Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples

<p>Abstract</p> <p>Background</p> <p>Pathogens such as fungi, bacteria and especially viruses, are highly variable even within an individual host, intensifying the difficulty of distinguishing and accurately quantifying numerous allelic variants co-existing in a single nucleic acid sample. The majority of currently available techniques are based on real-time PCR or primer extension and often require multiplexing adjustments that impose a practical limitation of the number of alleles that can be monitored simultaneously at a single locus.</p> <p>Results</p> <p>Here, we describe a novel method that allows the simultaneous quantification of numerous allelic variants in a single reaction tube and without multiplexing. Quantitative Single-letter Sequencing (QSS) begins with a single PCR amplification step using a pair of primers flanking the polymorphic region of interest. Next, PCR products are submitted to single-letter sequencing with a fluorescently-labelled primer located upstream of the polymorphic region. The resulting monochromatic electropherogram shows numerous specific diagnostic peaks, attributable to specific variants, signifying their presence/absence in the DNA sample. Moreover, peak fluorescence can be quantified and used to estimate the frequency of the corresponding variant in the DNA population.</p> <p>Using engineered allelic markers in the genome of <it>Cauliflower mosaic virus</it>, we reliably monitored six different viral genotypes in DNA extracted from infected plants. Evaluation of the intrinsic variance of this method, as applied to both artificial plasmid DNA mixes and viral genome populations, demonstrates that QSS is a robust and reliable method of detection and quantification for variants with a relative frequency of between 0.05 and 1.</p> <p>Conclusion</p> <p>This simple method is easily transferable to many other biological systems and questions, including those involving high throughput analysis, and can be performed in any laboratory since it does not require specialized equipment.</p

Large Bottleneck Size in Cauliflower Mosaic Virus Populations during Host Plant Colonization

The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Despite their high mutation rate and rapid evolution, this parameter is poorly documented experimentally in viruses, particularly plant viruses. All available studies, however, have demonstrated the existence of huge within-host demographic fluctuations, drastically reducing Ne upon systemic invasion of different organs and tissues. Notably, extreme bottlenecks have been detected at the stage of systemic leaf colonization in all plant viral species investigated so far, sustaining the general idea that some unknown obstacle(s) imposes a barrier on the development of all plant viruses. This idea has important implications, as it appoints genetic drift as a constant major force in plant virus evolution. By co-inoculating several genetic variants of Cauliflower mosaic virus into a large number of replicate host plants, and by monitoring their relative frequency within the viral population over the course of the host systemic infection, only minute stochastic variations were detected. This allowed the estimation of the CaMV Ne during colonization of successive leaves at several hundreds of viral genomes, a value about 100-fold higher than that reported for any other plant virus investigated so far, and indicated the very limited role played by genetic drift during plant systemic infection by this virus. These results suggest that the barriers that generate bottlenecks in some plant virus species might well not exist, or can be surmounted by other viruses, implying that severe bottlenecks during host colonization do not necessarily apply to all plant-infecting viruses

Uncovering the Underlying Mechanisms Blocking Replication of Bluetongue Virus Serotype 26 (BTV-26) in Culicoides Cells

At least 12 serotypes of ‘atypical’ bluetongue virus (BTV-25 to BTV-36) have been identified to date. These atypical serotypes fail to infect/replicate in Culicoides-derived cell lines and/or adult Culicoides vectors and hence can no longer be transmitted by these vectors. They appear to be horizontally transmitted from infected to in-contact ruminants, although the route(s) of infection remain to be identified. Viral genome segments 1, 2 and 3 (Seg-1, Seg2 and Seg-3) of BTV-26 were identified as involved in blocking virus replication in KC cells. We have developed Culicoides-specific expression plasmids, which we used in transfected insect cells to assess the stability of viral mRNAs and protein expression from full-length open reading frames of Seg-1, -2 and -3 of BTV-1 (a Culicoides-vectored BTV) or BTV-26. Our results indicate that the blocked replication of BTV-26 in KC cells is not due to an RNAi response, which would lead to rapid degradation of viral mRNAs. A combination of degradation/poor expression and/or modification of the proteins encoded by these segments appears to drive the failure of BTV-26 core/whole virus-particles to assemble and replicate effectively in Culicoides cells

Plant and Aphid Partners of Poleroviruses: Role in Virus Transmission by Aphids?

