Polyvalent Detection of Members of the Genus Potyvirus by Molecular Hybridization Using a Genus-Probe

[EN] The use of a unique riboprobe named polyprobe, carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the polyvalent detection of up to 10 different pathogens by using a nonradioactive molecular hybridization procedure. In the present analysis, we have developed a unique polyprobe with the capacity to detect all members of the genus Potyvirus, which we have named genus-probe. To do this, we have exploited the capacity of the molecular hybridization assay to cross-hybridize with related sequences by reducing the hybridization temperature. We observed that sequences showing a percentage similarity of 68% or higher could be detected with the same probe by hybridizing at 50 to 55 degrees C, with a detection limit of picograms of viral RNA comparable to the specific individual probes. According to this, we developed several polyvalent polyprobes, containing three, five, or seven different 500-nucleotide fragments of a conserved region of the NIb gene. The polyprobe carrying seven different conserved regions was able to detect all the 32 potyviruses assayed in the present work with no signal in the healthy tissue, indicating the potential capacity of the polyprobe to detect all described, and probably uncharacterized, potyviruses being then considered as a genus-probe. The use of this technology in routine diagnosis not only for Potyvirus but also to other viral genera is discussed.We thank L. Corachan for her excellent technical assistance and I. Font and A. O. Alfaro, from the Mediterranean Agroforestal Institute at the Polytechnic University of Valencia, for provide part of the potyvirus-infected field samples. This work was supported by grant BIO2017-88321-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT-MINECO).Sanchez Navarro, JA.; Cooper, C.; Pallás Benet, V. (2018). Polyvalent Detection of Members of the Genus Potyvirus by Molecular Hybridization Using a Genus-Probe. Phytopathology. 108(12):1522-1529. https://doi.org/10.1094/PHYTO-04-18-0146-RS152215291081

The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species

[EN] The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the `30K family¿. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior.We thank L. Corachan for her excellent technical assistance. This work was supported by grant BIO2014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT), the Prometeo Program GV2014/010 from the Generalitat Valenciana, CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), Capes (Conselho de Aperfeicoamento de Pessoal de Nivel Superior) and FAP-DF (Fundacao de Apoio a Pesquisa do Distrito Federal)Leastro, MO.; Pallás Benet, V.; Resende, RO.; Sanchez Navarro, JA. (2017). The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species. Virus Research. 227:57-68. https://doi.org/10.1016/j.virusres.2016.09.023S576822

Systemic transport of Alfalfa mosaic virus can be mediated by the movement proteins of several viruses assigned to five genera of the 30K family

We previously showed that the movement protein (MP) gene of Alfalfa mosaic virus (AMV) is functionally exchangeable for the cell-to-cell transport of the corresponding genes of Tobacco mosaic virus (TMV), Brome mosaic virus, Prunus necrotic ringspot virus, Cucumber mosaic virus and Cowpea mosaic virus. We have analysed the capacity of the heterologous MPs to systemically transport the corresponding chimeric AMV genome. All MPs were competent in systemic transport but required the fusion at their C terminus of the coat protein-interacting C-terminal 44 aa (A44) of the AMV MP. Except for the TMV MP, the presence of the hybrid virus in upper leaves correlated with the capacity to move locally. These results suggest that all the MPs assigned to the 30K superfamily should be exchangeable not only for local virus movement but also for systemic transport when the A44 fragment is present.We thank L. Corachan for her excellent technical assistance. This work was supported by the Spanish granting agency DGICYT via grant BIO2011-25018 and by the Generalitat Valenciana via grant PROMETEO 2011-003.Fajardo, TVM.; Peiró Morell, A.; Pallás Benet, V.; Sanchez Navarro, JA. (2013). Systemic transport of Alfalfa mosaic virus can be mediated by the movement proteins of several viruses assigned to five genera of the 30K family. Journal of General Virology. 94:677-681. https://doi.org/10.1099/vir.0.048793-0S6776819

The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism

[EN] Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.The authors thank L. Corachan-Valencia for technical assistance. This work was funded by grant BIO2017¿88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). J.A.N. and M.S.-B. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Ciencia, Innovacion y Universidades of Spain, respectively. Ministerio de Ciencia e Innovacion (PID2020-115571RB-I00), European Regional Development FundNavarro Bohigues, JA.; Sáiz-Bonilla, M.; Sanchez Navarro, JA.; Pallás Benet, V. (2021). The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism. The Plant Journal. 108(1):197-218. https://doi.org/10.1111/tpj.15435S197218108

