HIV-1 replication requires the integration of the viral genome into the cell genome. A viral-encoded enzyme, integrase (IN), performs this critical step of infection and is a promising target for anti-viral therapeutics. If the catalytic properties of INs are well characterized, the mechanisms responsible for their site selectivity are still under investigation. Several cellular proteins, such as the LEDFGF/p75 transcription regulator, the RNA polymerase II machinery, nuclear pore proteins and specific modified histones have been proposed to be involved in IN selectivity at a genomic level. In addition, structural parameters of the target DNA helix (curvature, flexibility and topology) are proposed to regulate IN selectivity at a local level. Our team is studying the role and molecular mechanisms associated with these various parameters (Botbol et al., 2008; Lesbats et al., 2011; Morchikh et al., 2013; Benleulmi et al., 2015; Naughtin et al.,). This project aims to define the role of cellular DNA topology during HIV-1 integration. We will first compare already mapped integration sites and superhelicity profiles and search for possible correlations between these two parameters. We will then modify topoisomerases activity in infected cells and study the consequences on viral replication and integration. Finally, we will study in vitro, the direct effects on integration of two parameters of DNA topology, the twist and writhe of the DNA helix. This project relies on complementary in vivo, in vitro and in silico approaches. Bio-informatics tools are crucial for the correlative and statistical analyses of integration sites and superhelicity maps.

Dengue is an arthropod-borne viral disease caused by dengue virus (DENV), which is transmitted by Aedes mosquitoes. Recent studies estimate that 100 million cases occur annually worldwide, making dengue the most prevalent mosquito-borne viral disease of human beings. DENV transmission results from interactions between humans, mosquitoes, viruses and their environment. If the human immune response against DENV is well studied, there are knowledge gaps in understanding how mosquitoes and DENV interact with each other. Moreover, it was shown that virus transmission does not only depend on the viral strain and mosquito population but also on the specific interaction between the two partners, named genotype-by-genotype (GxG) interaction. One aim of the team is to highlight factors at the mosquito and virus levels that regulate vector competence, i.e. the ability for a mosquito to acquire viral infection and subsequently transmit the virus to a new host. To this aim, we use field-derived Aedes aegypti populations that we challenge in the lab with virus isolates from DENV infected patients. This allows us to refine our understanding of the basic biology of virus transmission by mosquitoes.

The phylogenetic position and status of “giant viruses”, formerly called NucleoCytoplasmic Large DNA viruses (NCLDV) or putative order Megavirales, are controversial. Many preliminary phylogenetic analyses have been published, but their presentations are usually highly biased by the prejudice of the authors concerning the nature of giant viruses. Our own preliminary analyses suggest that giant viruses are indeed ancient (they predate the last universal eukaryotic ancestor) and have possibly provided important functions to emerging eukaryotic cells (e.g. DNA topoisomerase activities). The number of giant virus genomes has recently dramatically increased, opening new opportunity to study their position in the “universal tree of life” and their evolutionary relationships with eukaryotes. The aim of the project is to perform an exhaustive phylogenetic analysis of all giant virus proteins with eukaryotic (archaeal/bacterial) homologues to (i) test the monophyly of giant viruses, (ii) determine their contribution to early eukaryotic evolution, iii) determine if some giant virus proteins can be useful to root the eukaryotic tree. We need the help of a bioinformatics colleague with good expertise in building phylogenetic trees from large data sets using different methods of tree construction and robustness evaluation. This work will be complemented by the systematic search for significant indels (insertion/deletion) in the alignments obtained by two members of the BMGE team (Patrick Forterre and Morgan Gaia).

