CROSS-PROTECTION BETWEEN DIFFERENT PATHOTYPES OF Pepino mosaic virus REPRESENTING CHILEAN 2 GENOTYPE

Viral cross-protection in plants is a phenomenon, where a mild virus isolate can protect plants against damage caused by a severe challenge isolate of the same virus. Ithas been used on a large scale in cases where no resistant plants are available. We examined differences in cross-protection between pathotypes of Pepino mosaic virus representingChilean 2 genotype. The potential of a mild PepMV-P22 isolate to protect tomato against more aggressive challenge isolates causing yellowing and necrotic symptoms wasestablished. The challenge isolates were PepMV-P5-IY (yellowing), PepMV-P19 (necrotic) and PepMV-P22 K67E (artificial necrotic mutant of PepMV-P22 which differ from PepMV-P22 only by a point mutation). Efficient cross-protection was obtained using mild PepMV-P22 against PepMV-P5-IY. After a challenge inoculation with PepMV-P19 or PepMV-P22 K67E symptoms severity were significantly reduced in comparison to non-protected plants; however, necrotic symptoms appeared two months after coinfection. The real-time PCR analysis revealed that the level of accumulation of the necrotic isolate in tomato plants was even 5–7 times higher than that of PepMV-P22

Towards a conceptualization of the study of Africa’s indigenous manuscript heritage and tradition

This paper share experiences of th South African Conservation Technical Team of the Timbuktu Rare Manuscripts Project in the conservation and preservation of manuscripts in Timbuktu. A manuscript is always more than just its textual information – it is a living historical entity and its study a complex web of interrelated factors: the origins, production (that is, materials, formats, script, typography, and illustration), content, use and role of books in culture, educated and society in general. The widespread availability of paper made it easier to produce these manuscripts as some of the important vehicles for transmitting of knowledge in Islamic society. Islamic written culture, particularly during the time of the European middle ages was by all accounts incomparably more brilliant than anything known in contemporary Europe. The time for studying the African manuscript tradition has never been more appropriate given the recent renewed calls for the need to reappraise African history and achievements. It must be acknowledged, however, that the study of African manuscript heritage will not be without difficulty

Defective RNA particles derived from Tomato black ring virus genome interfere with the replication of parental virus

[EN] 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 Investigacion-FEDER (grant number BFU2015-65037P) (to S.F.E.). The funding bodies were not involved into the design of the study, analysis, and interpretation of data in the manuscript.Hasiow, B.; Minicka, J.; Zarzynska, A.; Budzynska, D.; Elena Fito, SF. (2018). Defective RNA particles derived from Tomato black ring virus genome interfere with the replication of parental virus. Virus Research. 250:87-94. https://doi.org/10.1016/j.virusres.2018.04.010S879425