Identification and genomic characterization of a novel tobamovirus from prickly pear cactus

[EN] In this work, we describe the complete sequence and genome organization of a novel tobamovirus detected in a prickly pear plant (Opuntia sp.) by high-throughput sequencing, tentatively named "opuntia virus 2". The full genome of opuntia virus 2 is 6,453 nucleotides in length and contains four open reading frames (ORFs) coding for the two subunits of the RNA polymerase, the movement protein, and the coat protein, respectively. Phylogenetic analysis using the complete nucleotide sequence revealed that the virus belongs to the genus Tobamovirus (family Virgaviridae), showing the highest nucleotide sequence identity (49.8%) with cactus mild mottle virus (CMMoV), being indicating that it belongs in the Cactaceae subgroup of tobamoviruses.This study was funded by project no. IN216317 of UNAM-Programa de Apoyo a Proyectos de Investigacion e Innovacion Tecnologica (PAPIIT) and by Grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER).Salgado-Ortiz, H.; De La Torre-Almaraz, R.; Sanchez Navarro, JA.; Pallás Benet, V. (2020). Identification and genomic characterization of a novel tobamovirus from prickly pear cactus. Archives of Virology. 165(3):781-784. https://doi.org/10.1007/s00705-020-04528-3S7817841653Sánchez LDL, López GC, Ávalos HI (2013) Nomenclatura vernácula, uso y manejo de Opuntia spp. en Santiago Bayacora, Durango, México. Revista Chapingo Serie Horticultura 19:367–380Alonso BB, Mora AG, Valdovinos PG, Ochoa MDL, Rodríguez LE, De La Torre AR (2015) Asociación de un Potexvirus como agente causal de manchas cloróticas en Opuntia ficus-indica. Rev Mex Fitopatol 33:75–86De La Torre AR, Salgado OH, Salazar SM, Pallás V, Sánchez NJA, Valverde RA (2016) First report of Schlumbergera virus X in prickly pear (Opuntia ficus-indica) in Mexico. Plant Dis 100(8):1799De La Torre AR, Salgado OH, Salazar SM, Pallás V, Sánchez NJA, Valverde RA (2016) First report of Rattail cactus necrosis-associated virus in prickly pear fruit (Opuntia albicarpa Scheinvar) in Mexico. Plant Dis 100(11):2339Min BE, Chung BN, Kim MJ, Ha JH, Lee BY, Ryu KH (2006) Cactus mild mottle virus is a new cactus-infecting tobamovirus. Arch Virol 151(1):13–21Kim NR, Hong JS, Song YS, Chung BN, Park JW, Ryu KH (2012) The complete genome sequence of a member of a new species of tobamovirus (rattail cactus necrosis-associated virus) isolated from Aporocactus flagelliformis. Arch Virol 157(1):185–187Adams MJ, Antoniw JF, Kreuze J (2009) Virgaviridae: a new family of rod-shaped plant viruses. Arch Virol 154(12):1967–1972. https://doi.org/10.1007/s00705-009-0506-6Lecoq H, Desbiez C (2012) Viruses of cucurbit crops in the Mediterranean region: an ever-changing picture. Adv Virus Res 84:67–126Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Regev A (2013) De novo transcript sequence reconstruction from RNA-Seq: reference generation and analysis with Trinity. Nat Protoc 8(8):1–43Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874Song YS, Min BE, Hong JS, Rhie MJ, Kim MJ, Ryu KH (2006) Molecular evidence supporting the confirmation of Maracuja mosaic virus as a species of the genus Tobamovirus and production of an infectious cDNA transcript. Arch Virol 151:2337–2348Gibbs AJ, Wood J, Garcia-Arenal F, Ohshima K, Armstrong JS (2015) Tobamoviruses have probably co-diverged with their eudicotyledonous hosts for at least 110 million years. Virus Evol 1(1):vev019Adams MJ, Adkins S, Blagard C et al (2018) International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Virgaviridae. http://www.ictv.global/report/virgaviridae. Accessed 19 Oct 201

Membrane Association and Topology of Citrus Leprosis Virus C2 Movement and Capsid Proteins