Comité de lecture : trueConférence invitée : falseDate de début de l'événement : 2011-07-11Date de fin de l'évenement : 2011-07-14Date de validation : Tue Aug 13 15:11:30 CEST 2013Diffusion de la pièce jointe : Publique, PubliqueIdentifiant : 200587Langue du titre : engNombre de consultation de la notice : 77Nombre de téléchargements de la pièce jointe : 8Pays de l'événement : BRAPublic visé : ScientifiqueType de communication avec actes : Présentation oraleType d'événement : SymposiumPoleroviruses are phloem limited viruses strictly transmitted by aphids in a circulative and non propagative manner. Virions are acquired by aphids when ingesting sap from infected plants. Virus particles cross the gut epithelium and the accessory salivary gland cells before being released, together with saliva, into the plant during a subsequent feed. This highly specific transcytosis mechanism relies on the presence of virus receptors on the surface of the aphid cells. We developed several approaches to identify virus partners in the plant and in the aphid to analyse their role in virus transmission by the vector. By screening different aphid cDNA libraries using a yeast two hybrid system, only few candidates were able to bind virus structural proteins. Among them, we found two nuclear proteins (GAR1 and ALY) which may not be the true virusreceptors but could be considered as virus-sensors. An Ephrin receptor-like protein was also found to interact with the viral proteins. Involvement of these candidates in virus transport through the aphid needs to be analyzed by developing in the insect RNAi-based techniques. These experiments are in progress. We also looked for plant virus-partners and identified several phloem proteins able to bind purified virions in vitro. We showed that these proteins could stimulate virus transmission by aphids when added together with purified virus to the aphid diet (Bencharki et al. 2010, M.P.M.I., 23: 799). By developing a yeast two hybrid system using a phloem specific cDNA library, we identified five additional proteins able to bind viral proteins. Among them, we found ALY proteins already identified as aphid virus-partners suggesting that orthologous plant and aphid proteins could be implicated in the virus cycle. So far, a direct implication of these proteins in aphid transmission has not been observed and experiments are on going to analyze their functions

Continuous Cell Lines from the European Biting Midge Culicoides nubeculosus (Meigen, 1830)

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens. View Full-Tex

Efficient detection of long dsRNA in vitro and in vivo using the dsRNA binding domain from FHV B2 protein

BM benefited from an IdEx postdoctoral fellowship from the Université de Strasbourg. Financial support to MI was provided in part by the INTERREG V Upper Rhine program Vitifutur, Transcending borders with every project. Work in the JT laboratory is funded by the U.K. Biotechnology and Biological Sciences Research Council (BB/M007200/1).Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.Publisher PDFPeer reviewe

Dynamics of the Multiplicity of Cellular Infection in a Plant Virus

Recombination, complementation and competition profoundly influence virus evolution and epidemiology. Since viruses are intracellular parasites, the basic parameter determining the potential for such interactions is the multiplicity of cellular infection (cellular MOI), i.e. the number of viral genome units that effectively infect a cell. The cellular MOI values that prevail in host organisms have rarely been investigated, and whether they remain constant or change widely during host invasion is totally unknown. Here, we fill this experimental gap by presenting the first detailed analysis of the dynamics of the cellular MOI during colonization of a host plant by a virus. Our results reveal ample variations between different leaf levels during the course of infection, with values starting close to 2 and increasing up to 13 before decreasing to initial levels in the latest infection stages. By revealing wide dynamic changes throughout a single infection, we here illustrate the existence of complex scenarios where the opportunity for recombination, complementation and competition among viral genomes changes greatly at different infection phases and at different locations within a multi-cellular host

FLUCTUATIONS DEMOGRAPHIQUES AU COURS DU CYCLE DE VIE DU CaMV (Cauliflower mosaic virus). Estimation de la taille efficace des populations virales lors de la colonisation des feuilles de la plante hôte, évaluation de la multiplicité d'infection cellulaire au sein de ces feuilles, et estimation de la taille des goulots d'étranglement lors de sa transmission d'hôte à hôte par vecteur

