Mixed infection of ITPase-encoding potyvirid and secovirid in Mercurialis perennis: evidences for a convergent euphorbia-specific viral counterstrike.

BACKGROUND: In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues. METHOD: Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated. RESULTS: While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules. CONCLUSION: Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts.

Nuances of Responses to Two Sources of Grapevine Leafroll Disease on Pinot Noir Grown in the Field for 17 Years.

Grapevine leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact of two different forms of GLD to a symptomless control treatment: a mildly symptomatic form infected with GLRaV-1 and a severe form with exceptionally early leafroll symptoms (up to six weeks before veraison) infected with GLRaV-1 and GLRaV-3. Vine physiology and fruit composition in 17-year-old Pinot noir vines were measured and a gradient of vigor, yield, and berry quality (sugar content and berry weight) was observed between treatments. Virome composition, confirmed by individual RT-PCR, was compared with biological indexing. Three divergent viromes were recovered, containing between four to seven viruses and two viroids. They included the first detection of grapevine asteroid mosaic-associated virus in Switzerland. This virus did not cause obvious symptoms on the indicators used in biological indexing. Moreover, the presence of grapevine virus B (GVB) did not cause the expected corky bark symptoms on the indicators, thus underlining the important limitations of the biological indexing. Transmission of GLRaV-3 alone or in combination with GVB by Planococcus comstocki mealybug did not reproduce the strong symptoms observed on the donor plant infected with a severe form of GLD. This result raises questions about the contribution of each virus to the symptomatology of the plant.

Identification and Molecular Characterization of a Novel Hordeivirus Associated With Yellow Mosaic Disease of Privet (Ligustrum vulgare) in Europe.

Wild plants serve as a large reservoir of known and yet-unknown viruses and as a source of viral pathogens of cultivated plants. Yellow mosaic disease of forest shrub Ligustrum vulgare (privet) was recurrently observed in Europe for more than 100 years. Using a universal virus identification approach based on deep sequencing and de novo assembly of viral small interfering (si)RNAs we identified a causative agent of this disease in Switzerland and reconstructed its complete 3-segmented RNA genome. Notably, a short 3'-terminal common region (CR) attached to each segment via a ∼53-71 nucleotide poly(A) tract, as determined by RT-PCR sequencing, was initially identified as an orphan siRNA contig with conserved tRNA-like secondary structure. Phylogenomic analysis classified this virus as a novel member in the genus Hordeivirus of family Virgaviridae, which we named ligustrum mosaic virus (LigMV). Similar to other hordeiviruses, LigMV formed rod-shape virions (visualized by electron microscopy), was transmitted through seeds and could also be mechanically transmitted to herbaceous hosts Chenopodium quinoa and Nicotiana benthamiana. Blot hybridization analysis identified genomic and subgenomic RNAs, sharing the 3'-CR and likely serving as monocistronic mRNAs for seven evolutionarily-conserved viral proteins including two subunits of viral RNA-dependent RNA polymerase, coat protein, triple gene block proteins mediating viral movement and cysteine-rich suppressor of RNA silencing. Analysis of size, polarity, and hotspot profiles of viral siRNAs suggested that they are produced by the plant antiviral Dicer-like (DCL) proteins DCL2 and DCL4 processing double-stranded intermediates of genomic RNA replication. Whole genome sequencing of French and Austrian isolates of LigMV revealed its genetic stability over a wide geographic range (>99% nucleotide identity to Swiss isolates and each other), suggesting its persistence and spread in Europe via seed dispersal.

Grapevine red blotch virus: Absence in Swiss Vineyards and Analysis of Potential Detrimental Effect on Viticultural Performance.

Grapevine red blotch virus (GRBV) is a recently described virus that infects grapevine. Little information is available on the possible occurrence and distribution outside North America. Therefore, we surveyed commercial vineyards from the three major grape-growing regions in Switzerland to determine the presence or absence of GRBV. In total, 3,062 vines were analyzed by polymerase chain reaction. None of the vines tested positive for GRBV, suggesting the absence of GRBV from Swiss vineyards. We also investigated whether GRBV was present in 653 grapevine accessions in the Agroscope grapevine virus collection at Nyon, including dominantly Swiss (457) but also international accessions. Only six referential accessions were infected by GRBV, all originating from the United States, whereas all others from 10 European and 8 non-European origins tested negative. High-throughput sequencing analysis of Zinfandel A2V13, in the collection since 1985, confirmed close similarity of GRBV isolate Z_A2V13 to American isolates according to genomes deposited in GenBank. Because the Zinfandel A2V13 reference was also maintained grafted on the leafroll virus indicator Vitis vinifera 'Gamay', we evaluated the effect of GRBV on viticultural performance over a 3-year period. Our results showed clear detrimental effects of GRBV on grapevine physiology (vine vigor, leaf chlorophyll content, and gas exchange) and fruit quality. These findings underscore the importance of implementation of GRBV testing worldwide in certification and quarantine programs to prevent the dissemination of this virus.

