A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies

Background: High-throughput sequencing technologies offer new perspectives for biomedical, agronomical and evolutionary research. Promising progresses now concern the application of these technologies to large-scale studies of genetic variation. Such studies require the genotyping of high numbers of samples. This is theoretically possible using 454 pyrosequencing, which generates billions of base pairs of sequence data. However several challenges arise: first in the attribution of each read produced to its original sample, and second, in bioinformatic analyses to distinguish true from artifactual sequence variation. This pilot study proposes a new application for the 454 GS FLX platform, allowing the individual genotyping of thousands of samples in one run. A probabilistic model has been developed to demonstrate the reliability of this method. Results: DNA amplicons from 1,710 rodent samples were individually barcoded using a combination of tags located in forward and reverse primers. Amplicons consisted in 222 bp fragments corresponding to DRB exon 2, a highly polymorphic gene in mammals. A total of 221,789 reads were obtained, of which 153,349 were finally assigned to original samples. Rules based on a probabilistic model and a four-step procedure, were developed to validate sequences and provide a confidence level for each genotype. The method gave promising results, with the genotyping of DRB exon 2 sequences for 1,407 samples from 24 different rodent species and the sequencing of 392 variants in one half of a 454 run. Using replicates, we estimated that the reproducibility of genotyping reached 95%. Conclusions: This new approach is a promising alternative to classical methods involving electrophoresis-based techniques for variant separation and cloning-sequencing for sequence determination. The 454 system is less costly and time consuming and may enhance the reliability of genotypes obtained when high numbers of samples are studied. It opens up new perspectives for the study of evolutionary and functional genetics of highly polymorphic genes like major histocompatibility complex genes in vertebrates or loci regulating self-compatibility in plants. Important applications in biomedical research will include the detection of individual variation in disease susceptibility. Similarly, agronomy will benefit from this approach, through the study of genes implicated in productivity or disease susceptibility trait

SESAME (SEquence Sorter & AMplicon Explorer): genotyping based on high-throughput multiplex amplicon sequencing

Summary: Characterizing genetic diversity through genotyping short amplicons is central to evolutionary biology. Next-generation sequencing (NGS) technologies changed the scale at which these type of data are acquired. SESAME is a web application package that assists genotyping of multiplexed individuals for several markers based on NGS amplicon sequencing. It automatically assigns reads to loci and individuals, corrects reads if standard samples are available and provides an intuitive graphical user interface (GUI) for allele validation based on the sequences and associated decision-making tools. The aim of SESAME is to help allele identification among a large number of sequences

Insights into Myodes glareolus / Puumala hantavirus interactions from rodent immunogenetics

Nephropathia epidemica (NE) is a mild form of hemorrhagic fever with renal syndrome (HFRS) caused by the hantavirus Puumala (PUUV). In Europe, its distribution is fragmented, whereas the bank vole Myodes glareolus, which is the reservoir of PUUV, is common all over the continent. Determining the causes underlying this heterogeneity is of main importance to better understand and prevent the risks of NE emergence. Besides climatic and ecological hypotheses, we have proposed that the geographic variability of bank vole immune responses to PUUV infection could shape differences in PUUV prevalence, and consequently NE incidence. We have tested this hypothesis by studying polymorphisms and / or expression levels of six candidate genes involved in the immune response to PUUV (DRB-MHC, TNF-alpha promoter, TLR4, TLR7, Mx2, Integrin bêta3) on ten populations of bank voles sampled in the French Ardennes, along a North-South transect including PUUV endemic and non-endemic areas. Signatures of selection have been evidenced for TNF-alpha and Mx2 genes using population genetics (FST scan) and genotype - phenotype association approaches. These genes have antiviral properties but also induce immunological damages, what make them central for driving a balance of resistance / tolerance to PUUV. Bank voles vary in their basal ability to tolerate/resist to PUUV. In high PUUV prevalence areas, TNF-alpha and Mx2 expression seemed down-regulated what suggest selection or phenotypic plasticity for higher tolerance to PUUV, at the benefit of lower immunopathological costs. Some of these results have been confirmed at the European scale.Le campagnol roussâtre Myodes glareolus est le réservoir de l’hantavirus Puumala (PUUV), responsable chez l’Homme d’une forme atténuée de Fièvre Hémorragique à Syndrome Rénal (FHSR), la Néphropathie Épidémique (NE). En Europe, l’incidence de la NE présente, malgré la distribution continue du réservoir, une forte variabilité géographique dont les causes ne sont à ce jour pas identifiées. Aux hypothèses climatiques et paysagères, nous proposons que des facteurs intrinsèques aux campagnols puissent également être impliqués. Une plus forte tolérance à l’infection par le virus PUUV, chez certains campagnols roussâtres, favoriserait la persistance et la transmission de ce virus, ce qui devrait accroître le risque de NE chez l’Homme. Nous avons testé cette hypothèse en étudiant les polymorphismes et/ou les niveaux d'expression de six gènes candidats impliqués dans la réponse immunitaire à PUUV (DRB-CMH, promoteur du TNF-alpha, TLR4, TLR7, Mx2, intégrine bêta3) chez dix populations de campagnols échantillonnées le long d’un axe nord/sud dans les Ardennes françaises, couvrant des zones endémiques et non endémiques à PUUV. Des signatures de sélection ont été détectées pour TNF-alpha et Mx2 grâce à des approches de génétique des populations (scan FST) et d’associations génotypes / phénotypes. Ces gènes codent des protéines dont les propriétés antivirales sont connues, mais qui induisent des coûts immunopathologiques importants. Ils pourraient donc jouer un rôle central dans une balance de tolérance / résistance à PUUV. De plus, dans les zones d’endémie, les gènes TNF-alpha et Mx2 sont sous-exprimés, ce qui suggère l’évolution d’une plus forte tolérance à PUUV, potentiellement au bénéfice d’un moindre coût immunopathologique. Certains de ces résultats ont été confirmés à l’échelle européenn