UMR BGPI Equipe 2 Diplôme : Dr. d'UniversiteCauliflower mosaic virus (CaMV), a double-strand DNA plant virus, is non-circulatively transmitted by aphid vectors. As for many other viruses, ample demographic fluctuations along the life cycle are poorly defined, though they may have considerable impact on their evolution. In order to monitor CaMV populations we have constructed 6 infectious clones, each containing a distinct genetic marker, and developed a new analysis method: Quantitative Single-letter Sequencing (QSS) allowing to quantify their relative frequency in infected plants and/or leaves. Through the evolution of markers' frequency, we could calculate the effective size of CaMV populations: hundreds to thousands of genomes are founding the population in every single new leaf during systemic infection. This evaluation is 10 to 100 fold higher than that previously published for all other plant viruses studied. Furthermore, we have demonstrated that the multiplicity of infection of host cells (MOI) by CaMV genomes is not constant, and increases during CaMV infection to reach a maximum value around 7, largely exceeding the rare other estimates available on animal viruses or phages. Finally, using the EPG technique, we have controlled the feeding behaviour of aphids vectors when acquiring CaMV, and evaluated the impact of this vector behaviour on the size of the bottleneck induced on viral population during plant-to-plant transmission.Le CaMV (Cauliflower mosaic virus) est un virus de plante à ADN transmis par pucerons. Comme pour tout autre virus, les larges fluctuations démographiques au cours du cycle de vie jouent un rôle prépondérant dans l'évolution, et pourtant, peu de données expérimentales sont disponibles à ce sujet. Afin de suivre l'évolution des populations de CaMV, nous avons construit 6 clones distincts marqués à un même locus, et développé une nouvelle méthode d'analyse : Quantitative Single-letter Sequencing (QSS). En quantifiant l'évolution de la fréquence des marqueurs, au sein d'une plante infectée, cette méthode nous a permis d'évaluer la taille efficace des populations du CaMV : plusieurs centaines à plusieurs milliers de génomes sont à l'origine de la colonisation de chaque feuille durant le développement de l'infection systémique ; une valeur 10 à 100 fois supérieure à celle estimé auparavant chez des phytovirus à ARN. Ensuite, nous avons poussé l'analyse au niveau cellulaire et montré que la multiplicité d'infection des cellules individuelles de l'hôte (MOI) n'est pas constante. Elle augmente au fil du temps pour culminer à une valeur proche de 7, qui dépasse amplement les données disponibles dans la littérature, quelle que soit l'espèce virale considérée. Il est très probable qu'une très forte MOI conditionne au moins partiellement la taille efficace élevé des populations du CaMV, mais cette hypothèse butte sur l'absence totale de donnée concernant la MOI chez d'autres virus de plantes. Enfin, connaissant la composition moyenne des populations mixtes de CaMV marqués, au niveau des feuilles et des cellules qui les composent, nous avons contrôlé le comportement alimentaire des pucerons vecteurs par la technique EPG, et évalué l'impact de ce comportement sur le goulot d'étranglement génétique induit sur la population virale lors de la transmission

Fluctuations démographiques au cours du cycle de vie du CaMV (Cauliflower mosaic virus) (estimation de la taille efficace des populations virales lors de la colonisation des feuilles de la plante hôte, évaluation de la multiplicité d'infection cellulaire au sein de ces feuilles et estimation de la taille des goulots d'étranglement lors de sa transmission d'hôte à hôte par vecteur)

Le CaMV (Cauliflower mosaic virus) est un virus de plante à ADN double brin transmis par des pucerons. Comme pour tous les virus avec un taux de mutation élevé, les larges fluctuations démographiques au cours du cycle de vie sont supposées avoir un fort impact sur l'évolution de la valeur sélective, du fait de la dérive génétique intense, et des effets associés du cliquet de Muller. Pourtant, de telles données démographiques sont rares. Afin de suivre l'évolution des populations de CaMV nous avons construit 6 clones infectieux contenant chacun un marqueur génétique distinct à un même locus. Nous avons développé une nouvelle méthode d'analyse : Quantitative Single-letter Sequencing (QSS) permettant de déterminer leur présence/absence et de suivre l'évolution de leur fréquence au sein d'une plante infectée. Tout d'abord, nous avons évalué la taille efficace des populations de CaMV durant l'invasion de l'hôte : plusieurs centaines à plusieurs milliers de génomes sont à l'origine de la colonisation de chaque feuille au cours de l'infection systémique. La diversité virale, au sein des populations du CaMV, est en conséquence distribuée de manière très homogène dans toutes les feuilles infectées de l'hôte. Cette évaluation est surprenante puisqu'elle est 10 à 100 fois supérieure à ce qui a été estimé auparavant chez tous les phytovirus étudiés. Elle démontre ainsi que la très faible taille efficace de population, et donc les larges fluctuations démographiques durant le cycle d'infection in planta, ne sont pas une règle générale chez les virus de plante. Ensuite, nous avons déterminé la multiplicité d'infection cellulaire naturelle (MOI, multiplicity of infection) qui se définit ici comme le nombre moyen de génomes qui pénètrent dans (et infectent) chaque cellule de l'hôte. Cette étude est inédite dans le sens où ce paramètre n'avait jamais été établi chez un virus de plante, et où nous avons montré pour la première fois que la MOI n'est pas une constante mais qu'elle augmente au fil du développement de l'infection pour atteindre un plateau, à une valeur proche de 7 dans le cas du CaMV. Cette valeur est très élevée ; elle dépasse amplement les données disponibles dans la littérature, quelle que soit l'espèce virale considérée. Des indices indirects semblent notamment suggérer que la MOI d'autres espèces de phytovirus puisse être très faible. Les causes et/ou conséquences de ces comportements contrastés (MOI forte ou faible) sur la biologie du virus représentent un champ de perspectives considérable pour l'équipe dans les années à venir. Enfin, à l'aide de la technique d'EPG, nous avons contrôlé le comportement alimentaire des pucerons vecteurs, et évalué l'impact de ce comportement sur le goulot d'étranglement induit sur la population virale lors de la transmissionThe Cauliflower mosaic virus (CaMV, a double-strand DNA plant virus) is non-circulatively transmitted by aphid vectors. As for many other viruses with a high mutation rate, ample demographic fluctuations along the life cycle, that are most often poorly defined, may have considerable impact on the evolution of fitness, due to intense genetic drift and associated Müller's ratchet. In order to monitor the CaMV populations we constructed 6 infectious clones, each containing a unique genetic marker at the same locus. We have developed and implemented a new analysis method: Quantitative Single-letter Sequencing (QSS) to determine the presence/absence and monitor the frequency of the markers in an infected plant. We first assessed the effective size of CaMV populations during host plant colonisation, and found that hundreds to thousands of genomes are founding the population in every single new leaf during systemic infection. This evaluation is 10 to 100 fold higher than that previously published for all other plant viruses, suggesting that ample demographic fluctuation during host infection is not a general rule for plant viruses. Then, we addressed the natural multiplicity of infection of cells (MOI) in CaMV infected plants, a trait that has never been investigated before in plant viruses, and very rarely in other virus species. We showed for the first time that the mean MOI value is not a constant, and increases along the course of the host infection, reaching a maximum value close to 7 for CaMV, largely exceeding the rare estimates available in the literature on animal viruses or phages. Indirect hints collected in the literature suggest that other plant viruses may have an oppositely very low MOI (close to 1), and the causes and/or consequences of this contrasted situation on the biology of viruses is discussed in depth. This represents a new field of investigation to be developed in the group in the next future. Finally, using the EPG technique, we have controlled the feeding behaviour of aphid vectors acquiring CaMV, and evaluated the impact of this vector behaviour on the size of the bottleneck induced on viral population during plant-to-plant transmission. Key words : Cauliflower mosaic virus, Bottleneck, Effective population size, Aphid transmission, Multiplicity of infectionMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Détermination et suivi quantitatif de la structure d'une population virale complexe par la technique d'analyse discriminante de signatures clonales (DACS® : Discriminative Analysis of Clone Signature)