The 2013-2015 Ebola virus disease epidemic is the largest outbreak so far described with 27 305 cases and 11 169 deaths. The virus spread by human to human contact throughout Western Africa and never before has a variant been transmitted for such a sustained period of time. Ebola virus are RNA virus so as other RNA viruses they could accumulate mutations during evolution. Therefore it is an emergency to monitor viral changes and adaptation within and between individuals in order to help researchers to better understand susceptibility to Ebola infections, to guide research on therapeutic targets and to ensure accurate diagnosis. New technologies can provide information about pathogen’s evolution and in our lab we have access to an Ion PGMTM sequencer. Thanks to the national reference center for viral hemorrhagic fever (VHF) we have at our disposal a large number of samples collected from Ebola infected patients especially from Guinea. We have developed an Amplicon approach using sixteen couples of specific primers for Ebola viruses and a RNA sequencing method based on randomly primed cDNA synthesis to product our libraries. Ion PGMTM Hi-Q sequencing kit will be used to sequence up to 400 bp inserts loaded onto 316v2TM or 318v2TM chip. Through high depth sequencing we would like to follow up the profiling of intra and inter host viral quasispecies at different time of the epidemic in the geographic area of the Ebola Treatment Centre in Macenta. Thanks to the activities of national reference center for VHF and the Biomics Pole one aim of the project is also to occasionally compare viral quasispecies and consensus sequences between patients who get uncommon symptoms from those who get classical illness and to study intra host quasispecies in different biological fluids (cerebrospinal fluid, sperm, urine) to see if there are differences between persistent species and viral quasispecies found during symptomatic step.

The goal of this project is to set sequence analysis tools to evaluate the frequency of mutations within a pool of bacteriophage genomes sequenced by NGS.

HIV-1 replication requires the integration of the viral genome into the cell genome. A viral-encoded enzyme, integrase (IN), performs this critical step of infection and is a promising target for anti-viral therapeutics. If the catalytic properties of INs are well characterized, the mechanisms responsible for their site selectivity are still under investigation. Several cellular proteins, such as the LEDFGF/p75 transcription co-activator, the RNA polymerase II machinery, nuclear pore proteins and specific modified histones have been proposed to be involved in IN selectivity at a genomic level but the underlying molecular mechanisms remain to be demonstrated. In addition, structural parameters of the target DNA helix (curvature, flexibility, topology) are proposed to regulate IN selectivity at a local level. Our aims are to study the role of these different parameters of IN selectivity, using both in vitro and in vivo approaches. In vitro, we will map integration sites on various target DNA substrates (naked DNA or chromatin, minicircles, plasmids with different topologies, transcribed templates) and will test the effect of purified proteins suspected to regulate IN selectivity. In vivo, integration sites will be mapped in cells depleted of these suspected regulators or in cells incubated with drugs targeting enzymes involved in transcription, DNA topology or histone modifications. Integration sites will be mapped using published or “home-made” protocols and the sites will be compared with DNA structural parameters, nucleosome positions, histone modifications or transcriptional parameters (published maps). Bio-informatics tools are crucial for these correlative and statistical analyses of integration sites. Our project relies on complementary in vivo, in vitro and in silico approaches. It should establish molecular and mechanistic rules of HIV-1 integration selectivity that could serve in the development of new antiviral strategies and of safer gene therapy vectors.

Controlling virus replication by generating a strong CD8+ T cell response against HIV is one of the major goals in the development of an effective HIV vaccine candidate. Indeed, during the chronic phase of infection, HIV-specific CD8+ T cells were shown to have impaired functionality and fail to control viral replication. Understanding the profile of CD8+ T cells able to efficiently tackle HIV is therefore much needed. HIV controllers (HIC) are individuals who control viremia without antiretroviral therapy. CD8+ T cells seems to play a major role in their HIV control. We hypothesized that HIV-specific CD8+ T cells associated with control of infection bear particular transcriptional signature (cell cycle, survival, activation, cytolytic function…) when compared to HIV-specific CD8+ T cells not associated with control of infection sharing the same memory phenotype.

Molecular markers of cervix lesions linked to HPV infections.

DNA topoisomerase IB (Topo IB) enzymes are ubiquitous in eukaryotes, where they represent the major DNA topoisomerase I activity. However, Topo IB sequences are also found in other phyla, such as archaea and bacteria, as well as viruses. Given the large amount of sequenced data available in public databases, this project aims to infer a robust Topo IB gene tree based on a representative set of homologous sequences gathered from a large taxonomic sample.

