Detection of Banana Mild Mosaic Virus in musa in vitro plants: High-throughput sequencing presents higher diagnostic sensitivity than (IC)-RT-PCR and identifies a new betaflexiviridae species

The banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus that infects Musa spp. and has a very wide geographical distribution. The current BanMMV indexing process for an accession requires the testing of no less than four plants culti-vated in a greenhouse for at least 6 months and causes a significant delay for the distribution of the germplasm. We evaluated the sensitivity of different protocols for BanMMV detection from in vitro plants to accelerate the testing process. We first used corm tissues from 137 in vitro plants and obtained a diagnostic sensitivity (DSE) of only 61% when testing four plants per accession. After thermotherapy was carried out to eliminate BanMMV infection, the meristem was recovered and further grown in vitro. The same protocol was evaluated in parallel on the corm tissue surrounding the meristem, as a rapid screening to evaluate virus therapy success, and was compared to the results obtained following the standard protocol. The obtained results showed 28% false negatives when conducting testing from corm tissues, making this protocol unsuitable in routine processes. Furthermore, RT-PCR and high-throughput sequencing (HTS) tests were applied on tissues from the base (n = 39) and the leaves (n = 36). For RT-PCR, the average DSE per sample reached 65% from either the base or leaves. HTS was applied on 36 samples and yielded 100% diagnostic specificity (DSP) and 100% DSE, whatever the sampled tissue, allowing the identification of a new Betaflex-iviridae species infecting Musa. These results suggest that a reliable diagnostic of BanMMV from in vitro plants using RT-PCR or HTS technologies might represent an efficient alternative for testing after greenhouse cultivation

Identification of divergent isolates of Banana Mild Mosaic Virus and development of a new diagnostic primer to improve detection

This study aims to describe the identification and genome sequencing of two isolates of Banana Mild Mosaic Virus, and, based on the virus sequences available in GenBank, to design and test a new diagnostic primer for a routine indexing use

A Survey Using High-Throughput Sequencing Suggests That the Diversity of Cereal and Barley Yellow Dwarf Viruses Is Underestimated

Worldwide, barley/cereal yellow dwarf viruses (YDVs) are the most widespread and damaging group of cereal viruses. In this study, we applied high-throughput sequencing technologies (HTS) to perform a virus survey on symptomatic plants from 47 cereal fields in Estonia. HTS allowed the assembly of complete genome sequences for 22 isolates of cereal yellow dwarf virus RPS, barley yellow dwarf virus GAV, barley yellow dwarf virus PAS (BYDV-PAS), barley yellow dwarf virus PAV (BYDV-PAV), and barley yellow dwarf virus OYV (BYDV-OYV). We also assembled a near-complete genome of the putative novel species BYDV-OYV from Swedish samples of meadow fescue. Previously, partial sequencing of the central part of the coat protein gene indicated that BYDV-OYV represented a putative new species closely related to BYDV-PAV-CN, which currently is recognized as a subtype of BYDV-PAV. The present study found that whereas the 3′gene block of BYDV-OYV shares the closest relationship with BYDV-PAV-CN, the 5′gene block of BYDV-OYV shows the closest relationships to that of BYDV-PAS. Recombination detection analysis revealed that BYDV-OYV is a parental virus for both. Analysis of complete genome sequence data indicates that both BYDV-OYV and BYDV-PAV-CN meet the species criteria of genus Luteovirus. The study discusses BYDV phylogeny, and through a systematic in silico analysis of published primers for YDV detection, the existing gaps in current diagnostic practices for detection of YDVs, proposing primer pairs based on the most recent genomic information for the detection of different BYDV species. Thanks to the rising number of sequences available in databases, continuous updating of diagnostic primers can improve test specificity, e.g., inclusivity and exclusivity at species levels. This is needed to properly survey the geographical and host distribution of the different species of the YDV complex and their prevalence in cereal/barley yellow dwarf disease epidemics

Semi-artificial datasets as a resource for validation of bioinformatics pipelines for plant virus detection

The widespread use of High-Throughput Sequencing (HTS) for detection of plant viruses and sequencing of plant virus genomes has led to the generation of large amounts of data and of bioinformatics challenges to process them. Many bioinformatics pipelines for virus detection are available, making the choice of a suitable one difficult. A robust benchmarking is needed for the unbiased comparison of the pipelines, but there is currently a lack of reference datasets that could be used for this purpose. We present 7 semi-artificial datasets composed of real RNA-seq datasets from virus-infected plants spiked with artificial virus reads. Each dataset addresses challenges that could prevent virus detection. We also present 3 real datasets showing a challenging virus composition as well as 8 completely artificial datasets to test haplotype reconstruction software. With these datasets that address several diagnostic challenges, we hope to encourage virologists, diagnosticians and bioinformaticians to evaluate and benchmark their pipeline(s)