[EN] Although citrus leprosis disease has been known for more than a hundred years, one of its causal agents, citrus leprosis virus C2 (CiLV-C2), is poorly characterized. This study described the association of CiLV-C2 movement protein (MP) and capsid protein (p29) with biological membranes. Our findings obtained by computer predictions, chemical treatments after membrane fractionation, and biomolecular fluorescence complementation assays revealed that p29 is peripherally associated, while the MP is integrally bound to the cell membranes. Topological analyses revealed that both the p29 and MP expose their N- and C-termini to the cell cytoplasmic compartment. The implications of these results in the intracellular movement of the virus were discussed.This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), proc. 2014/0845-9, 2017/50222-0, 2015/10249-1 and 2017/19898-8. This work was also supported by grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), and the Prometeo Program GV2015/010 from the Generalitat Valenciana.Oliveira Leastro, M.; Freitas-Astua, J.; Watanabe Kitajima, E.; Pallás Benet, V.; Sanchez Navarro, JA. (2021). Membrane Association and Topology of Citrus Leprosis Virus C2 Movement and Capsid Proteins. Microorganisms. 9(2):1-9. https://doi.org/10.3390/microorganisms9020418199

Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage

[EN] Previous results using a movement defective alfalfa mosaic virus (AMV) vector revealed that citrus leprosis virus C (CiLV-C) movement protein (MP) generates a more efficient local movement, but not more systemic transport, than citrus leprosis virus C2 (CiLV-C2) MP, MPs belonging to two important viruses for the citrus industry. Here, competition experiment assays in transgenic tobacco plants (P12) between transcripts of AMV constructs expressing the cilevirus MPs, followed by several biological passages, showed the prevalence of the AMV construct carrying the CiLV-C2 MP. The analysis of AMV RNA 3 progeny recovered from P12 plant at the second viral passage revealed the presence of a mix of progeny encompassing the CiLV-C2 MP wild type (MPWT) and two variants carrying serines instead phenylalanines at positions 72 (MPS72F) or 259 (MPS259F), respectively. We evaluated the effects of each modified residue in virus replication, and cell-to-cell and long-distance movements. Results indicated that phenylalanine at position 259 favors viral cell-to-cell transport with an improvement in viral fitness, but has no effect on viral replication, whereas mutation at position 72 (MPS72F) has a penalty in the viral fitness. Our findings indicate that the prevalence of a viral population may be correlated with its greater efficiency in cell-to-cell and systemic movements.This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant numbers 2014/0845-9, 2017/50222-0, 2015/10249-1, 2017/19898-8 and by the Spanish Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), grant number PID2020-115571RB-100.Oliveira Leastro, M.; Villar-Álvarez, D.; Freitas-Astúa, J.; Watanabe Kitajima, E.; Pallás Benet, V.; Sanchez Navarro, JA. (2021). Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage. Viruses. 13(12):1-16. https://doi.org/10.3390/v13122498S116131

Citrus Leprosis Virus C Encodes Three Proteins With Gene Silencing Suppression Activity