Strain-dependent mutational effects for Pepino mosaic virus in a natural host

[EN] Pepino mosaic virus (PepMV) is an emerging plant pathogen that infects tomatoes worldwide. Understanding the factors that influence its evolutionary success is essential for developing new control strategies that may be more robust against the evolution of new viral strains. One of these evolutionary factors is the distribution of mutational fitness effect (DMFE), that is, the fraction of mutations that are lethal, deleterious, neutral, and beneficial on a given viral strain and host species. The goal of this study was to characterize the DMFE of introduced nonsynonymous mutations on a mild isolate of PepMV from the Chilean 2 strain (PepMV-P22). Additionally, we also explored whether the fitness effect of a given mutation depends on the gene where it appears or on epistatic interactions with the genetic background. To address this latter possibility, a subset of mutations were also introduced in a mild isolate of the European strain (PepMV-P11) and the fitness of the resulting clones measured.This study was financially supported by grant 2011/01/D/NZ9/00279, from the Poland National Science Center, to B.H.J and by grants BFU2015-65037-P, from Spain Ministry of Economy and Competitiveness-FEDER, and PROMETEOII/2014/021, from Generalitat Valenciana, to S.F.E.Minicka, J.; Elena Fito, SF.; Borodynko-Filas, N.; Rubis, B.; Hasiów-Jaroszewska, B. (2017). Strain-dependent mutational effects for Pepino mosaic virus in a natural host. BMC Evolutionary Biology. 17:1-11. https://doi.org/10.1186/s12862-017-0920-4S11117Steinhauer DA, Domingo E, Holland JJ. Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase. Gene. 1992;122:281–8.Sanjuán R, Nebot MR, Chirico N, Mansky LM, Belshaw R. Viral mutation rates. J Virol. 2010;84:9733–48.Domingo E. Viruses at the edge of adaptation. 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Nature. 2014;505:686–90.Visher E, Whitefield SE, McCrone JT, Fitzsimmons W, Lauring AS. The mutational robustness of Influenza A virus. PLoS Pathog. 2016;12, e1005856.Carrasco P, de la Iglesia F, Elena SF. Distribution of fitness and virulence effects caused by single-nucleotide substitutions in Tobacco etch virus. J Virol. 2007;81:12979–84.Bernet GP, Elena SF. Distribution of mutational fitness effects and of epistasis in the 5′ untranslated region of a plant RNA virus. BMC Evol Biol. 2015;15:274.Domingo-Calap P, Cuevas JM, Sanjuán R. The fitness effects of random mutations in single-stranded DNA and RNA bacteriophages. PLoS Genet. 2009;5, e1000742.Peris JB, Davis P, Cuevas JM, Nebot MR, Sanjuán R. Distribution of fitness effects caused by single-nucleotide substitutions in bacteriophage f1. Genetics. 2010;185:603–9.Keightley PD, Ohnishi O. EMS-induced polygenic mutation rates for nine quantitative characters in Drosophila melanogaster. Genetics. 1998;148:753–66.Keightley PD, Davies EK, Peters AD, Shaw RG. Properties of ethylmethane sulfonate-induced mutations affecting life-history traits in Caenorhabditis elegans and inferences about bivariate distributions of mutation effects. Genetics. 2000;156:143–54.Koufopanou V, Lomas S, Tsai IJ, Burt A. Estimating the fitness effects of new mutations in the wild yeast Saccharomyces paradoxus. Genome Biol Evol. 2015;7:1887–95.Sanjuán R. Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis. Philos Trans R Soc B. 2010;365:1975–82.Keightley PD, Lynch M. Toward a realistic model of mutations affecting fitness. Evolution. 2003;57:683–5.Orr HA. The distribution of fitness effects among beneficial mutations. Genetics. 2003;163:1519–26.Miralles R, Gerrish PJ, Moya A, Elena SF. Clonal interference and the evolution of RNA viruses. Science. 1999;285:1745–7.Escarmís C, Dávila M, Charpentier N, Bracho A, Moya A, Domingo E. Genetic lesions associated with Muller’s ratchet in an RNA virus. J Mol Biol. 1996;264:255–67.Phillips PC. Epistasis - the essential role of gene interactions in the structure and evolution of genetic systems. Nat Rev Genet. 2008;9:855–67.De Visser JAGM, Krug J. Empirical fitness landscapes and the predictability of evolution. Nat Rev Genet. 2014;15:480–90.Lalić J, Elena SF. Magnitude and sign epistasis among deleterious mutations in a positive-sense plant RNA virus. Heredity. 2012;109:71–7.Lalić J, Elena SF. The impact of higher-order epistasis in the within-host fitness of a positive-sense plant RNA virus. J Evol Biol. 2015;28:2236–47.Hillung J, Cuevas JM, Elena SF. Evaluating the within-host fitness effects of mutations fixed during virus adaptation to different ecotypes of a new host. Philos Trans R Soc B. 2015;370:20140292.Cervera H, Lalić J, Elena SF. Effect of host species on the topography of the fitness landscape for a plant RNA virus. J Virol. 2016;90:10160–9.Cervera H, Lalić J, Elena SF. Efficient escape from local optima in a highly rugged fitness landscape by evolving RNA virus populations. Proc R Soc B. 2016;283:20160984.Blystad DR, van der Vlugt R, Alfaro-Fernandez A, Cordoba MD, Bese G, Hristova D, Pospieszny H, Mehle N, Ravnikar M, Tomassoli L, Varveri C, Nielsen SL. Host range and symptomatology of Pepino mosaic virus strains occurring in Europe. Eur J Plant Pathol. 2015;143:43–56.Mumford RA, Metcalfe EJ. The partial sequencing of the genomic RNA of a UK isolate of Pepino mosaic virus and the comparison of the coat protein sequence with other isolates from Europe and Peru. Arch Virol. 2001;146:2455–60.Roggero P, Masenga V, Lenzi R, Coghe F, Ena S, Winter S. First report of Pepino mosaic virus in tomato in Italy. Plant Dis. 2001;3:8.Cotillon AC, Girard M, Ducouret S. Complete nucleotide sequence of the genomic RNA of a French isolate of Pepino mosaic virus (PepMV). Arch Virol. 2002;147:2231–8.Maroon-Lango CJ, Guaragna MA, Jordan RL, Hammond J, Bandla M, Marquardt SK. Two unique US isolates of Pepino mosaic virus from a limited source of pooled tomato tissue are distinct from a third (European-like) US isolate. Arch Virol. 2005;150:1187–201.Pagán I, Córdoba-Selles MD, Martínez-Priego L, Fraile A, Malpica JM, Jorda C, García-Arenal F. Genetic structure of the population of Pepino mosaic virus infecting tomato crops in Spain. Phytopathology. 2006;96:274–9.Ling KS. Molecular characterization of two Pepino mosaic virus variants from imported tomato seed reveals high levels of sequence identity between Chilean and US isolates. Virus Genes. 2007;34:1–8.Hanssen IM, Paeleman A, Wittemans L, Goen K, Lievens B, Bragard C, Vanachter A, Thomma B. Genetic characterization of Pepino mosaic virus isolates from Belgian greenhouse tomatoes reveals genetic recombination. Eur J Plant Pathol. 2008;121:131–46.Hasiów B, Borodynko N, Pospieszny H. Complete genomic RNA sequence of the Polish Pepino mosaic virus isolate belonging to the US2 strain. Virus Genes. 2008;36:209–14.Hanssen IM, Paeleman A, Vandewoestijne E, Van Bergen L, Bragard C, Lievens B, Vanacher ACRC, Thomma BPHJ. Pepino mosaic virus isolates and differential symptomatology in tomato. Plant Pathol. 2009;58:450–60.Moreno-Pérez MG, Pagán I, Aragón-Caballero L, Cáceres F, Fraile A, García-Arenal F. Ecological and genetic determinants of Pepino mosaic virus emergence. J Virol. 2014;88:3359–68.Ling K, Li R, Bledsoe M. Pepino mosaic virus genotype shift in North America and development of a loop-mediated isothermal amplification for rapid genotype identification. 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Ultrastructural insights into tomato infections caused by three different pathotypes of Pepino mosaic virus and immunolocalization of viral coat proteins. Micron. 2015;79:84–92.Gómez P, Sempere RN, Aranda MA, Elena SF. Phylodynamics of Pepino mosaic virus in Spain. Eur J Plant Pathol. 2012;134:445–9.Vignuzzi M, Stone JK, Arnold JJ, Cameron CE, Andino R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature. 2006;439:344–8.Domingo E, Martíb V, Perales C, Grande-Pérez A, García-Arriaza Arias J. Viruses as quasispecies: biological implications. Curr Top Microbiol Immunol. 2006;299:51–82.Jones RAC, Koenig R, Lesemann DE. Pepino mosaic virus, a new potexvirus from pepino (Solanum muricatum). Ann Appl Biol. 1980;94:61–8.Domingo E, Sheldon J, Perales C. Viral quasispecies evolution. Microbiol Mol Biol Rev. 2012;76:159–216.Lough TJ, Emerson SJ, Lucas WJ, Forster RLS. 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Detection and genetic variability of newly identified dasheen mosaic virus in Poland