Dengue prevention relies primarily on controlling populations of the main mosquito vector, Aedes aegypti, which is failing in many parts of the world because of the lack of sustained commitment of resources and ineffective implementation. Novel entomological approaches to dengue control are being developed that aim at replacing or suppressing mosquito vector populations. Insufficient genomic resources for Ae. aegypti, however, have until now impeded progress in both basic and applied research on this medically important mosquito species. The only available reference genome for Ae. aegypti is a draft that consists of over 4,800 unassembled fragments with incomplete annotation. Moreover, the inbred Ae. aegypti laboratory strain that was sequenced does not universally represent the considerable genetic and ecological diversity of the species worldwide. The large size of the genome and its high content in repeat-rich sequences of transposable elements was a major difficulty to assemble the Ae. aegypti genome sequence. In the present project, we aim to overcome this difficulty using a novel strategy for genome sequencing and assembly. The ultimate goal is to produce several, fully assembled, well-annotated, new Ae. aegypti reference genomes from epidemiologically relevant populations. The expected outcome is a genome reference panel including a catalog of species-wide genetic variation that will significantly improve genomic resources for Ae. aegypti research and help address a broad range of biological questions related to Ae. aegypti vectorial capacity and dengue virus transmission.

The goal of this project is to set sequence analysis tools to evaluate the frequency of mutations within a pool of bacteriophage genomes sequenced by NGS.

L'extraction des protéines humaines qui interagir avec des protéines virales par le séquençage des barcodes associés avec les protéines humaines et virales. Analyses structurales des protéines virales et humain pour trouver les sites d’accrochage.

Analyses fonctionnel des protéines humains de type ubiquitin qui interagir avec les virales protéines de la grippe

L'Analyses phylogénétique par séquence protéique, temporal et géographique des échantillons grippales avant et après l’épidémie de 2009.

analyses et classification des échantillons grippales.

Collect statistics on variants found in different strains of influenza virus. Determine variant effects to the protein sequence.

We would like to uncover associations between transcriptomic features and dengue infection outcome. In order to do so, we want to take advantage not only of our data but also of all publicly available data. A main challenge is to translate the measurements across different transcriptomic technologies into a summary expression level per gene. For this C3BI project we would like to concentrate on the problem of mapping probe IDs into a common identifier for all experiments.

Analysis of the immune response to yellow fever 17D vaccination

Lassa virus (LASV) is an arenavirus causing hemorrhagic fever in human. 300 000 to 500 000 cases of LASV infection are reported every year in western Africa, including 5 000 to 6 000 deaths. LASV is highly pathogenic, and no vaccine or treatment is available in endemic areas. LASV pathogenesis mechanisms are not well documented, and further investigations are needed to understand viral and immunological factors involved during infection. As previously shown by studies conducted on patients and non-human primates infected by LASV, T-cell response and type I interferon (IFN-I) are important for an effective response to LASV infection. Among dendritic cells (DC), myeloid DC can induce T-cell activation and plasmacytoid DC are specialized in IFN-I response. DC are also the first target of LASV during the infection of a new host, and studies on in vitro differentiated DC suggest a role of DC in T-cell response to LASV infection. Therefore, plasmacytoid and myeloid DC could be important for an efficient response to LASV infection.

The objective of this study is to evaluate how the different programming of distinct CD4+ T cell subsets affected their susceptibility to HIV infection and the survival of infected cells. Dr. Saez-Cirion's Team evaluates how the metabolic state of CD4⁺ T cells and the regulation of cellular factors shape the distribution of the HIV reservoir in distinct CD4⁺ T cell subset. This project uses new technologies and reagents to analyze cells from uninfected donors, and from HIV patients in acute infection, chronic infection and HIV remission.

We wish to offer to the community of microbial virologists a place where they can store and find results of host range determination for microbial viruses

The aim of this project is to implement, at the level of the research units, a bioinformatics workflow dedicated to the analysis of the microbiome, and of the virome in particular, based on Next Generation Sequencing (NGS) data (Illumina technology). This workflow will be applied in different contexts, from the identification of nucleotide sequences belonging to a novel or emerging pathogen (mainly viruses) presente in a clinical sample (such as serum or cerebrospinal fluid), to the determination of the whole sequence genome of isolated and uncharacterized viruses (such as bacteriophages of Leptospira).  

