RNA2 of isolates from grapevines co-infected with GFLV field isolates and either GFLVGHu or ArMV-Ta were characterized to identify intra- and interspecies recombinants. Sequence analysis and phylogenetic relationships inferred intraspecies recombination among GFLV field isolates but not between field isolates and GFLV-GHu. SISCAN analysis confirmed a mosaic structure for two GFLV field isolates for which recombination siteswere located in the movement protein and coat protein genes. One of the recombinantswas found in eight grapevines thatwere in close spatial proximity within the vineyard site, suggesting its transmission by X. index. No interspecies recombination was detected between GFLV field isolates and ArMV-Ta. Altogether, our findings suggest that mild protective strains GFLV-GHu and ArMV-Ta did not assist the emergence of viable recombinants to detectable level during a 12-year crossprotection trial. To our knowledge, this is the first extensive characterization of the genetic structure and variability of virus isolates in cross-protected plants.

International audience; The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed “Secoviridae” to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae.

The nucleolytic ribozymes catalyse site-specific phosphodiester cleavage and ligation transesterification reactions in RNA. The hairpin ribozyme folds to generate an intimate loop-loop interaction to create the local environment in which catalysis can proceed. We have studied the ion-induced folding using single-molecule FRET experiments, showing that the four-way helical junction accelerates the folding 500-fold by introducing a discrete intermediate that juxtaposes the loops. Using FRET we can observe individual hairpin ribozyme molecules as they undergo multiple cycles of cleavage and ligation, and measure the rates of the internal reactions, free of uncertainties in the contributions of docking and substrate dissociation processes. On average, the cleaved ribozyme undergoes several docking-undocking events before a ligation reaction occurs. On the basis of these experiments, we have explored the role of the nucleobases G8 and A38 in the catalysis. Both cleavage and ligation reactions are pH dependent, corresponding to the titration of a group with pKa=6.2. We have used a novel ribonucleoside in which these bases are replaced by imidazole to investigate the role of acid-base catalysis in this ribozyme. We observe significant rates of cleavage and ligation, and a bell-shaped pH dependence for both.

The 5′ and 3′ non-translated regions (NTRs) of mRNAs of eukaryotes and their viruses often contain translational enhancers, including internal ribosomal entry sites (IRESs) comprised in the 5′ leaders of many uncapped viral mRNAs.Blackcurrant reversion virus(BRV) has a genome composed of two uncapped, polyadenylated RNAs with relatively short 5′ NTRs, almost devoid of secondary structure. In this work, a role of the RNA2 5′ NTR in translation was studied by using mono- and dicistronicPhotinus pyralisandRenilla reniformisluciferase reporter mRNAs in protoplasts ofNicotiana benthamiana. The RNA2 5′ leader was found to confer efficientin vivotranslation compared with the control 5′ NTR, and each half of the BRV leader was essential for stimulatory function. Such efficient translational enhancement was mediated, at least in part, through an IRES mechanism. Multiple RNA2 5′ NTR regions, complementary to a fragment of plant 18S rRNA demonstrated previously to be accessible for intermolecular mRNA–rRNA interactions and conserved between eukaryotes, were shown to be important for efficient translation. Similar mRNA–rRNA base-pairing potential was also predicted for the 5′ leaders of other nepoviruses.

Tobacco ringspot virus (TRSV, genus Nepovirus), causes severe diseases in soybean and tobacco plants. TRSV-induced bud blight disease significantly reduced both the yield and quality of soybeans. The function of the encoded viral gene product involved in TRSV infection was unclear due to the limitation of reverse genetics studies on the viral genome. Here, we represent the successful construction of infectious full-length cDNA clones of TRSV genome (RNA1 and RNA2). The cDNAs of TRSV RNA1 and RNA2 were cloned into the binary vector pPZP211 immediately downstream of a double cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator. Seven days after agrobacterium-mediated co-inoculation of these two constructs, Nicotiana benthamiana plants developed a systemic infection with necrotic ringspot symptoms and weak stunting of the leaves, similar to that induced by natural TRSV. The systemic infection was confirmed by transmission electron microscopy and Western blot analysis. Simultaneously, soybean, tomato, and Arabidopsis ecotype Estland were mechanically inoculated with sap prepared from TRSV-agroinfiltrated N. benthamiana leaves, showing typical symptoms of bud blight, necrotic spots, and lethal systemic necrosis, respectively. The system developed herein will be an appealing way to determine TRSV viral gene functions and study host-TRSV interactions.

