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We report the discovery of a new flavi-like virus identified in wild carrots (Daucus carota subsp. carota), using a double-stranded (ds)RNA high-throughput sequencing (HTS) approach. The new virus, tentatively named "carrot flavi-like virus 1" (CtFLV-1), has a large genome of 21.8 kb that harbours a single open reading frame encoding a 7,078-aa polyprotein with conserved RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains. The new virus is phylogenetically related to recently described flavi-like viruses from arthropods, but its closest relative is a plant-associated virus, gentian Kobu-sho-associated virus (GKSaV). A pairwise comparison showed that these two viruses share 38.4% amino acid (aa) sequence identity in their polyproteins and 73% and 47.8% aa sequence identity in their conserved RdRp and Hel domains, respectively. Based on their similar genome organization and phylogenetic relationship, GKSaV and CtFLV-1 could form the basis for a new genus of plant-associated viruses, possibly within the family Flaviviridae, for which the name "Koshovirus" is proposed.

International audience; In this study, we report the complete genome sequence of a novel luteovirus detected in almonds using high-throughput sequencing. The genome of the new luteovirus comprises 5,047 nucleotides, and its genomic organization is similar to that of the recently described nectarine stem pitting associated virus (NSPaV), with only four open reading frames encoding for replication related proteins, the coat protein (CP) and a CP readthrough protein involved in the aphid transmission of luteovirids. Phylogenic and pairwise distance analyses showed that this virus shares 79% and 57.8% amino acid identities in the P1-P2 fusion protein and the P3-P5 protein, respectively, with the most closely related luteovirus NSPaV, suggesting that it represents a novel species for which the name of Almond associated luteovirus 1 is proposed. To our knowledge, this is the first report of an almond-infecting luteovirus.

UMR BFP - Equipe Virologie; International audience; Double-stranded RNAs from an isolate of Neofusicoccum luteum collected from grapevines were analyzed by high-throughput sequencing. Contig annotations revealed the presence of a potential novel virus belonging to the newly proposed family Fusariviridae. Completion of the viral genome sequence was performed. The genome is 6,244 nucleotide long, excluding the poly(A) tail and contains two putative open reading frames (ORFs). The first one encodes a large polypeptide of 1,552 amino acids (aa) with conserved RNA-dependent RNA polymerase and helicase domains typical of viral replicases. The second ORF encodes a putative 475-aa-long polypeptide showing weak homology to the corresponding ORF of Macrophomina phaseolina single-stranded RNA virus 1, for which no function is known so far. Phylogenetic analyses indicated that this virus should be considered a novel mycovirus belonging to the proposed family Fusariviridae, for which the name "Neofusicoccum luteum fusarivirus 1" (NlFV1) is proposed.

UMR BFP - Equipe Virologie; International audience; The present work reports the discovery and the complete genome sequencing of a novel member of the genus Vitivirus in the family Betaflexiviridae (subfamily Trivirinae) from a symptomless grapevine of unknown variety from Portugal. Total RNAs extracted from phloem scrapings were sequenced using Illumina technology. Bioinformatic analysis of the RNA-seq data revealed a mixed infection involving three viruses and two viroids in addition to a novel vitivirus. Completion and analysis of the genome sequence (7446 nt excluding the polyA tail) showed a typical vitivirus genomic organization. Phylogenetic analysis of the various ORFs clearly showed the new virus to belong in the genus Vitivirus, but sequence divergence firmly establishes it as a member of a new species, for which the name "Grapevine virus H" is proposed.

UMR BFP - Equipe Virologie; International audience; Double-stranded RNAs purified from cultivated (Lactuca sativa) or wild (L. serriola) lettuce from southwest France were analyzed by high-throughput sequencing. For both samples, BLAST annotation revealed contigs with homology to Betaflexiviridae family members. The full genome sequence of the isolate from cultivated lettuce (JG1) was completed (8,536 nucleotides [nt], excluding the poly(A) tail). The sequence of the 3' half of the genome (4,800 nt) of a wild lettuce isolate (P22) was determined and found to share 95.1% nt sequence identity with the JG1 isolate. The JG1 genome contains four open reading frames, encoding a replicase, a movement protein, a capsid protein, and a protein of unknown function, respectively. Based on genome organization and phylogenetic relationships, the lettuce virus is most closely related to the recently described carrot chordoviruses 1 and 2 in the family Betaflexiviridae. Considering the species demarcation criteria in this family, the two lettuce viruses represent isolates of a new chordovirus species for which the name "lettuce chordovirus 1" (LeCV1) is proposed.

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; Lettuce mottle virus (LeMoV) and dandelion yellow mosaic virus (DaYMV) infect lettuce in South America and Europe, respectively. LeMoV and DaYMV possess isometric particles, occur at low concentrations in plants and have narrow host ranges. Partial genome sequences of both viruses were obtained using purified viral preparations and universal primers for members of the family Sequiviridae. DaYMV and LeMoV sequences were analyzed and showed identity with other members of the family. Universal primers that detect both viruses and specific primers for LeMoV and DaYMV were designed and used in RT-PCR-based diagnostic assays. These results provide the first molecular data on the LeMoV and DaYMV genomes and suggest that LeMoV is a member of the genus Sequivirus, probably distinct from DaYMV.