Poliomyelitis has been a major public health concern and currently, efforts are being made towards eradicating wild poliovirus type 2 (WPV2). A global switch from trivalent oral poliovirus vaccine (tOPV) to bivalent oral poliovirus vaccine (bOPV without PV2) is planned to take place this year in countries, like Madagascar, where epidemics of type 2 pathogenic circulating vaccine-derived polioviruses (cVDPVs) occur when low polio vaccine coverage allows the circulation and genetic drift of vaccine attenuated PV2 that can thus become pathogenic. In an attempt to monitor the presence and eventually absence of wild poliovirus 2, environmental and human surveillance will be conducted before, during, and after the switch in two regions in Madagascar. PV2, in particular cVDPVs and other enteroviruses will be genetically characterized. These data will support monitoring efforts and confirm the elimination of type 2 PV strains that have been implicated in outbreaks in this country.

Screening for NSm function in infected mammalian cells by proteomic analysis.

Les mutations de résistance aux traitements apparaissent sous l’effet de la sélection. Ces mutations sont transmises, les patients pouvant être infectés par des souches déjà résistantes à certains traitements. Ces mutations posent des problèmes considérables en limitant l’arsenal des traitements disponibles, pour les individus comme pour la population sur le long cours. Dans le cas du VIH, nous avons travaillé sur la transmission de ces mutations au sein de la population anglaise, et montré que la majorité de celles-ci étaient transmises par des patients naïfs ignorant leur infection (Mourad et al. AIDS 2015). La méthode employée était souvent ad-hoc et n’utilisait qu’une partie des données disponibles. L’objectif de ce projet est de mettre en place des modèles rigoureux et des méthodes efficaces d’estimation des paramètres (taux de transmission suivant les caractéristiques du donneur, taux de réversion, etc.).

Microbial discovery remains a challenging task for which there are a lot of unmet medical and public health needs. Deep sequencing has profoundly modified this field, which can be summarized in two questions : i) which pathogens or association of pathogens are associated with diseases of unknown etiology and ii) among microbes infecting animal (including arthropod) reservoirs, which ones are able to infect large vertebrates, including humans. We are currently addressing these two questions and our current request comes with the willingness for Institut Pasteur to increase its contribution and visibility of this thematic, in particular in relation with hospitals and the Institut Pasteur International network (IPIN).  We expect to identify new microbes associated with human diseases, and this is expected to pave the way for basic research programs focusing on virulence mechanisms and host specificity, and will also lead to phylogenetic and epidemiological studies (frequency of host infection, mode of transmission etc...), as well as the development of improved diagnostic tests for human infections. Our objective is also to contribute to the efforts of Institut Pasteur in the field of infectious diseases, by building a pipeline, from sample to microbial identification, able to manage large cohorts of samples. This project is currently supported by the LABEX IBEID and the CITECH, and critically requires a bioIT support, justifying this application. Partners include different hospitals including Necker-Enfants malades University Hospital regarding patients with progressive disease, different IPIN laboratories, as well as INRA and CIRAD regarding animal/arthropod reservoirs.

We would like to be able to use IgBlast on the Galaxy platform. We are studying B cells in adaptive immune response, and are particularly interested in the antibodies termed as broadly neutralizing antibodies (bNAbs). By definition, these antibodies can neutralize most known HIV-1 strains, and are produced by rare infected individuals several years post-infection. We are currently investigating the bNabs immunoglobulin repertoire by focusing our NGS (454 pyrosequencing) analysis on  immunoglobulin sequences (V-domains) from HIV-infected patients who developed bNAbs. As immunoglobulin sequences result from the combinatorial rearrangement of 3 gene segments : V , (D) and J gene segments, we need a specific tool to analyze these sequences. Indentifying the germline genes which are involved in the rearrangment is an essential step. Two main tools are being widely used to analyze Immunoglobulins: IMGT and IgBlast. IgBlast has several advantages; it is based on BLAST (it is then possible for the user to build his own database), open source, can use protein or nucleotide sequences as input, and most of all, IgBlast is already installed on the Institut Pasteur's cluster as well as the germline genes database. As it would be very convenient for us to use the bic cluster and galaxy platform to run our analyzes, we would be grateful if IgBlast could be implemented in the Pasteur Galaxy Platform. In this regard, we are of course fully disposed to help in any ways. We also believe that it would be very useful to people working on immunoglobulin sequences in the immunology department by building specific pipelines. Thank you very much.