Microevolution of bank voles (Myodes glareolus) at neutral and immune-related genes during multiannual dynamic cycles : Consequences for Puumala hantavirus epidemiology

Understanding howhost dynamics, including variations of population size and dispersal, may affect the epidemiology of infectious diseases through ecological and evolutionary processes is an active research area. Here we focus on a bank vole (Myodes glareolus) metapopulation surveyed in Finland between 2005 and 2009. Bank vole is the reservoir of Puumala hantavirus (PUUV), the agent of nephropathia epidemica (NE, a mild form of hemorrhagic fever with renal symptom) in humans. M. glareolus populations experience multiannual density fluctuations that may influence the level of genetic diversity maintained in bank voles, PUUV prevalence and NE occurrence. We examine bank vole metapopulation genetics at presumably neutral markers and immunerelated genes involved in susceptibility to PUUV (Tnf-promoter, Tlr4, Tlr7 and Mx2 gene) to investigate the links between population dynamics, microevolutionary processes and PUUV epidemiology. We show that genetic drift slightly and transiently affects neutral and adaptive genetic variability within the metapopulation. Gene flow seems to counterbalance its effects during the multiannual density fluctuations. The low abundance phase may therefore be too short to impact genetic variation in the host, and consequently viral genetic diversity. Environmental heterogeneity does not seem to affect vole gene flow, which might explain the absence of spatial structure previously detected in PUUV in this area. Besides, our results suggest the role of vole dispersal on PUUV circulation through sex-specific and density-dependent movements. We find little evidence of selection acting on immune-related genes within this metapopulation. Footprint of positive selection is detected at Tlr-4 gene in 2008 only. We observe marginally significant associations between Mx2 genotype and PUUV genogroups. These results show that neutral processes seem to be the main factors affecting the evolution of these immune-related genes at a contemporary scale, although the relative effects of neutral and adaptive forces could vary temporally with density fluctuations. Immune related gene polymorphism may in turn partly influence PUUV epidemiology in this metapopulation. (C) 2016 Published by Elsevier B.V.Peer reviewe

Concomitant influence of helminth infection and landscape on the distribution of Puumala hantavirus in its reservoir, Myodes glareolus

Peer reviewe

Variabilité de la résistance/tolérance des campagnols roussâtres à l'hantavirus Puumala et conséquences épidémiologiques