[EN] Citrus leprosis virus C (CiLV-C) belongs to the genusCilevirus, familyKitaviridae, and is considered the most devastating virus infecting citrus in Brazil, being the main viral pathogen responsible for citrus leprosis (CL), a severe disease that affects citrus orchards in Latin America. Here, proteins encoded by CiLV-C genomic RNA 1 and 2 were screened for potential RNA silencing suppressor (RSS) activity by five methods. Using the GFP-based reporter agroinfiltration assay, we have not found potential local suppressor activity for the five CiLV-C encoded proteins. However, when RSS activity was evaluated using the alfalfa mosaic virus (AMV) system, we found that the p29, p15, and p61 CiLV-C proteins triggered necrosis response and increased the AMV RNA 3 accumulation, suggesting a suppressive functionality. From the analysis of small interfering RNAs (siRNAs) accumulation, we observed that the ectopic expression of the p29, p15, and p61 reduced significantly the accumulation of GFP derived siRNAs. The use of the RSS defective turnip crinkle virus (TCV) system revealed that only thetrans-expression of the p15 protein restored the cell-to-cell viral movement. Finally, the potato virus X (PVX) system revealed that the expression of p29, p15, and p61 increased the PVX RNA accumulation; in addition, the p29 and p15 enhanced the pathogenicity of PVX resulting in the death of tobacco plants. Furthermore, PVX-p61 infection resulted in a hypersensitive response (HR), suggesting that p61 could also activate a plant defense response mechanism. This is the first report describing the RSS activity for CiLV-C proteins and, moreover, for a member of the familyKitaviridae.This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), proc. 2014/0845-9, 2017/50222-0, 2015/10249-1, and 2017/19898-8. This work was also supported by Instituto para la Formacion y Aprovechamiento de Recursos Humanos, Becas IFARHU-SENACYT, contrato 270-2018-361, grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI), Fondo Europeo de Desarrollo Regional (FEDER), and the Prometeo Program GV2015/010 from the Generalitat Valenciana.Leastro, MO.; Ortega Castro, DY.; Freitas-Astúa, J.; Kitajima, EW.; Pallás Benet, V.; Sanchez Navarro, JA. (2020). Citrus Leprosis Virus C Encodes Three Proteins With Gene Silencing Suppression Activity. Frontiers in Microbiology. 11:1-16. https://doi.org/10.3389/fmicb.2020.01231S11611Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H., & Vance, V. B. (1998). A viral suppressor of gene silencing in plants. Proceedings of the National Academy of Sciences, 95(22), 13079-13084. doi:10.1073/pnas.95.22.13079Aravin, A. A., Naumova, N. M., Tulin, A. V., Vagin, V. V., Rozovsky, Y. M., & Gvozdev, V. A. (2001). Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. Current Biology, 11(13), 1017-1027. doi:10.1016/s0960-9822(01)00299-8Arena, G. D., Ramos-González, P. L., Nunes, M. A., Ribeiro-Alves, M., Camargo, L. E. A., Kitajima, E. W., … Freitas-Astúa, J. (2016). Citrus leprosis virus C Infection Results in Hypersensitive-Like Response, Suppression of the JA/ET Plant Defense Pathway and Promotion of the Colonization of Its Mite Vector. Frontiers in Plant Science, 7. doi:10.3389/fpls.2016.01757Borges, F., & Martienssen, R. A. (2015). The expanding world of small RNAs in plants. Nature Reviews Molecular Cell Biology, 16(12), 727-741. doi:10.1038/nrm4085Brigneti, G. (1998). Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. The EMBO Journal, 17(22), 6739-6746. doi:10.1093/emboj/17.22.6739Burgyán, J., & Havelda, Z. (2011). Viral suppressors of RNA silencing. Trends in Plant Science, 16(5), 265-272. doi:10.1016/j.tplants.2011.02.010Cañizares, M. C., Navas-Castillo, J., & Moriones, E. (2008). Multiple suppressors of RNA silencing encoded by both genomic RNAs of the crinivirus, Tomato chlorosis virus. Virology, 379(1), 168-174. doi:10.1016/j.virol.2008.06.020Castel, S. E., & Martienssen, R. A. (2013). RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nature Reviews Genetics, 14(2), 100-112. doi:10.1038/nrg3355Chiba, M., Reed, J. C., Prokhnevsky, A. I., Chapman, E. J., Mawassi, M., Koonin, E. V., … Dolja, V. V. (2006). Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon. Virology, 346(1), 7-14. doi:10.1016/j.virol.2005.09.068Delgadillo, M. O., Sáenz, P., Salvador, B., García, J. A., & Simón-Mateo, C. (2004). Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants. Journal of General Virology, 85(4), 993-999. doi:10.1099/vir.0.19735-0Ding, S.-W., & Voinnet, O. (2007). Antiviral Immunity Directed by Small RNAs. Cell, 130(3), 413-426. doi:10.1016/j.cell.2007.07.039Freitas-Astúa, J., Ramos-González, P. L., Arena, G. D., Tassi, A. D., & Kitajima, E. W. (2018). Brevipalpus-transmitted viruses: parallelism beyond a common vector or convergent evolution of distantly related pathogens? Current Opinion in Virology, 33, 66-73. doi:10.1016/j.coviro.2018.07.010García, J. A., & Pallás, V. (2015). Viral factors involved in plant pathogenesis. Current Opinion in Virology, 11, 21-30. doi:10.1016/j.coviro.2015.01.001Garita, L. C., Tassi, A. D., Calegario, R. F., Freitas-Astúa, J., Salaroli, R. B., Romão, G. O., & Kitajima, E. W. (2014). Experimental host range of Citrus leprosis virus C (CiLV-C). Tropical Plant Pathology, 39(1), 43-55. doi:10.1590/s1982-56762014005000004Guilley, H., Bortolamiol, D., Jonard, G., Bouzoubaa, S., & Ziegler-Graff, V. (2009). Rapid screening of RNA silencing suppressors by using a recombinant virus derived from beet necrotic yellow vein virus. Journal of General Virology, 90(10), 2536-2541. doi:10.1099/vir.0.011213-0Guo, Q., Liu, Q., A. Smith, N., Liang, G., & Wang, M.-B. (2016). RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops. Current Genomics, 17(6), 476-489. doi:10.2174/1389202917666160520103117Gupta, A. K., Hein, G. L., Graybosch, R. A., & Tatineni, S. (2018). Octapartite negative-sense RNA genome of High Plains wheat mosaic virus encodes two suppressors of RNA silencing. Virology, 518, 152-162. doi:10.1016/j.virol.2018.02.013Hamilton, A. (2002). Two classes of short interfering RNA in RNA silencing. The EMBO Journal, 21(17), 4671-4679. doi:10.1093/emboj/cdf464Hamilton, A. J., & Baulcombe, D. C. (1999). A Species of Small Antisense RNA in Posttranscriptional Gene Silencing in Plants. Science, 286(5441), 950-952. doi:10.1126/science.286.5441.950Hammond, S. M., Bernstein, E., Beach, D., & Hannon, G. J. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 404(6775), 293-296. doi:10.1038/35005107Jones, L., Hamilton, A. J., Voinnet, O., Thomas, C. L., Maule, A. J., & Baulcombe, D. C. (1999). RNA–DNA Interactions and DNA Methylation in Post-Transcriptional Gene Silencing. The Plant Cell, 11(12), 2291-2301. doi:10.1105/tpc.11.12.2291Kakumani, P. K., Ponia, S. S., S, R. K., Sood, V., Chinnappan, M., Banerjea, A. C., … Bhatnagar, R. K. (2013). Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor. Journal of Virology, 87(16), 8870-8883. doi:10.1128/jvi.02774-12Kuchibhatla, D. B., Sherman, W. A., Chung, B. Y. W., Cook, S., Schneider, G., Eisenhaber, B., & Karlin, D. G. (2013). Powerful Sequence Similarity Search Methods and In-Depth Manual Analyses Can Identify Remote Homologs in Many Apparently «Orphan» Viral Proteins. Journal of Virology, 88(1), 10-20. doi:10.1128/jvi.02595-13LAEMMLI, U. K. (1970). Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227(5259), 680-685. doi:10.1038/227680a0Leastro, M. O., Kitajima, E. W., Silva, M. S., Resende, R. O., & Freitas-Astúa, J. (2018). Dissecting the Subcellular Localization, Intracellular Trafficking, Interactions, Membrane Association, and Topology of Citrus Leprosis Virus C Proteins. Frontiers in Plant Science, 9. doi:10.3389/fpls.2018.01299Leastro, M. O., Pallás, V., Resende, R. O., & Sánchez-Navarro, J. A. (2015). The movement proteins (NSm) of distinct tospoviruses peripherally associate with cellular membranes and interact with homologous and heterologous NSm and nucleocapsid proteins. Virology, 478, 39-49. doi:10.1016/j.virol.2015.01.031Leastro, M. O., Pallás, V., Resende, R. O., & Sánchez-Navarro, J. A. (2017). The functional analysis of distinct tospovirus movement proteins (NS M ) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species. Virus Research, 227, 57-68. doi:10.1016/j.virusres.2016.09.023Li, F., & Ding, S.