National audienceComment obtenir des informations quantitatives - sans l'hybridation moléculaire et sans la Q-PCR - sur la structure d'une population virale complexe (mélange de plusieurs clones, variants, ou souches) et son évolution ? Notre but était de pouvoir créer des marqueurs génétiques, puis de les identifier et de suivre leur fréquence relative dans une population virale complexe. Différents clones viraux du CaMV (Cauliflower mosaic virus) ont été construits. Chaque construction diffère par une cassette spécifique de 40 pb, insérée à une position identique dans le génome viral. Des solutions virales mixtes, comportant 5 à 6 clones viraux en proportion variable, ont été préparées et soit analysées directement, soit inoculées à des plantes hôtes. Pour l'analyse de ces différentes populations mixtes, nous avons mis en place une technique originale dérivée du DACS (technologie propriétaire développée par GENOME express). Cette technique consiste en un séquençage sur une seule lettre (par exemple A) des produits PCR correspondant à la région de l'ADN viral contenant les cassettes. Du fait de la faible homologie de séquence des cassettes, chaque clone viral possède alors une signature spécifique à ce niveau, avec présence ou absence de A à chaque position de nucléotide. Il existe donc sur l'électrophorégramme un ou plusieurs « pic(s) » spécifique(s) pour chacun de nos marqueurs (signature). Ainsi, nous pouvons très simplement déterminer, par la présence ou l'absence de ces pics spécifiques, la présence/absence de chacun des marqueurs et ainsi, la composition de populations virales complexes. L'obtention de données quantitatives s'effectue par une simple analyse complémentaire du même électrophorégramme. En effet, chaque pic spécifique à un marqueur donné aura une hauteur proportionnelle à la fréquence relative de ce marqueur dans la population. Nous pouvons ainsi, sur un simple produit de PCR amplifié à partir d'une population virale contenant plusieurs clones marqués, obtenir rapidement des informations fiables et reproductibles sur la présence/absence des marqueurs dans la population et sur leur fréquence relative. La mise au point de cette méthode sera présentée sur la base de l'étude des goulots d'étranglement que subissent les populations du CaMV lors de l'invasion de la plante hôte et lors de la transmission par pucerons vecteurs. Nous pensons que cette méthode d'analyse originale pourrait servir au suivi quantitatif de compétitions entre clones viraux de laboratoire (notre cas), ainsi qu'entre des variants ou des souches naturelles de séquence connue, voir même dans certains cas entre différentes espèces virales. Elle peut certainement aussi être appliquée à d'autres parasites et plus généralement à l'analyse populationnelle de divers microorganismes