15 research outputs found
Identification et caractérisation de virus aviaires par des approches de séquençage à haut débit
En mĂ©decine humaine et vĂ©tĂ©rinaire, les agents pathogĂšnes reprĂ©sentent la cause de mortalitĂ© principale Ă travers la planĂšte. Les mĂ©thodes de diagnostic de ces pathogĂšnes ont considĂ©rablement changĂ© et Ă©voluĂ© particuliĂšrement depuis lâapparition du sĂ©quençage haut dĂ©bit. Les nouvelles mĂ©thodes de sĂ©quençage massif ont considĂ©rablement diminuĂ© le prix dâune sĂ©quence permettant de rendre accessible cette technologie rĂ©volutionnaire. Dans le cadre de mes travaux de thĂšse, nous avons mis en place un protocole pour lâutilisation du sĂ©quençage IlluminaÂź (avec le sĂ©quenceur MiSeq) comme mĂ©thode de diagnostic lors de diffĂ©rents cas pathologiques aviaires. Lâutilisation de cette mĂ©thode nous a permis dans un premier temps dâidentifier lâagent Ă©tiologique de la maladie foudroyante de la pintade. Cette Ă©tude nous a permis de valider lâutilisation de ce genre de mĂ©thode pour des cas ciblĂ©s, ici lors dâun Ă©pisode clinique particulier nâimpliquant vraisemblablement quâun seul candidat pathogĂšne. Ce nouveau coronavirus a fait lâobjet dâĂ©tudes complĂ©mentaires afin de le caractĂ©riser. Nous avons Ă©largis les cibles recherchĂ©es en analysant dans un deuxiĂšme temps lâensemble des virus ARN chez le canard lors dâĂ©pisodes cliniques respiratoires et/ou de chute de ponte. Lâanalyse des donnĂ©es a mis en Ă©vidence une importante diversitĂ© virale et a permis dâidentifier des candidats responsables potentiels. Lâensemble des rĂ©sultats obtenus nous permet de valider lâutilisation du sĂ©quençage Ă haut dĂ©bit comme un outil puissant de diagnostic. ABSTRACT : Infectious diseases are considered the most prevalent cause of mortality in humans as well as other animals worldwide. Since the advent of high throughput sequencing technologies, diagnostic methods for these conditions have quickly changed and evolved, as the continuously decreasing cost of mass sequencing is making this tool available to larger numbers of people. As part of my thesis project, an IlluminaÂź-based sequencing method (on a MiSeq machine) was designed for diagnostic purposes in clinical cases in poultry. We first used this method to identify the causative agent of the fulminating disease of guinea fowl. This validated the use of our protocol to identify the pathogenic infectious agent behind a specific condition. This newly identified Coronavirus was further analysed and characterised. In a second study we used an unbiased mass sequencing approach to describe the RNA virus populations present in the duck respiratory tract during clinical episodes (respiratory illness or egg drops). Data showed an important viral diversity and we identified some candidate pathogens. Taken together, these results validate the use of high throughput sequencing as a powerful diagnostic tool
Identification and characterisation of avian viruses using high throughput sequencing
En mĂ©decine humaine et vĂ©tĂ©rinaire, les agents pathogĂšnes reprĂ©sentent la cause de mortalitĂ© principale Ă travers la planĂšte. Les mĂ©thodes de diagnostic de ces pathogĂšnes ont considĂ©rablement changĂ© et Ă©voluĂ© particuliĂšrement depuis lâapparition du sĂ©quençage haut dĂ©bit. Les nouvelles mĂ©thodes de sĂ©quençage massif ont considĂ©rablement diminuĂ© le prix dâune sĂ©quence permettant de rendre accessible cette technologie rĂ©volutionnaire. Dans le cadre de mes travaux de thĂšse, nous avons mis en place un protocole pour lâutilisation du sĂ©quençage IlluminaÂź (avec le sĂ©quenceur MiSeq) comme mĂ©thode de diagnostic lors de diffĂ©rents cas pathologiques aviaires. Lâutilisation de cette mĂ©thode nous a permis dans un premier temps dâidentifier lâagent Ă©tiologique de la maladie foudroyante de la pintade. Cette Ă©tude nous a permis de valider lâutilisation de ce genre de mĂ©thode pour des cas ciblĂ©s, ici lors dâun Ă©pisode clinique particulier nâimpliquant vraisemblablement quâun seul candidat pathogĂšne. Ce nouveau coronavirus a fait lâobjet dâĂ©tudes complĂ©mentaires afin de le caractĂ©riser. Nous avons Ă©largis les cibles recherchĂ©es en analysant dans un deuxiĂšme temps lâensemble des virus ARN chez le canard lors dâĂ©pisodes cliniques respiratoires et/ou de chute de ponte. Lâanalyse des donnĂ©es a mis en Ă©vidence une importante diversitĂ© virale et a permis dâidentifier des candidats responsables potentiels. Lâensemble des