-W. (2006). Virus Counterdefense: Diverse Strategies for Evading the RNA-Silencing Immunity. Annual Review of Microbiology, 60(1), 503-531. doi:10.1146/annurev.micro.60.080805.142205Li, W. X., & Ding, S. W. (2001). Viral suppressors of RNA silencing. Current Opinion in Biotechnology, 12(2), 150-154. doi:10.1016/s0958-1669(00)00190-7Locali-Fabris, E. C., Freitas-Astúa, J., Souza, A. A., Takita, M. A., Astúa-Monge, G., Antonioli-Luizon, R., … Machado, M. A. (2006). Complete nucleotide sequence, genomic organization and phylogenetic analysis of Citrus leprosis virus cytoplasmic type. Journal of General Virology, 87(9), 2721-2729. doi:10.1099/vir.0.82038-0Sue Loesch-Fries, L., Jarvis, N. P., Krahn, K. J., Nelson, S. E., & Hall, T. C. (1985). Expression of Alfalfa Mosaic virus RNA 4 cDNA transcripts in Vitro and in Vivo. Virology, 146(2), 177-187. doi:10.1016/0042-6822(85)90002-9Lu, R., Folimonov, A., Shintaku, M., Li, W.-X., Falk, B. W., Dawson, W. O., & Ding, S.-W. (2004). Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome. Proceedings of the National Academy of Sciences, 101(44), 15742-15747. doi:10.1073/pnas.0404940101Lu, R., Maduro, M., Li, F., Li, H. W., Broitman-Maduro, G., Li, W. X., & Ding, S. W. (2005). Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans. Nature, 436(7053), 1040-1043. doi:10.1038/nature03870Lu, R. (2003). High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance. The EMBO Journal, 22(21), 5690-5699. doi:10.1093/emboj/cdg546Lucy, A. P. (2000). Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus. The EMBO Journal, 19(7), 1672-1680. doi:10.1093/emboj/19.7.1672Mallory, A. C., Reinhart, B. J., Bartel, D., Vance, V. B., & Bowman, L. H. (2002). A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco. Proceedings of the National Academy of Sciences, 99(23), 15228-15233. doi:10.1073/pnas.232434999Mann, K. S., Johnson, K. N., Carroll, B. J., & Dietzgen, R. G. (2016). Cytorhabdovirus P protein suppresses RISC-mediated cleavage and RNA silencing amplification in planta. Virology, 490, 27-40. doi:10.1016/j.virol.2016.01.003Martínez-Pérez, M., Navarro, J. A., Pallás, V., & Sánchez-Navarro, J. A. (2019). A sensitive and rapid RNA silencing suppressor activity assay based on alfalfa mosaic virus expression vector. Virus Research, 272, 197733. doi:10.1016/j.virusres.2019.197733Matranga, C., & Zamore, P. D. (2007). Small silencing RNAs. Current Biology, 17(18), R789-R793. doi:10.1016/j.cub.2007.07.014Mérai, Z., Kerényi, Z., Kertész, S., Magna, M., Lakatos, L., & Silhavy, D. (2006). Double-Stranded RNA Binding May Be a General Plant RNA Viral Strategy To Suppress RNA Silencing. Journal of Virology, 80(12), 5747-5756. doi:10.1128/jvi.01963-05Moissiard, G., & Voinnet, O. (2004). Viral suppression of RNA silencing in plants. Molecular Plant Pathology, 5(1), 71-82. doi:10.1111/j.1364-3703.2004.00207.xMoon, J. Y., & Park, J. M. (2016). Cross-Talk in Viral Defense Signaling in Plants. Frontiers in Microbiology, 07. doi:10.3389/fmicb.2016.02068Nakanishi, K. (2016). Anatomy of RISC  : how do small RNAs and chaperones activate Argonaute proteins? WIREs RNA, 7(5), 637-660. doi:10.1002/wrna.1356Pallás, V., Más, P., & Sánchez-Navarro, J. A. (1998). Detection of Plant RNA Viruses by Nonisotopic Dot-Blot Hybridization. Plant Virology Protocols, 461-468. doi:10.1385/0-89603-385-6:461Pascon, R. C., Kitajima, J. P., Breton, M. C., Assumpção, L., Greggio, C., Zanca, A. S., … da Silva, A. C. R. (2006). The Complete Nucleotide Sequence and Genomic Organization of Citrus Leprosis Associated Virus, Cytoplasmatic type (CiLV-C). Virus Genes, 32(3), 289-298. doi:10.1007/s11262-005-6913-1Peiro, A., Martinez-Gil, L., Tamborero, S., Pallas, V., Sanchez-Navarro, J. A., Mingarro, I., & Simon, A. (2013). The Tobacco mosaic virus Movement Protein Associates with but Does Not Integrate into Biological Membranes. Journal of Virology, 88(5), 3016-3026. doi:10.1128/jvi.03648-13Pfeffer, S., Dunoyer, P., Heim, F., Richards, K. E., Jonard, G., & Ziegler-Graff, V. (2002). P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing. Journal of Virology, 76(13), 6815-6824. doi:10.1128/jvi.76.13.6815-6824.2002Pisacane, P., & Halic, M. (2017). Tailing and degradation of Argonaute-bound small RNAs protect the genome from uncontrolled RNAi. Nature Communications, 8(1). doi:10.1038/ncomms15332Powers, J. G., Sit, T. L., Qu, F., Morris, T. J., Kim, K.