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

The Needs for Developing Experiments on Reservoirs in Hantavirus Research: Accomplishments, Challenges and Promises for the Future

Due to their large geographic distribution and potential high mortality rates in human infections, hantaviruses constitute a worldwide threat to public health. As such, they have been the subject of a large array of clinical, virological and eco-evolutionary studies. Many experiments have been conducted in vitro or on animal models to identify the mechanisms leading to pathogenesis in humans and to develop treatments of hantavirus diseases. Experimental research has also been dedicated to the understanding of the relationship between hantaviruses and their reservoirs. However, these studies remain too scarce considering the diversity of hantavirus/reservoir pairs identified, and the wide range of issues that need to be addressed. In this review, we present a synthesis of the experimental studies that have been conducted on hantaviruses and their reservoirs. We aim at summarizing the knowledge gathered from this research, and to emphasize the gaps that need to be filled. Despite the many di_culties encountered to carry hantavirus experiments, we advocate for the need of such studies in the future, at the interface of evolutionary ecology and virology. They are critical to address emerging areas of research, including hantavirus evolution and the epidemiological consequences of individual variation in infection outcomes

Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife

Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations.Methodology/Principal Findings : We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance :We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of bacterial prevalence in host populations, and generation of intra-reservoir patterns of bacterial interactions. Lastly, the number of bacterial reads obtained with the 16S-MiSeq could be a good proxy for bacterial prevalence

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

Detection of Orientia sp. DNA in rodents from Asia, West Africa and Europe

Article Open AccessInternational audienceOrientia bacterium is the agent of the scrub typhus, a seriously neglected life-threatening disease in Asia. Here, we report the detection of DNA of Orientia in rodents from Europe and Africa. These findings have important implications for public health. Surveillance outside Asia, where the disease is not expected by sanitary services, needs to be improved

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

Three-way relationships between gut microbiota, helminth assemblages and bacterial infections in wild rodent populations

Despite its central role in host fitness, the gut microbiota may differ greatly between individuals. This variability is often mediated by environmental or host factors such as diet, genetics, and infections. Recently, particular attention has been given to the interactions between gut bacteriota and helminths, as these latter could affect host susceptibility to other infections. Further studies are still required to better understand the three-way interactions between gut bacteriota, helminths and other parasites, especially because previous findings have been very variable, even for comparable host-parasite systems. In our study, we used the V4 region of the 16S rRNA gene to assess the variability of gut bacteriota diversity and composition in wild populations of a small mammal, the bank vole Myodes glareolus. Four sites were sampled at a regional geographical scale (100 km) along a North-South transect in Eastern France. We applied analyses of community and microbial ecology to evaluate the interactions between the gut bacteriota, the gastro-intestinal helminths and the pathogenic bacteria detected in the spleen. We identified important variations of the gut bacteriota composition and diversity among bank voles. They were mainly explained by sampling localities and reflected the North/South sampling transect. In addition, we detected two main enterotypes, that might correspond to contrasted diets. We found geographic variations of the Firmicutes/Bacteroidetes ratio, that correlated positively with body mass index. We found positive correlations between the specific richness of the gut bacteriota and of the helminth community, as well as between the composition of these two communities, even when accounting for the influence of geographical distance. The helminths Aonchotheca murissylvatici, Heligmosomum mixtum and  the bacteria Bartonella sp were the main taxa associated with the whole gut bacteriota composition. Besides, changes in the relative abundance of particular gut bacteriota taxa were specifically associated with other helminths (Mastophorus muris, Catenotaenia henttoneni, Paranoplocephala omphalodes and Trichuris arvicolae) or pathogenic bacteria. Especially, infections with Neoehrlichia mikurensis, Orientia sp, Rickettsia sp and P. omphalodes were associated with lower relative abundance of the family Erysipelotrichaceae (Firmicutes), while coinfections with higher number of bacterial infections were associated with lower relative abundance of a Bacteroidales family (Bacteroidetes). These results emphasize complex interlinkages between gut bacteriota and infections in wild animal populations. They remain difficult to generalize due to the strong impact of the environment on these interactions, even at regional geographical scales. Abiotic features, as well as small mammal community composition and within host parasite coinfections, should now be considered to better understand the spatial variations observed in the relationships between gut bacteriota, gastro-intestinal helminths and bacterial infections

Immunogenetic Factors Affecting Susceptibility of Humans and Rodents to Hantaviruses and the Clinical Course of Hantaviral Disease in Humans

Peer reviewe

Parasites and invasions: changes in gastrointestinal helminth assemblages in invasive and native rodents in Senegal

Understanding why some exotic species become widespread and abundant in their colonised range is a fundamental issue that still needs to be addressed. Among many hypotheses, newly established host populations may benefit from a parasite loss ("enemy release" hypothesis) through impoverishment of their original parasite communities or reduced infection levels. Moreover, the fitness of competing native hosts may be negatively affected by the acquisition of exotic taxa from invaders ("parasite spillover") and/or by an increased transmission risk of native parasites due to their amplification by invaders ("parasite spillback"). We focused on gastrointestinal helminth communities to determine whether these predictions could explain the ongoing invasion success of the commensal house mouse (Mus musculus domesticus) and black rat (Rattus rattus), as well as the associated decrease in native Mastomys spp., in Senegal. For both invasive species, our results were consistent with the predictions of the enemy release hypothesis. A decrease in overall gastrointestinal helminth prevalence and infracommunity species richness was observed along the invasion gradients as well as lower specific prevalence/abundance (Aspiculuris tetraptera in Mus musculus domesticus, Hymenolepis diminuta in Rattus rattus) on the invasion fronts. Conversely, we did not find strong evidence of GIH spillover or spillback in invasion fronts, where native and invasive rodents co-occurred. Further experimental research is needed to determine whether and how the loss of gastrointestinal helminths and reduced infection levels along invasion routes may result in any advantageous effects on invader fitness and competitive advantage