International audience; The recent observation of the frequent occurrence of natural recombinant Plum pox virus (PPV) isolates has led to the identification of a distinct PPV subgroup, named PPV-Rec. The diversity, origin and geographical spread of the recombinant PPV isolates belonging to this subgroup remain, however, relatively poorly known. In an effort to further our understanding of these isolates, eight PPV isolates from Serbia, the country from which the first such recombinant (PPV-o6) originated, were characterized. Depending on the genomic region targeted by different typing assays, seven of the eight isolates tested presented discrepancies in their typing behavior. Sequence analysis of the (Cter)NIb-(Nter)CP region confirmed the recombinant nature of these seven isolates which all presented an identical recombination breakpoint identical to previously characterized PPV-Rec isolates. Biological indexing and immunoblot analysis provided indications that asymptomatic infection of the GF305 peach indicator and migration of the coat protein as a double-band in immunoblots may represent conserved and discriminating properties of PPV-Rec isolates. The genetic diversity of PPV-Rec isolates from former Yugoslavia (Serbia, Bosnia and Herzegovina) was estimated to be twice as large as that of the PPV-Rec isolates obtained from all other countries to date (Albania, Bulgaria, Czech republic, Germany, Hungary and Slovakia). These last results are consistent with the hypothesis that former Yugoslavia is the center of dispersion of PPV-Rec. Taken together, the results presented here provide further evidence for the wide distribution and temporal genetic stability of these natural PPV recombinant isolates and provide for the first time a possible scenario for their dispersion throughout central and eastern Europe.

Plant cryptic viruses, are intriguing agents very tightly associated with their host plants. They form isometric particles which encapsidate a double stranded RNA (dsRNA) genome with 2 or 3 components. Like other members of the Partitiviridae, which include viruses of fungi, they do not seem to cause symptoms in their hosts. They are transmitted by the ovule and by pollen to the seed embryo but there is no graft-transmission and apparently no cell-to-cell transport, except at cell division. The only known mode of transmission is by seed. The cryptic viruses are classified in two genera, Alphacryptovirus and Betacryptovirus, according to differences in particle morphology and size of the genomic dsRNAs [5]. White clover cryptic virus 1 (WCCV1) is the type species of the genus Alphacryptovirus but the sequences of its two genome segments and their encoded proteins have not been published. We here report on these, as regards RNA1; the following paper [2] reports the full sequence of RNA2.

The new plant virus family Flexiviridae is described. The family is named because its members have flexuous virions and it includes the existing genera Allexivirus, Capillovirus, Carlavirus, Foveavirus, Potexvirus, Trichovirus and Vitivirus, plus the new genus Mandarivirus together with some related viruses not assigned to any genus. The family is justified from phylogenetic analyses of the polymerase and coat protein (CP) sequences. To help to define suitable molecular criteria for demarcation of species, a complete set of pairwise comparisons was made using the nucleotide (nt) and amino acid (aa) sequences of each fully-sequenced gene from every available accession in the family. Based on the distributions and on inspection of the data, it was concluded that, as a general rule, distinct species have less than ca. 72% identical nt or 80% identical aa between their entire CP or replication protein genes.

International audience; Recently obtained molecular and biological information has prompted the revision of the taxonomic structure of the family Closteroviridae. In particular, mealybug-transmitted species have been separated from the genus Closterovirus and accommodated in a new genus named Ampelovirus (from ampelos, Greek for grapevine). Thus, the family now comprises three genera. Their major properties are (i) Closterovirus: type species Beet yellows virus, genome monopartite, 15.5–19.3 kb in size, a 22–25 kDa major coat protein (CP), the gene encoding the divergent CP analogue (CPd) upstream of the CP cistron, transmission by aphids, a membership of 8 definitive and 4 tentative species; (ii) Ampelo-virus: type species Grapevine leafroll virus 3, genome monopartite 16.9–19.5 kb in size, a 35–37 kDa major CP, a CPd cistron generally located downstream of the CP gene, transmission by pseudococcid and coccid mealybugs, a membership of 6 definitive and 5 tentative species; (iii) Crinivirus: type species Lettuce infectious yellows virus, genome essentially bipartite 15.3–19 kb in size, a 28–33 kDa CP, a CPd cistron downstream of the CP gene, transmission by whiteflies (Bemisia, Trialeurodes), a membership of 7 definitive and 3 tentative species. There are five unassigned species in the family.