-H., & Lommel, S. A. (2008). A Versatile Assay for the Identification of RNA Silencing Suppressors Based on Complementation of Viral Movement. Molecular Plant-Microbe Interactions®, 21(7), 879-890. doi:10.1094/mpmi-21-7-0879Pumplin, N., & Voinnet, O. (2013). RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nature Reviews Microbiology, 11(11), 745-760. doi:10.1038/nrmicro3120Qu, F., & Morris, T. J. (2005). Suppressors of RNA silencing encoded by plant viruses and their role in viral infections. FEBS Letters, 579(26), 5958-5964. doi:10.1016/j.febslet.2005.08.041Rodamilans, B., Valli, A., Mingot, A., San León, D., López-Moya, J. J., & García, J. A. (2018). An atypical RNA silencing suppression strategy provides a snapshot of the evolution of sweet potato-infecting potyviruses. Scientific Reports, 8(1). doi:10.1038/s41598-018-34358-yRoth, B. (2004). Plant viral suppressors of RNA silencing. Virus Research, 102(1), 97-108. doi:10.1016/j.virusres.2004.01.020Roy, A., Stone, A., Otero-Colina, G., Wei, G., Choudhary, N., Achor, D., … Brlansky, R. H. (2013). Genome Assembly of Citrus Leprosis Virus Nuclear Type Reveals a Close Association with Orchid Fleck Virus. Genome Announcements, 1(4). doi:10.1128/genomea.00519-13Samuel, G. H., Wiley, M. R., Badawi, A., Adelman, Z. N., & Myles, K. M. (2016). Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA. Proceedings of the National Academy of Sciences, 113(48), 13863-13868. doi:10.1073/pnas.1600544113Sanchez-Navarro, J., Miglino, R., Ragozzino, A., & Bol, J. F. (2001). Engineering of Alfalfa mosaic virus RNA 3 into an expression vector. Archives of Virology, 146(5), 923-939. doi:10.1007/s007050170125Silhavy, D. (2002). A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. The EMBO Journal, 21(12), 3070-3080. doi:10.1093/emboj/cdf312Taschner, P. E. M., Van Der Kuyl, A. C., Neeleman, L., & Bol, J. F. (1991). Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes. Virology, 181(2), 445-450. doi:10.1016/0042-6822(91)90876-dThomas, C. L., Leh, V., Lederer, C., & Maule, A. J. (2003). Turnip crinkle virus coat protein mediates suppression of RNA silencing in nicotiana benthamiana. Virology, 306(1), 33-41. doi:10.1016/s0042-6822(02)00018-1Van Dun, C. M. P., Van Vloten-Doting, L., & Bol, J. F. (1988). Expression of alfalfa mosaic virus cDNA1 and 2 in transgenic Tobacco plants. Virology, 163(2), 572-578. doi:10.1016/0042-6822(88)90298-xVanitharani, R., Chellappan, P., Pita, J. S., & Fauquet, C. M. (2004). Differential Roles of AC2 and AC4 of Cassava Geminiviruses in Mediating Synergism and Suppression of Posttranscriptional Gene Silencing. Journal of Virology, 78(17), 9487-9498. doi:10.1128/jvi.78.17.9487-9498.2004Voinnet, O., Lederer, C., & Baulcombe, D. C. (2000). A Viral Movement Protein Prevents Spread of the Gene Silencing Signal in Nicotiana benthamiana. Cell, 103(1), 157-167. doi:10.1016/s0092-8674(00)00095-7Voinnet, O., Pinto, Y. M., & Baulcombe, D. C. (1999). Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants. Proceedings of the National Academy of Sciences, 96(24), 14147-14152. doi:10.1073/pnas.96.24.14147Voinnet, O., Vain, P., Angell, S., & Baulcombe, D. C. (1998). Systemic Spread of Sequence-Specific Transgene RNA Degradation in Plants Is Initiated by Localized Introduction of Ectopic Promoterless DNA. Cell, 95(2), 177-187. doi:10.1016/s0092-8674(00)81749-3Vuorinen, A. L., Kelloniemi, J., & Valkonen, J. P. T. (2011). Why do viruses need phloem for systemic invasion of plants? Plant Science, 181(4), 355-363. doi:10.1016/j.plantsci.2011.06.008Yaegashi, H., Isogai, M., & Yoshikawa, N. (2012). Characterization of Plant Virus-Encoded Gene Silencing Suppressors. Antiviral Resistance in Plants, 113-122. doi:10.1007/978-1-61779-882-5_8Yang, X., Ren, Y., Sun, S., Wang, D., Zhang, F., Li, D., … Zhou, X. (2018). Identification of the Potential Virulence Factors and RNA Silencing Suppressors of Mulberry Mosaic Dwarf-Associated Geminivirus. Viruses, 10(9), 472. doi:10.3390/v10090472Yelina, N. E., Savenkov, E. I., Solovyev, A. G., Morozov, S. Y., & Valkonen, J. P. T. (2002). Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families. Journal of Virology, 76(24), 12981-12991. doi:10.1128/jvi.76.24.12981-12991.200

