Approches phylogénétiques de la taxonomie du genre badnavirus

De nouvelles séquences entières ou partielles de badnavirus sont produites régulièrement et mises à disposition sur Genbank, certaines remettant en cause la classification liée au type de plante hôte. La variabilité importante des badnavirus rend peu aisé le choix de la partie du génome à étudier qui "résumerait" la variabilité globale. En effet, il existe environ 47% identité entre une espèce de badnavirus et le Rice tungro bacilliform virus (RTBV) représentant le genre des tungrovirus et il existe seulement autour de 50% entre deux badnavirus éloignés. L'existence de motifs consensus sur la 3ème partie de l'ORF3 et sur le site d'accrochage au tRNAmet a permis le design de différents couples d'amorces badnavirus "universelles" badna 2/badnaT (1993), Badna2.1/3.1 (1996), Badna1/4 (2002), BadnaF/R (2003). Les motifs de type RTase et RNaseH, correspondant aux sites d'accrochage de la plupart de ces amorces, sont, malheureusement, également présents sur des séquences de rétrotransposons. Les séquences amplifiées par ces amorces ont néanmoins permis de générer de nombreuses séquences de badnavirus libres ou intégrées qui ont abouti à la construction de nombreuses phylogénies de badnavirus. Ces phylogénies ne sont cependant pas toujours cohérentes avec les phylogénies obtenues d'après l'alignement des séquences entières. Il n'y a en fait pas eu de réflexion sur le choix d'une zone adaptée à la classification taxonomique du genre badnavirus puisque la région du génome utilisée pour la classification correspond par défaut à la région du génome des badnavirus disponible majoritairement sur Genbank et il est impossible d'obtenir systématiquement des séquences entières pour classifier ce genre de virus. Afin de mieux comprendre la diversité des badnavirus voire proposer des réaménagements de leur taxonomie, il est nécessaire de se baser sur des phylogénies utilisant des régions du génome représentatives du génome complet. Des études phylogénétiques plus poussées qui permettent d'implémenter des taux d'évolution différents selon les groupes de séquences ont également besoin de ce type de résultat. Pour remédier à ce manque d'informations phylogénétiques pertinentes, le génome des badnavirus entièrement séquencés (20 séquences) a été découpé en morceaux et des phylogénies ont été construites en alignant les différentes zones obtenues. Les phylogénies construites avec le logiciel Phyml (maximum de vraisemblance) sont présentées et discutées; une portion du génome ne produisant que peu de distorsions par rapport aux phylogénies basées sur les séquences entières est proposée. (Texte intégral

Sequential integrations of badnaviruses into the M. acuminata and M. balbisiana genomes

Banana streak virus (BSV) is a double stranded DNA pararetrovirus belonging to the genus Badnavirus and triggering necrotic mosaic lesions on banana plants. BSV exhibits a higher biodiversity than other badnaviruses as a consequence of two phenomena: the epidemic process of the disease through contamination of plant to plant by vector and the release of infectious viral genomes by the banana genome harboring endogenous sequences. BSV sequences described so far are spread among the three groups of the badnavirus diversity. BSV-like sequences belonging to group 2 seem to be all integrated into the banana genome since no infectious episomal particles have been identified so far. We describe here the characterization of such integrations in thirty-four plant samples belonging to different families of the Zingiberale order with a banana sampling representative of the Musa diversity. We have firstly looked for group 2 sequences using PCR with 7 sets of primers which are specific of the 7 BSV-like species identified so far in this group. We then used Southern blot approach with viral probes corresponding to each BSV-like species. Sequenced PCR products of the RT-RNase H region of the viral genome have been used for the construction of a phylogenetic tree. We found all the 7 BSV-like species within the A genomes whereas only 4 species were within B genomes. Interestingly, two new species have been discovered in M. balbisiana genomes only. Additionally, four BSV-like species seem to be integrated in M. schizocarpa, M. basjoo, M. ornata and M. itinerans but these results have to be confirmed. The integration patterns observed by Southern blot analysis show multiple and different integrations of BSV-like species into M. acuminata plants conversely to M. balbisiana plants where patterns are highly conserved. The data suggest that BSV-like integrations likely occurred for some of them before the speciation M. acuminata/M. balbisiana whereas others occurred afterwards. (Texte intégral

Molecular evidence for the association of a strain of Uganda variant of East African cassava mosaic virus to symptom severity in cassava (Manihot esculenta Crantz) fields in Togo