Testing the 'zero-sum game' hypothesis: An examination of school health policy and practice and inequalities in educational outcomes

Background: There is recognition that health and education are intrinsically linked, through for example the World Health Organizations' Health Promoting Schools' (HPS) framework. Nevertheless, promoting health via schools is seen by some as a 'zero-sum game'; that is, schools have nothing to gain, and in fact may experience detriments to the core business of academic attainment as a result of focussing resources on health. Crucially, there is a paucity of evidence around the impacts of health and well-being policy and practice on attainment, with recent Cochrane reviews highlighting this gap. This study explored the 'zero-sum game' hypothesis among schools with varying levels of deprivation; that is, the role of health and wellbeing interventions in schools in reducing, or widening, socioeconomic inequality in educational attainment. Methods: Wales-wide, school-level survey data on health policies and practices, reflective of the HPS framework, were captured in 2016 using the School Environment Questionnaire (SEQ). SEQ data were linked with routinely collected data on academic attainment. Primary outcomes included attendance and attainment at Key Stages 3 and 4. Interaction terms were fitted to test whether there was an interaction between FSM,overall HPS activity, and outcomes. Linear regression models were constructed separately for high (>15% of pupils) and low (<15%) Free School Meal (FSM) schools, adjusting for confounders. Findings: The final analyses included 48 low and 49 high FSM secondary schools. Significant interactions were observed between FSM and overall HPS for KS3 attainment (b=0.28; 95% CI: 0.09, 0.47) and attendance(b=0.05; 95% CI: 0.02, 0.09), reflecting an association between health improvement activities and education outcomes among high, but not low FSM schools. There was no significant interaction for KS4 attainment (b=0.18; 95% CI: -0.22, 0.57).Interpretation: Our findings did not support the 'zero-sum game' hypothesis; in fact, among more deprived schools, there was a tendency for better attendance and attainment at Key Stage 3. Schools must equip students with the skills required for good physical, mental health and well-being in addition to academic and cognitive skills. The study included a large, nationally representative sample of secondary schools;however, the cross-sectional nature has implications for causality

Adaptation des populations virales aux résistances variétales et exploitation des ressources génétiques des plantes pour contrôler cette adaptation

Plants carrying major resistance genes have been widely used to fight against diseases. However, the pathogensability to overcome the resistance after a few years of usage requires the search for efficient and durable resistances.The objectives of this thesis were (i) to identify plant genomic regions limiting pathogen evolution by inducinggenetic drift effects and (ii) to study the impact of the evolutionary forces imposed by the plant on the pathogenability to adapt to resistance, the goal being to further use these forces to limit pathogen evolution. The pepper(Capsicum annuum) – PVY (Potato virus Y) pathosystem has been mainly used to conduct these researches.Regarding the first objective, quantitative trait loci (QTL) were mapped on a biparental pepper population andthrough genome-wide association on a pepper core-collection. These approaches have allowed the detection ofgenomic regions on chromosomes 6, 7 and 12 controlling viral effective population size during the inoculationstep. Some of these QTLs were common to PVY and CMV (Cucumber mosaic virus) while other were virusspecific.Moreover, the QTL detected on chromosome 6 colocalizes with a previously identified QTL controllingPVY accumulation and interacting with a QTL affecting the breakdown frequency of a major resistance gene.Regarding the second objective, a correlation analysis between the evolutionary forces imposed by the plant andan experimental estimation of the durability of a major resistance gene has been done. Experimental evolution ofPVY populations on plants contrasted for the levels of genetic drift, selection and virus accumulation they imposedhas also been performed. Both studies demonstrated that a plant inducing a strong genetic drift combined to areduction in virus accumulation limits virus evolution and could even lead to the extinction of the virus population.These results open new perspectives to deploy plant genetic factors directly controlling pathogen evolutionarypotential and could help to preserve the durability of major resistance genes.L’utilisation de variétés de plantes porteuses de gènes majeurs de résistance a longtemps été une solution privilégiée pour lutter contre les maladies des plantes. Cependant, la capacité des agents pathogènes à s’adapter à ces variétés après seulement quelques années de culture rend nécessaire la recherche de résistances à la fois efficaces et durables. Les objectifs de cette thèse étaient (i) d’identifier chez la plante des régions génomiques contraignant l’évolution des agents pathogènes en induisant des effets de dérive génétique et (ii) d’étudier l’impact des forces évolutives induites par la plante sur la capacité d’adaptation des pathogènes aux résistances variétales, l’ambition étant par la suite d’employer au mieux ces forces pour limiter l’évolution des pathogènes. Le pathosystème piment (Capsicum annuum) – PVY (Potato virus Y) a été principalement utilisé pour mener ces travaux de recherche. Afin de répondre au premier objectif, une cartographie de QTL (quantitative trait loci) sur une population biparentale de piment et une étude de génétique d’association sur une core-collection de piments ont été réalisées. Ces deux approches ont permis de mettre en évidence des régions génomiques sur les chromosomes 6, 7 et 12 impliquées dans le contrôle de la taille efficace des populations virales lors de l’étape d’inoculation du virus dans la plante. Certains de ces QTL ont montré une action vis-à-vis du PVY et du CMV (Cucumber mosaic virus) tandis que d’autres se sont révélés être spécifiques d’une seule espèce virale. Par ailleurs,le QTL détecté sur le chromosome 6 co-localise avec un QTL précédemment identifié comme contrôlant l’accumulation virale et interagissant avec un QTL affectant la fréquence de contournement d’un gène majeur de résistance. Pour répondre au second objectif, une analyse de la corrélation entre l’intensité des forces évolutives induites par la plante et une estimation expérimentale de la durabilité du gène majeur a été réalisée. De l’évolution expérimentale de populations de PVY sur des plantes induisant des effets de dérive génétique, de sélection et d’accumulation virale contrastés a également été effectuée. Ces deux études ont démontré qu’une plante induisant une forte dérive génétique associée à une réduction de l’accumulation virale permettait de contraindre l’évolution des populations virales, voire d’entraîner leur extinction. Ces résultats ouvrent de nouvelles perspectives pour le déploiement de déterminants génétiques de la plante qui influenceraient directement le potentiel évolutif du pathogène et permettraient de préserver la durabilité des gènes majeurs de résistance