The bank vole Myodes glareolus is the main reservoir of hantavirus Puumala (PUUV)responsible of the nephropathia epidemica (NE) in Europe. This work aims to describe theresistance/tolerance variability of M. glareolus to PUUV and to explore its role in the distribution andtransmission of the virus. We hypothesized that tolerance to PUUV favours its persistence andtransmission, which could increase the NE emergent risk.We developed candidate gene approach to determine the role of three immune genes inresistance / tolerance to PUUV. The existence of positive association between Drb gene allele andPUUV infection in one hand and the negative relationship detected between the Tnf-_ expression leveland PUUV prevalence in the other hand corroborates the evolution of tolerance in NE endemic area. Itcould be selected in response to the cost of inflammatory response which is activated againstPUUV.The population genetic approach demonstrated the role of population dynamic of reservoir inPUUV epidemiology. The comparison of Drb and Tnf-_ polymorphism and neutral genetic patternseems to indicate a weak influence of selection in the polymorphism of these immune genes. Thisresult suggests a potential important effect of phenotypic plasticity in the levels of resistance/toleranceobserved. The study of helminth communities confirmed this hypothesis showing the impact ofparticular species on the risk of PUUV infection.Le campagnol roussâtre Myodes glareolus est le réservoir principal de l’hantavirus Puumala(PUUV) responsable de la néphropathie épidémique (NE) en Europe. L’objectif de cette thèse est dedécrire la variabilité de la résistance / tolérance de M. glareolus à PUUV et d’explorer son rôle dans ladistribution et la transmission de ce virus. Nous émettons l’hypothèse que la tolérance à PUUVfavorise sa persistance et sa transmission, ce qui devrait accroître le risque d’émergence de la NE.Nous avons développé une approche gène candidat pour déterminer le rôle de trois gènes del’immunité dans la résistance / tolérance à PUUV. L’existence d’associations positives entre les allèlesdu gène Drb et l’infection par PUUV d’une part et les relations négatives détectées entre le niveaud’expression du gène Tnf-_ et la prévalence en PUUV d’autre part corroborent l’évolution d’unetolérance en zones d’endémie de la NE. Elle pourrait être sélectionnée en réponse au coût de laréponse inflammatoire développée contre PUUV. L’approche de génétique des populations a démontréle rôle de la dynamique des réservoirs dans l’épidémiologie de PUUV. La comparaison dupolymorphisme des gènes Drb et Tnf-_ et des patrons génétiques neutres semble cependant indiquerune faible influence de la sélection dans le polymorphisme de ces gènes. Ce résultat suggère un effetpotentiellement important de la plasticité phénotypique dans les niveaux de résistance / toléranceobservés. L’étude de la communauté d’helminthes a confirmé cette hypothèse en révélant l’impact decertaines espèces sur le risque d’infection à PUUV

Variability of resistance/tolerance to Puumala hantavirus virus in bank voles and epidemiological consequences

Le campagnol roussâtre Myodes glareolus est le réservoir principal de l'hantavirus Puumala (PUUV) responsable de la néphropathie épidémique (NE) en Europe. L'objectif de cette thèse est de décrire la variabilité de la résistance / tolérance de M. glareolus à PUUV et d'explorer son rôle dans la distribution et la transmission de ce virus. Nous émettons l'hypothèse que la tolérance à PUUV favorise sa persistance et sa transmission, ce qui devrait accroître le risque d'émergence de la NE. Nous avons développé une approche gène candidat pour déterminer le rôle de trois gènes de l'immunité dans la résistance / tolérance à PUUV. L'existence d'associations positives entre les allèles du gène Drb et l'infection par PUUV d'une part et les relations négatives détectées entre le niveau d'expression du gène Tnf-α et la prévalence en PUUV d'autre part corroborent l'évolution d'une tolérance en zones d'endémie de la NE. Elle pourrait être sélectionnée en réponse au coût de la réponse inflammatoire développée contre PUUV. L'approche de génétique des populations a démontré le rôle de la dynamique des réservoirs dans l'épidémiologie de PUUV. La comparaison du polymorphisme des gènes Drb et Tnf-α et des patrons génétiques neutres semble cependant indiquer une faible influence de la sélection dans le polymorphisme de ces gènes. Ce résultat suggère un effet potentiellement important de la plasticité phénotypique dans les niveaux de résistance/tolérance observés. L'étude de la communauté d'helminthes a confirmé cette hypothèse en révélant l'impact de certaines espèces sur le risque d'infection à PUUV.The bank vole Myodes glareolus is the main reservoir of Puumala hantavirus (PUUV), the agent of nephropathia epidemica (NE) in Europe. This work aims at describing the variability of M. glareolus resistance / tolerance to PUUV and at exploring its role in the distribution and transmission of the virus. We hypothesized that tolerance to PUUV should favour its persistence and transmission, what could increase the risk of NE emergence.We developed a candidate gene approach to determine the role of three immune genes in the resistance / tolerance to PUUV. Both the detection of positive associations between Drb alleles and PUUV infection and the negative relationship observed between Tnf-α gene expression and PUUV prevalence corroborated the evolution of tolerance in NE endemic areas. The costly inflammatory response activated against PUUV infection could mediate this evolution.Using landscape population genetics, we revealed the role of M. glareolus population dynamics in PUUV epidemiology. The comparison of Drb and Tnf-α genetic differentiation with the neutral pattern detected at microsatellites indicated that selection weakly acted on these immune genes. This result suggested the potential effect of phenotypic plasticity in the balance of resistance/tolerance to PUUV. The study of helminth communities confirmed this hypothesis as it revealed the impact of two nematode species on the risk of PUUV infectio