Integration of the viral reverse-transcribed genome into the genome of infected cells is an essential step of retroviral replication and is performed by a viral-encoded enzyme, named integrase (IN). In the case of HIV-1, IN is a new and efficient anti-viral target. The selectivity of this enzyme for its cellular genomic sites is also a major parameter of HIV replication and is regulated by several cellular parameters. One of them is chromatin, and different levels of this nucleoprotein complex are involved in the regulation of IN selectivity. Using in vitro integration assays, established by our team and collaborators, we have studied this regulation at two levels of chromatin architecture: large poly-nucleosome templates (Botbol et al., 2008; Lesbats et al., 2011; Benleulmi et al., 2015; Naughtin et al., 2015) or nucleosome-induced DNA curvature mimicked by DNA minicircles (Pasi et al., 2016). Our present project is to study IN selectivity into mononucleosomes (MN). These MNs will be used as target substrates of integration and the role of MN structure, histone modifications and IN cofactors will be studied. Results obtained in vitro, will be confronted to structural data obtained by molecular modeling and to integration sites observed in infected cells. This project will benefit from our expertise in integration in chromatin templates and a previous collaboration with the C3BI on the analysis of integration sites (Pasi, M., Mornico, D., S. Volant, S., et al., 2016). This project is funded by the ANRS.

The PhageTermini software was developed to allow biologists acquire valuable information on bacteriophage termini sequences and packaging mode using phage sequenced with the Illumina TruSeq technology.

See below  

We are interested to look at evolution of bacteria in presence of bacteriophages within the gastrointestinal tract of animals.    

Lassa virus (LASV) is an arenavirus causing hemorrhagic fever in human. 300 000 to 500 000 cases of LASV infection are reported every year in western Africa, including 5 000 to 6 000 deaths. LASV is highly pathogenic, and no vaccine or treatment is available in endemic areas. LASV pathogenesis mechanisms are not well documented, and further investigations are needed to understand viral and immunological factors involved during infection. As previously shown by studies conducted on patients and non-human primates infected by LASV, type I interferon (IFN-I) is important for an effective response to LASV infection. As plasmacytoid DC are specialized in IFN-I response, they could be important for an efficient response to LASV infection.

We previously showed that humanized immune system (HIS) mice generated in Balb/c Rag2^-/-γ_c^-/- SIRP^NOD (BRGS) recipients are susceptible to HIV-1 infection (X4 and R5 isolates) and maintain circulating HIV-1 in the plasma, resulting in a dramatic depletion of human CD4⁺ T cells. We also characterized features of HIV physiopathology in this model. Human thymocyte subsets developing in the thymus of HIS mice appear phenotypically normal, but in the periphery the T cell repertoire is restricted compared with that of human peripheral blood T cells. This negatively impacts on the ability of HIS mice to generate antigen-specific human immune responses when mice are vaccinated with protein antigens or following infection with lymphotropic viruses such as HIV. One likely explanation for these functional deficiencies involves the fact that human T cells are selected intrathymically by mouse MHC molecules and that naïve T cells in peripheral lymphoid organs interact primarily with mouse DC (as human DC development in HIS mice is limited). As a first line of improvement, we recently generated a novel mouse model by crossing our BRGS mice with the HLA-A*02-HHD class I transgenic mice and the HLA-DRB1*15 class II transgenic mice, resulting in BRGS-A2DR2 mice. Following intra-hepatic injection of these mice with MHC-matched CD34⁺ stem cells we observed increased engraftment, with faster kinetics. Moreover BRGS-A2DR2 HIS mice have an increased T cell development leading to a more equilibrated B/T and CD4/CD8 phenotype. We showed that BRGS-A2DR2 HIS mice were able to sustain replication of HIV R5 virus as the BRGS hosts. Viremia was similar in a first phase and then lower in a second phase in BRGS-A2DR2 compared to BRGS HIS mice, which could be a consequence of a better quality of the immune response. However, the viremia reached a similar plateau in the last phase. We propose to study the impact of the immune res