Adaptation of viral populations to plant resistance and exploitation of plant genetic resources to control this adaptation

L’utilisation de variétés de plantes porteuses de gènes majeurs de résistance a longtemps été une solution privilégiée pour lutter contre les maladies des plantes. Cependant, la capacité des agents pathogènes à s’adapter à ces variétés après seulement quelques années de culture rend nécessaire la recherche de résistances à la fois efficaces et durables. Les objectifs de cette thèse étaient (i) d’identifier chez la plante des régions génomiques contraignant l’évolution des agents pathogènes en induisant des effets de dérive génétique et (ii) d’étudier l’impact des forces évolutives induites par la plante sur la capacité d’adaptation des pathogènes aux résistances variétales, l’ambition étant par la suite d’employer au mieux ces forces pour limiter l’évolution des pathogènes. Le pathosystème piment (Capsicum annuum) – PVY (Potato virus Y) a été principalement utilisé pour mener ces travaux de recherche. Afin de répondre au premier objectif, une cartographie de QTL (quantitative trait loci) sur une population biparentale de piment et une étude de génétique d’association sur une core-collection de piments ont été réalisées. Ces deux approches ont permis de mettre en évidence des régions génomiques sur les chromosomes 6, 7 et 12 impliquées dans le contrôle de la taille efficace des populations virales lors de l’étape d’inoculation du virus dans la plante. Certains de ces QTL ont montré une action vis-à-vis du PVY et du CMV (Cucumber mosaic virus) tandis que d’autres se sont révélés être spécifiques d’une seule espèce virale. Par ailleurs,le QTL détecté sur le chromosome 6 co-localise avec un QTL précédemment identifié comme contrôlant l’accumulation virale et interagissant avec un QTL affectant la fréquence de contournement d’un gène majeur de résistance. Pour répondre au second objectif, une analyse de la corrélation entre l’intensité des forces évolutives induites par la plante et une estimation expérimentale de la durabilité du gène majeur a été réalisée. De l’évolution expérimentale de populations de PVY sur des plantes induisant des effets de dérive génétique, de sélection et d’accumulation virale contrastés a également été effectuée. Ces deux études ont démontré qu’une plante induisant une forte dérive génétique associée à une réduction de l’accumulation virale permettait de contraindre l’évolution des populations virales, voire d’entraîner leur extinction. Ces résultats ouvrent de nouvelles perspectives pour le déploiement de déterminants génétiques de la plante qui influenceraient directement le potentiel évolutif du pathogène et permettraient de préserver la durabilité des gènes majeurs de résistance.Plants carrying major resistance genes have been widely used to fight against diseases. However, the pathogensability to overcome the resistance after a few years of usage requires the search for efficient and durable resistances.The objectives of this thesis were (i) to identify plant genomic regions limiting pathogen evolution by inducinggenetic drift effects and (ii) to study the impact of the evolutionary forces imposed by the plant on the pathogenability to adapt to resistance, the goal being to further use these forces to limit pathogen evolution. The pepper(Capsicum annuum) – PVY (Potato virus Y) pathosystem has been mainly used to conduct these researches.Regarding the first objective, quantitative trait loci (QTL) were mapped on a biparental pepper population andthrough genome-wide association on a pepper core-collection. These approaches have allowed the detection ofgenomic regions on chromosomes 6, 7 and 12 controlling viral effective population size during the inoculationstep. Some of these QTLs were common to PVY and CMV (Cucumber mosaic virus) while other were virusspecific.Moreover, the QTL detected on chromosome 6 colocalizes with a previously identified QTL controllingPVY accumulation and interacting with a QTL affecting the breakdown frequency of a major resistance gene.Regarding the second objective, a correlation analysis between the evolutionary forces imposed by the plant andan experimental estimation of the durability of a major resistance gene has been done. Experimental evolution ofPVY populations on plants contrasted for the levels of genetic drift, selection and virus accumulation they imposedhas also been performed. Both studies demonstrated that a plant inducing a strong genetic drift combined to areduction in virus accumulation limits virus evolution and could even lead to the extinction of the virus population.These results open new perspectives to deploy plant genetic factors directly controlling pathogen evolutionarypotential and could help to preserve the durability of major resistance genes