Abstract: Problem statement: This study was carried out to demonstrate that the severity of Cassava Mosaic Disease (CMD) in Togo, is not only influenced by synergism between cassava Begomoviruses in presence, but essentially by recombination between the different Begomoviruses infecting cassava. Approach: Foliar samples presenting typical biological features of Begomoviruses infection were collected from cassava and wild infected plants from different regions of Togo and analysed by PCR targeting the Coat Protein (CP). The PCR products obtained from different isolates of two major Begomoviruses species infecting cassava in Togo were then sequenced and compared with the sequenced of the African cassava mosaic Begomoviruses identified to date and available in NCBI GenBank database by phylogenetic analysis. Results: The results indicate that not only the two major Begomoviruses could be in synergistic interaction in infected cassava in Togo as it has been shown between African Cassava Mosaic Virus (ACMV) and East African Cassava Mosaic Virus (EACMV) elsewhere, but could also create recombinants which would be highly interfering in the development of symptom severity in the country. Conclusion/Recommendations: The study confirmed the assumption that the symptom severity in cassava fields in Togo is rather caused by recombination between different Begomoviruses in presence than by synergistic interaction. More investigations should be done to give insight to this founding. (Résumé d'auteur

A novel approach to the synthesis of nanostructured metal-organic films: X-ray radiolysis of silver ions using a Langmuir monolayer as a template

An application of the radiolysis method using X-ray synchrotron beam is developed as a novel approach to the synthesis of metal-organic films with controlled shapes and thickness. We demonstrate that a Langmuir monolayer deposited onto a silver ion-containing subphase, irradiated by an incident beam impinging below the critical angle for total reflection, induces the synthesis of a stable nanostructured silver-organic ultrathin film at the air-water interface. The X-ray scattering is also used to monitor in situ the structure of the silver layer during the synthesis process. The layer is observed by AFM after its transfer onto a silicon substrate. One observes a film thickness of 4.6 nm, in good agreement with the X-ray penetration depth, about 4.5 nm. The silver structure is oriented by the initial organic film phase. This experiment demonstrates the considerable potential of this approach to produce various controlled metal-organic films with a surfactant self-assembly as a template.Comment: 12 pages, 4 figure

Origine de la biodiversité des séquences BSV détectées en Afrique de l'Est

Le Banana streak virus (BSV), du genre Badnavirus présente une structuration polyphylétique en trois clades bien distincts. Cette structuration complexe demande à être élucidée sachant que le BSV peut exister à la fois sous forme libre et intégrée au génome de son hôte. Des échantillons ont été prélevés en Ouganda, seul pays d'épidémie ancienne où la variabilité moléculaire du BSV est particulièrement importante puisque les trois clades y ont été détectés, contrairement à d'autres régions du monde. Afin d'étudier la variabilité moléculaire du BSV dans cette zone en lien avec son origine intégrée ou pas, le virus a été recherché par immuno-captures PCR avec des amorces spécifiques des espèces BSV du clade I, puis par PCR sur les extraits d'ADN totaux avec des amorces spécifiques des espèces BSV du clade II et III. Par ailleurs, la nature libre ou intégrée des séquences BSV dans le génome de bananier a été étudiée par hybridation en Southern Blot. (Texte intégral

A scenario of co-evolution between badnaviruses and Musa sp.. [P.22]

The banana streak disease is due to a complex of distinct Banana streak viruses (BSVs) species showing a wide genetic diversity. Banana streak viruses (BSVs) are double stranded DNA pararetroviruses belonging to the family Caulimoviridae, genus Badnavirus. Outbreaks of BSVs causing banana streak disease have been recorded worldwide where Musa spp. is grown during the last 20 years with no convincing evidence of epidemics. Epidemics were previously reported in Uganda where BSV is currently endemic. In addition, the banana genome (Musa sp.) is invaded by numerous badnavirus sequences. The majority of these viral integrants is mostly defective as a result of pseudogenisation driven by the host genome evolution. They are just called BEV (banana endogenous virus sequences) because episomal particles corresponding to the integrated counterparts have not been identified so far. Conversely, only few viral integrants named endogenous BSV (eBSVs) can release a functional BSV genome following stresses. All the badnaviral sequences described so far are spread among the three main clades of the badnavirus genus diversity. Our group established that BSVs are distributed among Clade I and Clade III. Clade I gathers BSV species distributed worldwide whereas Clade III is dedicated to BSV species only present in Uganda. eBSVs exclusively correspond to BSV species of Clade I and are endogenous to the Musa balbisiana (B) genomes only. We elucidated their sequence and organization for three BSV species (BSOLV, BSGFV and BSIMV) present in the seedy banana diploid PKW (BB). In addition, we established that all BEVs sequences belong to Clade II. All together our results allowed us to propose an evolutionary scheme of badnavirus and banana co-evolution, which is presented here. (Résumé d'auteur

Study on the origin of cacao swollen shoot virus and its dispersal on cacao trees in West Africa [W112]