Molecular characterization of artemisia virus A, a new sobemovirus isolated from Artemisia annua.

A distinct sobemovirus was isolated from diseased Artemisia annua plants grown in experimental culture plots in Switzerland. Electron microscopy performed on extracts of leaf and root samples of a diseased A. annua plant revealed icosahedral-30 nm viral particles. The complete nucleotide sequence of the viral genome was determined. The single positive-strand RNA of 4138 nt encodes four open reading frames with an organization similar to that described for sobemoviruses. Phylogenetic analysis revealed a close relationship to ryegrass mottle virus. The virus was efficiently acquired by healthy A. annua from contaminated soil samples. "Artemisia virus A" is tentatively proposed as a name for this new candidate member of the unassigned genus Sobemovirus.

Genome organization, serology and phylogeny of Grapevine leafroll-associated viruses 4 and 6: taxonomic implications.

Complete nucleotide sequences of the type isolate of Grapevine leafroll-associated virus 4 (GLRaV-4) and of an isolate of GLRaV-6 from cv 'Estellat' (GLRaV-6Est) were generated and compared mutually and with related viruses. The genome organization of both viruses resembled that of members of Subgroup I in the genus Ampelovirus (fam. Closteroviridae). The availability of these sequences, along with previously existing data on related GLRaVs, allowed critical review of the taxonomy and nomenclature of these viruses. In phylogenetic analyses, GLRaV-4 and -6Est consistently grouped with GLRaV-5, -9, and -Pr forming a poorly resolved sub-cluster ("GLRaV-4 group") within the genus Ampelovirus. In-depth study showed that genetic distances between these viruses do not exceed the intra-species diversity observed in other closteroviruses. In Western blots, partially purified preparations of GLRaVs -4, -5, -6 and -9 reacted only with homologous monoclonal antibodies, but were all recognized by polyclonal antisera to GLRaV-5 and GLRaV-9. Serological relatedness among these viruses was further confirmed in DAS-ELISA. In immuno-electron microscopy, GLRaV-6 particles appeared uniformly decorated with homologous monoclonal antibodies, whereas GLRaV-2, used as a control, showed "bipolar" morphology of the virion. Results of this study challenge taxonomy and nomenclature of several GLRaVs suggesting that they are divergent isolates of Grapevine leafroll-associated virus 4 and not, as has been assumed, distinct species (definitive and/or putative) in the genus Ampelovirus.

Rapid identification of potato virus Y strains by one-step triplex RT-PCR.

A one-step triplex RT-PCR method was characterised that allows rapid, strain-specific detection of potato virus Y (PVY) occurring on potato: PVY(N), PVY(O), PVY(NTN) (recombinant isolates), PVY(N)Wi and PVY(C). Three specific primer pairs were designed on aligned PVY sequences available from genomic data banks. The specificity of the selected primers was first examined by simplex RT-PCR with a large number of PVY reference isolates. Two fragments of 0.44 and 1.11kb were amplified for PVY(N) and non-recombinant PVY(NTN) isolates, two fragments of 0.53 and 0.66kb for PVY(O) isolates, a single fragment of 0.44kb for recombinant PVY(NTN) isolates, a 0.66kb fragment for PVY(C) isolates and a 0.53kb fragment for PVY(N)Wi isolate. The primers were then combined in a one-step triplex RT-PCR reaction, optimised stepwise and validated with the reference isolates. The great similarity between the genomes of PVY(N) and non-recombinant PVY(NTN) prevented their differentiation using this method. No fragments were amplified with samples infected by non-related potato viruses, as well as with samples from healthy tobacco and potato plants. The one-step triplex RT-PCR described here fastens specific detection of PVY strains that are otherwise only distinguishable by combined serological and biological assays.