Virus evolution faced to multiple host targets: The potyvirus—pepper case study

International audienceThe wealth of variability amongst genes controlling immunity against potyviruses in pepper (Capsicum spp.) has been instrumental in understanding plant-virus co-evolution and major determinants of plant resistance durability. Characterization of the eukaryotic initiation factor 4E1 (eIF4E1), involved in mRNA translation, as the basis of potyvirus resistance in pepper initiated a large body of work that showed that recessive resistance to potyviruses and other single-stranded positive-sense RNA viruses resulted from mutations in eukaryotic initiation factors in many plant crop species. Combining mutations in different eIF4Es in the same pepper genotype had complex effects on the breadth of the resistance spectrum and on resistance durability, revealing a trade-off between these two traits. In addition, combining eIF4E1 mutations with a quantitatively resistant genetic background had a strong positive effect on resistance durability. Analysing the evolutionary forces imposed by pepper genotypes onto virus populations allowed identifying three key factors improving plant resistance durability: the complexity of mutational pathways involved in virus adaptation to the plant resistance, the decrease of competitivity induced by these mutations on the virus and the intensity of genetic drift imposed by plant genotypes on the virus during its infection cycle

Identification, molecular and biological characterization of two novel secovirids in wild grass species in Belgium

International audienceHigh throughput sequencing was performed on virion-associated nucleic acids (VANA) from a pool of fifty asymptomatic rough bluegrasses (Poa trivialis L.) collected in a Belgian grazed pasture. Bioinformatics analyses produced some contigs presenting similarities with secovirid genomes, in particular nepoviruses and waikaviruses. Three distinct positive-sense single-stranded RNAs including 5? and 3? UTR were reconstructed and they represented two novel viruses infecting rough bluegrass, for which the provisional names poaceae Liege nepovirus A (PoLNVA, 7298 nt for RNA1 and 4263 nt for RNA2) and poaceae Liege virus 1 (PoLV1, 11,623 nt) were proposed. Compared to other Secoviridae members, the highest amino acid identity reached 90.7 % and 66.7 % between PoLNVA and nepoviruses for the Pro-Pol and CP regions respectively, while PoLV1 presented the highest amino acid identity with waikaviruses but with lower identities, i.e. 41.2 % for Pro-Pol and 25.8 % for CP regions, far below the ICTV demarcation criteria for novel secovirid. Based on sequence identity and phylogenetic analyses, PoLNVA was proposed to belong to the genus Nepovirus and PoLV1 as an unclassified secovirids. Detection of the two novel viruses was confirmed in high prevalence in rough bluegrass and ten other wild Poaceae species (Agropyron repens, Agrostis capillaris, Apera spica-venti, Anthoxanthum odoratum, Cynosorus cristatus, Festuca rubra, Holcus lanatus, Lolium perenne, Phleum bertolini and Phleum pratense) by RT-PCR and Sanger sequencing, revealing a diverse host range within Poaceae for these novel secovirids. Seed transmission was evaluated and confirmed for PoLNVA