Cacao swollen shoot virus (CSSV) is a member of the family Caulimoviridae, genus Badnavirus naturally transmitted to Theobroma cacao by several mealybug species. The virus, restricted to West Africa whereas the cacao tree originates from the Western Hemisphere, could therefore most probably have an indigenous origin on the West African subcontinent. The disease has caused enormous economic damage in Ghana since the1930s but was only restricted to small areas in Togo and Côte d'Ivoire until recently. Now, renewed outbreaks in the main producing areas in Côte d'Ivoire, Ghana and Togo cause serious problems. CSSV populations in West African countries are genetically structured in twelve groups according to the diversity in the first part of ORF3 and the 20% threshold of nucleotide divergence. However, according to ICTV recommendations taking into account the nucleotide diversity in the RTAse region, we could describe seven different species. The high variability observed within CSSV populations compared to its very short evolutionary history on cocoa trees, suggests the existence of many emergences from native hosts to cacao trees in the various countries of West Africa. Moreover, based on the geographical dispersal of the different species, we could propose the existence at different times of parallel emergences in each of the West African countries. As a perspective of this work, a newly accepted project funded by European Cocoa Association will be presented. (Texte intégral

Study on the origin of cacao swollen shoot virus and its dispersal on cacao trees in West Africa

BACKGROUND and OBJECTIVES Cacao swollen shoot virus (CSSV) is a member of the family Caulimoviridae, genus Badnavirus naturally transmitted to Theobroma cacao by several mealybug species. Typical symptoms of the disease on cocoa trees are red vein banding of young leaves, mosaic on older leaves and swelling of the orthotropic shoots. The virus, restricted to West Africa whereas the cacao tree originates from the Western Hemisphere, could therefore most probably have an indigenous origin on the West African subcontinent. The disease has caused enormous economic damage in Ghana since the1930s but was only restricted to small areas in Togo and Côte d'Ivoire until recently. Now, renewed outbreaks in the main producing areas in Côte d'Ivoire, Ghana and Togo cause serious problems. The knowledge of the viral biodiversity in the different outbreaks will in turn help to provide a better understanding of the development of the epidemics, and of the evolution of viral populations and may permit to retrace the emergence and dispersal of CSSV. MATERIAL and METHODS Prospections were made over several successive years in Ivory Coast, Togo and Ghana. Virus variability was studied by PCR amplification with CSSV primers, direct sequencing, sequence alignment and phylogenetic studies. RESULTS CSSV diversity is genetically structured in twelve groups according to the diversity in the first part of ORF3 and the 20% threshold of nucleotide divergence. However, according to ICTV recommendations which consider the nucleotide diversity in the RTAse region, we could describe at least seven different species. CONCLUSIONS The high variability observed within CSSV populations compared to its very short evolutionary history on cocoa trees, suggests the existence of many emergences from native hosts to cacao trees in the various countries of West Africa. Moreover, based on the geographical dispersal of the different species, we could propose the existence at different times of parallel emergences in each of the West African countries. (Texte intégral

Towards a better characterization of endogenous badnavirus sequences of yams (Dioscorea spp.)

Yams, and more generallytubers, are very important crops for food security in tropical and subtropical countries.They are propagated vegetativelytherefore they accumulate viruses over long periods of time. Viruses are currently the main constraint for yam production and yam germplasm conservation and distribution. A wide range of badnavirus sequences belonging to 13 distinct viral species were amplified from genomic DNA of severalyam species when using badnavirus degenerate primers [1; 2].However, we consistently observed that the proportion of amplification products raised by PCR performed on total genomic DNA is significantly higher than that raised by direct binding PCR, which has been designed to detect episomal forms of yam badnaviruses.Both observations have fueled suspicion that yams might host endogenous badnavirus sequences, and possibly infectious ones like bananas [3]. Therefore, search for endogenous badnavirus sequences was undertaken in yam accessions conserved in the germplasm collection of the Guadeloupe Tropical Plant Biological Ressource Center (CRB-PT) and the yam quarantine facility in Montpellier (France). Southern blots performed on genomic DNA extracted from uninfected Dioscoreatrifidaand using parts of yam badnavirus genomes as probes confirmed the suspicion of endogenous badnavirus sequences in yam genomes. Furthermore, PCR performed on genomic DNA extracted from healthy seedlings of D. alata and D. rotundata using badnavirus degenerate primers raised amplification products whose sequences fit in the current phylogeny of badnaviruses. Amplification products raised from several of these DNA samples by long-PCR displayedrearrangements such asduplications andreversions which are reminiscent of endogenous badnavirus sequences encountered in the genome of other crops such as banana. Similarly rearranged sequences were raised by rolling circle amplification, which is known to sometime amplify chromosomal sequences. These results suggest that yams do host endogenous sequences of several distinct badnavirus species. (Texte intégral