Dasheen mosaic virus (DsMV) is one of the most important viral pathogens of aroids and can cause major economic losses for ornamental crops. Here, we present the detection and molecular characterisation of DsMV isolates originating from Monstera adansonii plants in Poland. Moreover, the genetic variability of DsMV isolates was analyzed based on the coat protein gene ( CP) of the Polish and other DsMV isolates described to date. The presence of DsMV was confirmed by transmission electron microscopy (TEM) and reverse transcription polymerase chain reaction (RT-PCR) with specific, diagnostic primers in three out of ten examined plants. To obtain full-length sequences of CP, two pairs of primers were designed and used in the RT-PCR. The specificity of obtained products was confirmed by Sanger sequencing. The obtained sequences of CP were compared with 44 other DsMV sequences retrieved from the GenBank. Analyses revealed that DsMV population is very diverse. The variability of DsMV isolates was confirmed by low sequence identity and pervasive recombination events. The phylogenetic analysis was performed based on 37 non-recombinant CP sequences. The maximum-likelihood reconstruction revealed that the Polish isolates are distinct and grouped separately from other DsMV isolates. Due to the high genetic diversity, detecting the virus could be difficult. Nonetheless disease management relies strongly on a fast and accurate identification of the causal agent. To our knowledge this is the first report of DsMV in Poland