Variability of resistance/tolerance to Puumala hantavirus virus in bank voles and epidemiological consequences

Le campagnol roussâtre Myodes glareolus est le réservoir principal de l'hantavirus Puumala (PUUV) responsable de la néphropathie épidémique (NE) en Europe. L'objectif de cette thèse est de décrire la variabilité de la résistance / tolérance de M. glareolus à PUUV et d'explorer son rôle dans la distribution et la transmission de ce virus. Nous émettons l'hypothèse que la tolérance à PUUV favorise sa persistance et sa transmission, ce qui devrait accroître le risque d'émergence de la NE. Nous avons développé une approche gène candidat pour déterminer le rôle de trois gènes de l'immunité dans la résistance / tolérance à PUUV. L'existence d'associations positives entre les allèles du gène Drb et l'infection par PUUV d'une part et les relations négatives détectées entre le niveau d'expression du gène Tnf-a et la prévalence en PUUV d'autre part corroborent l'évolution d'une tolérance en zones d'endémie de la NE. Elle pourrait être sélectionnée en réponse au coût de la réponse inflammatoire développée contre PUUV. L'approche de génétique des populations a démontré le rôle de la dynamique des réservoirs dans l'épidémiologie de PUUV. La comparaison du polymorphisme des gènes Drb et Tnf-a et des patrons génétiques neutres semble cependant indiquer une faible influence de la sélection dans le polymorphisme de ces gènes. Ce résultat suggère un effet potentiellement important de la plasticité phénotypique dans les niveaux de résistance/tolérance observés. L'étude de la communauté d'helminthes a confirmé cette hypothèse en révélant l'impact de certaines espèces sur le risque d'infection à PUUV.The bank vole Myodes glareolus is the main reservoir of Puumala hantavirus (PUUV), the agent of nephropathia epidemica (NE) in Europe. This work aims at describing the variability of M. glareolus resistance / tolerance to PUUV and at exploring its role in the distribution and transmission of the virus. We hypothesized that tolerance to PUUV should favour its persistence and transmission, what could increase the risk of NE emergence.We developed a candidate gene approach to determine the role of three immune genes in the resistance / tolerance to PUUV. Both the detection of positive associations between Drb alleles and PUUV infection and the negative relationship observed between Tnf-a gene expression and PUUV prevalence corroborated the evolution of tolerance in NE endemic areas. The costly inflammatory response activated against PUUV infection could mediate this evolution.Using landscape population genetics, we revealed the role of M. glareolus population dynamics in PUUV epidemiology. The comparison of Drb and Tnf-a genetic differentiation with the neutral pattern detected at microsatellites indicated that selection weakly acted on these immune genes. This result suggested the potential effect of phenotypic plasticity in the balance of resistance/tolerance to PUUV. The study of helminth communities confirmed this hypothesis as it revealed the impact of two nematode species on the risk of PUUV infectionMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Data from: Helminth interaction with the host immune system: short-term benefits and costs in relation to the infectious environment

Chronic infections imply that the parasite and the host immune system closely interact for a long time without a fatal outcome. Environmental changes encountered by hosts and parasites, such as coinfections, can deeply affect the stability of this apparent equilibrium. Our study aimed to determine the effect of the infectious environment on the costs and benefits of chronic infection with the gut nematode Heligmosomoides polygyrus in mice. Heligmosomoides polygyrus is known for its capacity to actively interfere with the host immune response by secreting molecules that can dampen immunity. We simulated bacterial coinfection of H. polygyrus-infected CBA-strain mice during the chronic phase of the infection by injecting them with Escherichia coli lipopolysaccharide. We found that infection by H. polygyrus induced only weak costs for the host (in terms of reproductive investment) and was characterized by the upregulation of both Th1 (interferon-γ) and anti-inflammatory (transforming growth factor-β) cytokines, which is favorable to parasite persistence. However, when co-occurring with the simulated bacterial infection, H. polygyrus infection was associated with a pronounced shift toward a pro-inflammatory status, which was deleterious to both the parasite and the host. Our study highlights the dynamic equilibrium reached during chronic infection, where a rapid environmental change, such as a concomitant bacterial infection, can deeply affect the outcome of the host-parasite interaction