The 3′ non-translated regions (NTRs) of mRNAs of eukaryotes and their viruses often contain translational enhancers (TEs). Blackcurrant reversion nepovirus (BRV) has a genome composed of two uncapped polyadenylated RNAs with very long 3′ NTRs, nucleotide sequences of which are very conserved between different BRV isolates. In this work, we studied a role of the RNA2 3′ NTR in translation, using mutagenesis of the firefly luciferase reporter mRNA, in protoplasts of Nicotiana benthamiana. The RNA2 3′ NTR was found to contain a cap-independent TE (3′ CITE), which must base pair with the 5′ NTR to facilitate translation. The BRV 3′ CITE and poly(A) tail provided a major contribution to translational efficiency, with less input from other 3′ NTR parts. The BRV 3′ CITE does not share similarity in nucleotide sequence and secondary structure with other viruses and thus represents a new class of 3′ CITE.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Cherry leaf roll virus (CLRV) belongs to the Nepovirus genus within the family Comoviridae. It has a host range which includes a number of wild tree and shrub species. The serological and molecular diversity of CLRV was assessed using a collection of isolates and samples recovered from woody and herbaceous host plants from different geographical origins. Molecular diversity was assessed by sequencing a short (375-bp) region of the 3' noncoding region (NCR) of the genomic RNAs while serological diversity was assessed using a panel of seven monoclonal antibodies raised initially against a walnut isolate of CLRV. The genomic region analyzed was shown to exhibit a significant degree of molecular variability with an average pairwise divergence of 8.5% (nucleotide identity). Similarly, serological variability proved to be high, with no single monoclonal antibody being able to recognize all isolates analyzed. Serological and molecular phylogenetic reconstructions showed a strong correlation. Remarkably, the diversity of CLRV populations is to a large extent defined by the host plant from which the viral samples are originally obtained. There are relatively few reports of plant viruses for which the genetic diversity is structured by the host plant. In the case of CLRV, we hypothesize that this situation may reflect the exclusive mode of transmission in natural plant populations by pollen and by seeds. These modes of transmission are likely to impose barriers to host change by the virus, leading to rapid biological and genetic separation of CLRV variants coevolving with different plant host species.

The nucleotide sequence of RNA-2 of Grapevine Anatolian ringspot virus (GARSV) and Grapevine deformation virus (GDefV), two recently described nepoviruses, has been determined. These RNAs are 3753 nt (GDefV) and 4607 nt (GARSV) in size and contain a single open reading frame encoding a polyprotein of 122 kDa (GDefV) and 150 kDa (GARSV). Full-length nucleotide sequence comparison disclosed 71-73% homology between GDefV RNA-2 and that of Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), and 62-64% homology between GARSV RNA-2 and that of Grapevine chrome mosaic virus (GCMV) and Tomato black ring virus (TBRV). As previously observed in other nepoviruses, the 5' non-coding regions of both RNAs are capable of forming stem-loop structures. Phylogenetic analysis of the three proteins encoded by RNA-2 (i.e. protein 2A, movement protein and coat protein) confirmed that GDefV and GARSV are distinct viruses which can be assigned as definitive species in subgroup A and subgroup B of the genus Nepovirus, respectively.

The complete nucleotide sequence of the genomic RNA 1 of the grapevine isolate NW (Neustadt an der Weinstrasse) of Arabis mosaic virus (ArMV) was determined. It is 7334 nucleotides long excluding the poly(A) tail, and contains one long open reading frame encoding a polypeptide of 2284 amino acids. The 5' and 3' non-coding regions were 227 and 252 nucleotides long respectively, and showed stretches of high identity with the corresponding 5' and 3' non-coding regions of ArMV-NW RNA 2. The analysis of the amino acid sequence of the polyprotein encoded by the RNA 1 of the ArMV-NW showed that the conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cystein protease, and the RdRp core domains, were all present. Amino acid sequence comparisons between the polyproteins encoded by the RNAs 1 of ArMV-NW and other nepoviruses showed 75% identity with the GFLV-F13, and up to 36% with other nepoviruses.