First detection of Grapevine rupestris stem pitting-associated virus and Grapevine rupestris vein feathering virus, and new phylogenetic groups for Grapevine fleck virus and Hop stunt viroid isolates, revealed from grapevine field surveys in Spain

[EN] Evaluation of the prevalence of virus and viroid infections was conducted in a grapevine field collection in Valencia, Spain. Samples of autochthonous and traditional grapevine cultivars were collected during November 2011 and tested for the presence of fourteen viruses and five viroids, using RT-PCR. The prevalent viruses were Grapevine rupestris stem pitting-associated virus (GRSPaV: 49% infected samples) and Grapevine leafroll-associated virus 2 (GLRaV-2: 15% of samples). GLRaV-1, GLRaV-3, GLRaV-4 (variants 4 and 5), Grapevine fanleaf virus, Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV) and Grapevine virus A were also detected. Hop stunt viroid (HSVd: 92% of plants infected) and Grapevine yellow speckle viroid 1 (6% of plants) were also detected. Mixed infections with two, and up to six different viruses and/or viroids were common. Only five samples (4%) were free from 19 pathogens tested. This is the first report of GLRaV-4 (variants 4 and 5) in the Valencia region of Spain, and the first record of GRSPaV and GRVFV in this country. Phylogenetic analyses performed with the sequences of these viruses showed that the Spanish isolates of GLRaV-4, GFkV and HSVd belong to new phylogenetic groups.This study was supported by Projects Consejo Superior de Investigaciones Científicas CSIC (2010CL0021) and BIO2011-25018 from the Spanish MINECO / UNIVERSIDAD DE CHILE 04/11-2 and 2010CL0021Fiore, N.; Zamorano, A.; Sánchez Diana, N.; González, X.; Pallás Benet, V.; Sanchez Navarro, JA. (2016). First detection of Grapevine rupestris stem pitting-associated virus and Grapevine rupestris vein feathering virus, and new phylogenetic groups for Grapevine fleck virus and Hop stunt viroid isolates, revealed from grapevine field surveys in Spain. Phytopathologia Mediterranea. 55(2):225-238. https://doi.org/10.14601/Phytopathol_Mediterr-15875S22523855

Factors que incideixen en la inclusió sociolaboral després del desinternament

El present treball va sorgir en el marc de la comissió de CRAEs (Centres Residencials d'Atenció Educativa) de la FEDAIA (Federació d'Entitats d'Atenció i Educació a la Infància i l'Adolescència). La comissió estava treballant en un nou model d'intervenció de qualitat. Davant del fet de que ens mancaven dades sobre els resultats de la intervenció actual, es va plantejar una col·laboració amb la Universitat Autònoma de Barcelona (UAB) per tal que un grup d'investigadors realitzessin aquesta feina, i posteriorment van incorporar-se investigadores de la Universitat de Lleida i la Ramon Llul