A pathogen was transmitted from apricot trees showing symptoms of viral infection to GF305 peach seedlings which reacted by stunting, shortened internodes and chlorotic mottling. The agent was transmitted to cherry, apricot, peach and plum by grafting and to several herbaceous hosts by mechanical inoculation. Isometric nepovirus-like particles of 30–31 nm diameter extracted from infected Chenopodium quinoa sedimented as two peaks in sucrose gradients. These particles contained two single stranded RNAs of approximately 5.9 and 7.9 kb, and a single coat protein subunit of 53.7 kDa. No cross-reactions were observed with a number of nepoviruses infecting fruit trees. Inoculation of purified particles to herbaceous or woody hosts reproduced the same symptoms caused by the original isolate. Sequencing of a 2.2 kbp cDNA clone covering the 3 � end of the small genomic RNA identified an open reading frame encoding a 317 aa N-truncated protein exhibiting significant similarities with the coat protein of nepoviruses. The 1257 nt long 3 � non-coding region showed up to about 65% homology to the equivalent region of members of the subgroup C of nepoviruses. The properties of this pathogen do not match those of any previously described nepovirus. It should therefore be considered as a new member of the subgroup C of nepoviruses, for which the name of Apricot latent ringspot virus (ALRSV) is proposed. The nucleotide sequence reported in this work has been deposited in the EMBL databank under the accession number AJ278875.

Field-grown citrus trees often harbor complex mixtures of 4–5 different viroid species, and the presence of citrus viroid III (CVd-III) has been shown to reduce the rate of tree growth without inducing disease. To more fully define the structure of its quasi-species, we have examined nine citrus viroid complexes for the presence of previously undescribed sequence variants of CVd-III. Analysis of 86 full-length cDNAs generated from these nine viroid complexes by RT-PCR revealed the presence of 20 new CVd-III variants. Chain lengths ranged from 293–297 nucleotides, and sequence changes were confined largely to the lower portions of the central conserved region and variable domain. The previously described variants CVd-IIIa (297 nt) and CVd-IIIb (294 nt) were clearly predominant, but phylogenetic analysis indicated that certain isolates may contain representatives of two additional fitness peaks. At least one group of CVd-III variants appears to have arisen as a result of RNA recombination. Populations recovered from diseased/declining trees were the most diverse, but even dwarfing isolates originating from old line Shamouti trees showed considerable variability.

International audience; Lettuce mosaic virus (LMV) is an economically important Potyvirus causing a severe disease of commercial lettuce crops. Based on molecular data, three phylogenetic groups of isolates have previously been discriminated, reflecting their geographical origin (Western Europe-California, Greece, or Yemen). Sequence information for the entire coat protein domain was only available for one of the Western Europe-California phylogenetic group. We have now sequenced the 3′ terminal region of the genome LMV-Gr4, -Gr5 and -GrB, isolates which belong to the Greek phylogenetic group and of LMV-Yar, the sole known representative of the third LMV phylogenetic group. The region sequenced encodes the last 62 amino-acids of the polymerase and the entire coat protein of the four isolates, plus the 3′ non-translated region of LMV-Gr5 and -Yar. The Greek and Yemenite isolates studied are all very aggressive on lettuce, are able to overcome the resistance genes mo1 1 and mo1 2 and belong to the two phylogenetic groups which have so far been only partially characterised. As for other Potyviruses, the core and the C-terminal regions of the coat protein are highly conserved among all isolates whereas the N-terminus is more variable. No amino acid change in the coat protein or carboxy-terminal part of the polymerase could be related to the resistance-breaking properties of the isolates analysed. The sequences obtained provide the basis for the rapid typing of LMV isolates using the restriction pattern of segments of cDNA amplified by PCR.

International audience; A full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator. This construct was found infectious when inoculated to lettuce plants. The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity. The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E. coli. Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out. The symptoms induced in lettuce by either the intron-less or the intron-containing constructs were identical to those caused by the wild-type virus. However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA.

International audience; The genome of the Balaton1 severe cherry isolate of apple chlorotic leaf spot trichovirus (ACLSV-Bal1) has been cloned and sequenced. The genomic RNA is 7 549 nucleotide long, excluding the poly A tail. The genomic organization, with three overlapping open reading frames (ORF), is similar to that of the other sequenced ACLSV isolates. Sequence comparisons indicate a high variability between ACLSV isolates, with overall nucleotide sequence homology levels between 76 and 82%. The coat protein, encoded internally inside a larger ORF, is the most conserved protein (identity levels between 87 and 93%) while the central ORF, encoding the putative movement protein, is the most divergent (77 to 85% identity).

International audience; A virus-induced, viral RNA-specific, RNA-dependent-RNA-poly-merase activity has been observed in vitro associated with membrane extracts of plants of three different species after infection with grapevine chrome mosaic nepovirus (GCMV). The products of this activity are full-length, positive sense GCMV RNAs present in double-stranded structures. As has been demonstrated for a relative to nepoviruses, cowpea mosaic comovirus, this activity probably corresponds to the nepoviral replication complex.

International audience