Genomic and structural study of Toscana virus

La première partie de mon travail a consisté (i) en une étude génomique des souches de TOSV avec séquençage de 10 nouvelles souches afin i) d’enrichir les données disponibles dans Genbank (au début de ce travail, 6 séquences complètes); (ii) d’utiliser les 16 séquences complètes pour définir et évaluer le premier système de typage par technique de RT-q PCR en temps réel afin de discriminer les souches de LA et de LB. Dans la deuxième partie de ce projet, on s’est intéressée à des études structurales et fonctionnelles de la nucléoproteine (N) de TOSV afin d’élucider le mécanisme d’encapsidation d’ARN virale. La N est une protéine de 28 KD, sont rôle majeur est l’encapsidation de l’ARN génomique et sert en tant que co-facteur de la transciption/replication de TOSV. Les études crystallographique de la protéine N , du complexe protéique (N-ARN) ont été déterminé par Olal D et al 2014 au moment que nous avons obtenu la diffraction de crystal de la N sans ARN à 3,7 Å (code PDB: 5FVA). Ces études montrent la protéine N ainsi le complexe (N-ARN) en forme d’un anneau hexamèrique fermé alors encapside l'ARN dans une organisation filamenteux. Nous avons donc décidé de combiner ces résultats avec des études structurales complémentaires en solution , telles que la microscopie électronique (EM) et des études de « Diffusion des rayons X aux petits angles »(SAXS) pour de proposer un modèle décrivant correctement le mécanisme d'encapsidation en solution . Le protéine N se comporte principalement en pentamère déformé et ouvert, qui met en lumière la façon dont nucléoprotéine peut être organisée en filaments.The first part of my work consisted (i) of genomic sequencing of 10 TOSV strains to increase the total number of complete sequences (at the outset, 6 complete sequences were accessible in Genbank). (ii) to use the 16 sequences to design and evaluated the first lineage-specific real-time RT-PCR assay (Lisp-TOSV) to discriminate between strains of lineages A and B Complete sequencing of the 10 strains was achieved. The second part of this project was dedicated for structural and functional studies of the TOSV N protein in order to decipher viral RNA encapsidation. TOSV Nucleoprotein (N) is a protein of 28 KD, is encapsidating the viral RNA genome and serves as a co-factor the RNA trancription/replication. The crystal structures of TOSV N protein , and the complex N-RNA were already determined (Olal D and al; 2014) while we just obtained diffracting crystal of the N free RNA at 3,7 Å (PDB code : 5FVA ). Crystallographic structures show an hexameric rings whereas N encapsidates the RNA in a filamentous organization. We therefore decided to combine these results with complementary studies, such as electron microscopy (EM) and samll angle X-ray scattering to build a low resolution structure model in solution describing properly the encapsidation mechanism. The N behaves mainly as an opened, deformed pentamer that shed light on how the N can be organized in filaments

Structural characterization of the N-terminal part of the MERS-CoV nucleocapsid by X-ray diffraction and small-angle X-ray scattering

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Toscana virus nucleoprotein oligomer organization observed in solution.

International audienceToscana virus (TOSV) is an arthropod-borne virus belonging to the Phlebovirus genus within the Bunyaviridae family. As in other bunyaviruses, the genome of TOSV is made up of three RNA segments. They are encapsidated by the nucleoprotein (N), which also plays an essential role in virus replication. To date, crystallographic structures of phlebovirus N have systematically revealed closed-ring organizations which do not fully match the filamentous organization of the ribonucleoprotein (RNP) complex observed by electron microscopy. In order to further bridge the gap between crystallographic data on N and observations of the RNP by electron microscopy, the structural organization of recombinant TOSV N was investigated by an integrative approach combining X-ray diffraction crystallography, transmission electron microscopy, small-angle X-ray scattering, size-exclusion chromatography and multi-angle laser light scattering. It was found that in solution TOSV N forms open oligomers consistent with the encapsidation mechanism of phlebovirus RNA

2,4-D induction of somaclonal variations in in vitro grown date palm (Phoenix dactylifera L. cv Barhee)

International audienceKey message: Results demonstrate that the 2,4-D can affect physiological and molecular parameters of vitrocultures when used at high concentrations without hampering their morphogenetic capacities. It was found to be efficient at inducing genetic and epigenetic variations.Abstract: The present study is a part of a program designed at improving the date palm, Phoenix dactylifera L. cv. Barhee, through induced somaclonal variation. In this work, caulogenic cultures were subcultured on MS media supplemented with 0, 1, 5, 10, 20 and 40 mg L-1 2,4-D in order to induce genetic and epigenetic variations. The highest doses of 2,4-D were found to induce severe negative effects on in vitro cultures, although some tissues were able to survive and to produce calli with high morphogenetic capacities. Our analysis showed some significant effect of 2,4-D on several physiological parameters. Indeed, chlorophyll and growth rates were found to drastically decrease while proline content increased from 535 to 2973 nmol g(-1) FW when 40 mg L-1 2,4-D were used. In vitro cultures showed several signs of oxidative stress, such as high levels of hydrogen peroxide and malondialdehyde; likewise, the specific activity of several antioxidant enzyme was found to increase. Plant regeneration from in vitro cultures treated with 2,4-D was obtained after subculturing explants onto PGR-free media. The ISSR analysis of 2,4-D-treated material showed that this plant growth regulator (PGR) induced measurable genetic variations. The global DNA methylation rates (GMR) as estimated through the HPLC analysis of nucleosides also confirmed the presence of epigenetic changes caused by 2,4-D as GMRs increased from 13.8 to 18.93%

Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part I: Important Points to Consider Ante Field Work

International audienceThe purpose of this review is to provide practical information to help researchers intending to perform “from field to laboratory” studies on phleboviruses transmitted by sandflies. This guideline addresses the different steps to be considered starting from the field collection of sandflies to the laboratory techniques aiming at the detection, isolation, and characterization of sandfly-borne phleboviruses. In this guideline article, we address the impact of various types of data for an optimal organization of the field work intending to collect wildlife sandflies for subsequent virology studies. Analysis of different data sets should result in the geographic positioning of the trapping stations. The overall planning, the equipment and tools needed, the manpower to be deployed, and the logistics to be anticipated and set up should be organized according to the objectives of the field study for optimal efficiency

Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part II: Important Points to Consider for Fieldwork and Subsequent Virological Screening

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Mary Ann Liebert/Society for Zoonotic Ecology and Epidemiology (SocZEE)

In this series of review articles entitled "Practical guidelines for studies on sandfly-borne phleboviruses," the important points to be considered at the prefieldwork stage were addressed in part I, including parameters to be taken into account to define the geographic area for sand fly trapping and how to organize field collections. Here in part II, the following points have been addressed: (1) factors influencing the efficacy of trapping and the different types of traps with their respective advantages and drawbacks, (2) how to process the trapped sand flies in the field, and (3) how to process the sand flies in the virology laboratory. These chapters provide the necessary information for adopting the most appropriate procedures depending on the requirements of the study. In addition, practical information gathered through years of experience of translational projects is included to help newcomers to fieldwork studies.The work of all authors was carried under the frame of EurNegVec COST Action TD1303. This work was supported, in part, by (1) the European Virus Archive goes Global (EVAg) project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 653316 and by (2) the EDENext FP7-no. 261504 EU project; this article is catalogued by the EDENext Steering Committee as EDENext463 (www.edenext.eu).info:eu-repo/semantics/publishedVersio