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants underlying this response, we characterized the TCR repertoire directed at the most immunodominant CD4 epitope in HIV-1 capsid, Gag293. HIV Controllers from the ANRS CO21 CODEX cohort showed a highly skewed TCR repertoire characterized by a predominance of the TRAV24 and TRBV2 variable gene families. Controllers shared public clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control (Benati D. et al., J Clin Invest 2016).   We propose to test the generality of these findings by characterizing the TCRs specific for a series of immunodominant HIV Gag and Env epitopes, and comparing the frequencies of public clonotypes in groups of HIV Controllers and treated patients. We will then assay the functions of the most prevalent public clonotypes through lentivector-based TCR transfer, and correlate the panel of T cell functions to TCR affinity and frequency.

HIV infects and depletes CD4+ T cells, leading to a progressive loss of adaptive immune responses and ultimately to AIDS. In addition, HIV preferentially targets HIV-specific CD4+ T cells, resulting in an attrition of the very cells that should orchestrate the antiviral immune response. Due to this preferential depletion, HIV-specific CD4+ T cells are few and remain incompletely characterized. The emergence of single cell technologies opens the opportunity for an in depth analysis of the rare specific CD4+ T cells that persist in the circulation of chronically infected patients. We have sorted single CD4+ T cells specific for the most immunodominant epitope in HIV-1 capsid, using an optimized MHC class II tetramer labeling protocol. We now propose to analyze these single cells by multiplexed real time PCR in a microfluidics device, to define their transcriptional profile. We will analyze the differentiation status of HIV-specific CD4+ T cells in rare patients who naturally control HIV infection and in progressor patients who control HIV replication due to antiretroviral therapy. This project will help define the parameters of an efficient T cell response against HIV, and may provide insights into the type of responses that should be induced by candidate HIV vaccines.

In order to study a differential regulation of RNA and protein expression level during Rift valley fever virus (RVFV) infection in a murine model, we want to compare transcriptomic and proteomic analysis.  

Despite effective prevention against HBV infection, 300 million people worldwide are chronic HBV carriers, of whom 25% will die of liver cirrhosis or hepatocellular carcinoma (HCC). Current treatments for chronic hepatitis B (CHB) are inefficient to completely clear the virus and liver cancer is a lethal disease, thus representing an area of highly unmet medical need. Viral persistence is due to the maintenance, in the nuclei of infected cells, of the viral nuclear DNA : the cccDNA that is not targeted by the antiviral treatment and to the impairment of both the innate and adaptive immune responses that accompanies CHB infection. Viral replication depends on a balance between factors that benefit and those that restrict viral infection. However little is known about cellular factors that repress HBV replication. Using quantitative temporal viromics approach developed by P. Lenher in Cambridge we have performed a quantitative analysis of temporal changes in host and viral proteins in primary human hepatocytes through the course of HBV infection. We will in particular search for cellular factors involved in virus restriction and uncover the mechanism of viral escape.