rĂ©sultats obtenus nous permet de valider lâutilisation du sĂ©quençage Ă haut dĂ©bit comme un outil puissant de diagnostic.Infectious diseases are considered the most prevalent cause of mortality in humans as well as other animals worldwide. Since the advent of high throughput sequencing technologies, diagnostic methods for these conditions have quickly changed and evolved, as the continuously decreasing cost of mass sequencing is making this tool available to larger numbers of people. As part of my thesis project, an IlluminaÂź-based sequencing method (on a MiSeq machine) was designed for diagnostic purposes in clinical cases in poultry. We first used this method to identify the causative agent of the fulminating disease of guinea fowl. This validated the use of our protocol to identify the pathogenic infectious agent behind a specific condition. This newly identified Coronavirus was further analysed and characterised. In a second study we used an unbiased mass sequencing approach to describe the RNA virus populations present in the duck respiratory tract during clinical episodes (respiratory illness or egg drops). Data showed an important viral diversity and we identified some candidate pathogens. Taken together, these results validate the use of high throughput sequencing as a powerful diagnostic tool
Serological cross-reactivity between Merkel cell polyomavirus and two closely related chimpanzee polyomaviruses.
Phylogenetic analyses based on the major capsid protein sequence indicate that Merkel cell polyomavirus (MCPyV) and chimpanzee polyomaviruses (PtvPyV1, PtvPyV2), and similarly Trichodysplasia spinulosa-associated polyomavirus (TSPyV) and the orangutan polyomavirus (OraPyV1) are closely related. The existence of cross-reactivity between these polyomaviruses was therefore investigated. The findings indicated serological identity between the two chimpanzee polyomaviruses investigated and a high level of cross-reactivity with Merkel cell polyomavirus. In contrast, cross-reactivity was not observed between TSPyV and OraPyV1. Furthermore, specific antibodies to chimpanzee polyomaviruses were detected in chimpanzee sera by pre-incubation of sera with the different antigens, but not in human sera
Molecular detection and characterization of duck respiratory viruses using unbiased high-throughput sequencing
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Phylogenetic relationships between human and closely related simian polyomaviruses based on VP1 protein.
<p>Human polyomaviruses are in black and simian polyomaviruses are in gray. Sequence accession numbers used are NC_001538 for BKPyV, NC_001699 for JCPyV, NC_009238 for KIPyV, NC_009539 for WuPyV, NC_010277 for MCPyV, NC_014406 for HPyV6, NC_014407 for HPyV7, NC_014361 for TSPyV, HQ696595 for HPyV9, NC_018102 for MWPyV, JX463183 for STLPyV, JX308829 for HPyV12, NC_001669 for SV40, NC_004763 for LPyV, AY691168 for ChPyV, HQ385747 for PtvPyV1b, HQ385750 for PtvPyV2c, FN356900 for OraPyV1 and FN356901 for OraPyV2.</p
Détection moléculaire et caractérisation de virus respiratoires du canard par une approche de séquençage à haut débit
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Human seroreactivity distribution Venn diagrams.
<p>(A) Negative (â), simple, and multiple seropositive (+) sera for 828 sera investigated for MCPyV, PtvPyV1 and PtvPyV2. (B) Negative (â), simple and double seropositive (+) sample for the 300 sera investigated for TSPyV and OraPyV.</p
Correlation between MCPyV and PtvPyV reactivity and between TSPyV and OraPyV1 reactivity.
<p>Correlation between ELISA reactivity of human serum samples against the different VLPs. Each point represents one serum sample. Correlation coefficients (Spearman) were determined using XLStat software.</p
Electron micrographs of VP1 virus-like particles for MCPyV, PtvPyV1b, PtvPyV2c, TSPyV and OraPyV1.
<p>VP1 were produced using recombinant baculoviruses. The preparations were applied to carbon-coated grids, negatively stained with 1.5% uranyl acetate and observed at 50,000 nominal magnification with a JEOL 1011 electron microscope (Scale bars, 100 nm).</p
Competitive inhibition of seroreactivity between MCPyV, PtvPyV1 and PtvPyV2.
<p>ELISA Reactivity against MCPyV, PtvPyV1 and PtvPyV2 was determined for five human (A) and five chimpanzee (B) sera after preincubation with or without MCPyV, PtvPyV1 or PtvPyV VLPs. Percentage of inhibition after preincubation is indicated above bars.</p