The transcriptomics of an experimentally evolved plant-virus interaction

[EN] Models of plant-virus interaction assume that the ability of a virus to infect a host genotype depends on the matching between virulence and resistance genes. Recently, we evolved tobacco etch potyvirus (TEV) lineages on different ecotypes of Arabidopsis thaliana, and found that some ecotypes selected for specialist viruses whereas others selected for generalists. Here we sought to evaluate the transcriptomic basis of such relationships. We have characterized the transcriptomic responses of five ecotypes infected with the ancestral and evolved viruses. Genes and functional categories differentially expressed by plants infected with local TEV isolates were identified, showing heterogeneous responses among ecotypes, although significant parallelism existed among lineages evolved in the same ecotype. Although genes involved in immune responses were altered upon infection, other functional groups were also pervasively over-represented, suggesting that plant resistance genes were not the only drivers of viral adaptation. Finally, the transcriptomic consequences of infection with the generalist and specialist lineages were compared. Whilst the generalist induced very similar perturbations in the transcriptomes of the different ecotypes, the perturbations induced by the specialist were divergent. Plant defense mechanisms were activated when the infecting virus was specialist but they were down-regulated when infecting with generalist.We thank Francisca de la Iglesia and Paula Agudo for excellent technical assistance and our labmates for useful discussions and suggestions. This work was supported by grants BFU2012-30805 from the Spanish Ministry of Economy and Competitiveness (MINECO), PROMETEOII/2014/021 from Generalitat Valenciana and EvoEvo (ICT610427) from the European Commission 7th Framework Program to SFE, and grant PROMETEOII/2014/025 to JD. JMC was supported by a JAE-doc postdoctoral contract from CSIC. JH was recipient of a predoctoral contract from MINECO.Hillung, J.; García-García, F.; Dopazo, J.; Cuevas Torrijos, JM.; Elena Fito, SF. (2016). The transcriptomics of an experimentally evolved plant-virus interaction. Scientific Reports. 6:1-19. https://doi.org/10.1038/srep24901S1196Duffy, S., Shackelton, L. A. & Holmes, E. C. Rates of evolutionary change in viruses: patterns and determinants. Nat. Rev. Genet. 9, 267–276 (2008).Parrish, C. R. et al. Cross-species virus transmission and the emergence of new epidemic diseases. Microbiol. Mol. Biol. Rev. 72, 457–470 (2008).Holmes, E. C. The comparative genomics of viral emergence. Proc. Natl. 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Safegaurding Africa's Literary Heritage : timbuktu rare manuscripts project

I would like to first set the context for this presentation: that is the place of Timbuktu – it is a city of legend and myth, as much as of actual history. The city of Timbuktu is located in modern-day West African country of Mali close to the river Niger at its northern-most bend, on the fringes of the Sahara desert. It is though that Timbuktu was founded some time around 1100 CE (Bovill 1958:88; Hunwick 2003:1;Saad 1983:4). The fortuitous placement of Timbuktu at the crossing of the Niger River and a major caravan route that continues to Marrakech (Morocco) in the north and swings towards the modern-day state of Sudan across the Sahara desert; as well as one of the major routes for pilgrimage to Mecca is surely a large part of the reason for its success as a centre of commerce – which brought with it both wealth and culture (Bovill 1958:105; De Villiers & Hirtle 2003:212; Saad 1983:6). Part of the legend of Timbuktu’s manuscripts is due to the reputed vast number of manuscripts to be found in Timbuktu; literature on the subject ascribe anything between one to five million manuscripts in Timbuktu and its immediate environs. Timbuktu’s most celebrated scholar, Ahmed Baba (1564-1627 CE) claimed that his personal library contained some 1 600 volumes (Hunwick 2003:3), and that his was the smallest library within his family. His family, the Aqit, were the leading scholarly family during the 16th century in Timbuktu. The 16th century traveller Leo Africanus, noted that books were the most valued among the various articles of trade and wrote that: “… hither are brought divers manuscripts or written books out of Barbary, which are sold for more money than any other merchandise.” (De Villiers & Hirtle 2003:212; Saad 1983:88). In addition scholars returning from pilgrimage further augmented Timbuktu’s manuscript collections over the centuries and study in other centres of Islamic learning, often copied by their own hands. Within Timbuktu there existed an active copying and scribal industry (Hunwick 2003:3) that ensured a continual production of manuscripts for the consumption of scholars, students and literate citizens

Towards a conceptualization of the study of Africa’s indigenous manuscript heritage and tradition

This paper share experiences of th South African Conservation Technical Team of the Timbuktu Rare Manuscripts Project in the conservation and preservation of manuscripts in Timbuktu. A manuscript is always more than just its textual information – it is a living historical entity and its study a complex web of interrelated factors: the origins, production (that is, materials, formats, script, typography, and illustration), content, use and role of books in culture, educated and society in general. The widespread availability of paper made it easier to produce these manuscripts as some of the important vehicles for transmitting of knowledge in Islamic society. Islamic written culture, particularly during the time of the European middle ages was by all accounts incomparably more brilliant than anything known in contemporary Europe. The time for studying the African manuscript tradition has never been more appropriate given the recent renewed calls for the need to reappraise African history and achievements. It must be acknowledged, however, that the study of African manuscript heritage will not be without difficulty.</jats:p