Because of their increasing incidence, dramatic severity, lack of treatment or vaccine, complicated diagnosis, misreading of the pathogenesis, and need for a maximum containment, Viral Hemorrhagic Fevers (VHF) constitute a major public health problem. There is therefore an urgent need to further study VHF to understand the pathogenesis of the severe disease and the host responses involved in their control or in the dramatic damages. Among VHF, Lassa fever (LF) is probably the most worrying one because of its endemicity and the large number of cases. LF is caused by the Old-World arenavirus Lassa virus (LASV). It is endemic to West Africa and is responsible for 300,000 cases and 5,000 to 6,000 deaths each year. We propose here to study the pathogenesis of VHF by using LF in cynomolgus monkeys as a paradigm, with a particular emphasis on the very early events. The viral tropism, pathophysiological mechanisms, and immune responses will be studied during the course of infection, including the incubation period. Powerful approaches will be used to (1) identify early biological markers of infection, to be able to confirm infection and isolate patients; (2) determine the viral tropism and dynamics during the course of infection to understand the natural history of virus into its host. (3) characterize the early pathogenic events that lead to the severe hemorrhagic syndrome to fully understand the pathophysiogenesis of VHF and identify new therapeutic targets. (4) identify the immune responses involved in the control of infection or in the fatal outcome, to reveal the involvement of immunopathological mechanisms and help to design a vaccine approach. This ambitious and unprecedented project will allow to develop therapeutic and prophylactic approaches but also to identify early biological markers of infection and improve the early diagnosis to optimize the management of outbreaks in the field and increase the survival rate in patients.

We have identified a candidate gene associated with increased resistance to a pathogen. This gene is poorly annotated in public databases. To get insight into its function we are focusing on genes that are co-expressed with this candidate gene in various datasets, including microarray collection on various mouse tissues or in a given tissue across inbred strains of mice. Indeed, coexpression is one of the central idea in gene expression analysis. The 'Guilt by association' principle states that gene coexpression might indicate shared regulatory mechanisms and roles in related biological processes. We have established lists of genes that are co-expressed in various tissues, and in collection of individuals with different genotypes. We are willing to get graphical representation giving a compact overview of genes that are coexpressed with our candidate gene.

Notre objectif est d'étudier comment la recombinaison génétique génère de la diversité au sein des écosystèmes d'entérovirus.

Our lab is interested in the assembly of the inner viral membrane of Vaccinia virus and what are the molecular regulators (proteins and lipids) playing a role in this complex and unique process of virion morphogenesis.

Blood cells were collected in the field from patients admited in the Ebola treatment center of Macenta (Guinea). After red cells lysis, blood cells were frozen and RNA was extracted after the shipment of samples in France. Despite poor RNA quality, Affimetrix chips have been performed and transcriptomic data are available. The aim of this study is to compare the transcriptomic data from patients who survived and those who died and also to perform a kinetic analysis of the infection in the two groups of patients.  Samples from febrile patients who were not infected with Ebola virus have been used as negative controls.

Samples were collected in the field from infected patients admited in Ebola treatment Center of Macenta (ETC Guinea). Deep sequencing was performed in order to look for majority and minority variants. The aim of this study is to compare quasispecies of patients who survived and those who died and also to get detailed description of EBOV evolution in an ETC to identify phylogenetic clusters or transmission chain.

Yellow fever virus (YFV), a Flavivirus transmitted by mosquitoes causes a severe hemorrhagic fever in humans. Despite the availability of a safe and effective vaccine (17D), YFV is still a public health problem in tropical Africa and South America. In the Americas, the massive campaign of mosquito control during the first half of the 20th century led to the eradication of Aedes aegypti from most American countries, and as a consequence, urban outbreaks of YF were no longer observed. However, the relaxation of vector control led to the reinfestation of urban areas by Ae. aegypti and the subsequent establishment of the Asian tiger mosquito Aedes albopictus. In Brazil, while human cases are sporadically detected in the Amazonian basin where sylvatic YFV strains circulate between non-human primates and arboreal canopy-dwelling mosquitoes (Haemagogus sp.), they are increasingly reported outside the jungle moving towards the Atlantic coast, the most populated area. In the absence of routine immunization programs, YF may come back in the American towns as it was in the past. The causes leading to the current YF resurgence are multifactorial. From a mosquito vector viewpoint, changes in vector densities, distribution, vector competence or vector as a site of selection for epidemic YFV strains, can be regarded as critical factors. Our project aims to address the contribution of the invasive mosquito Ae. albopictus as a missing link to allow a selvatic YF strain (1D) to become adapted for a transmission in urban areas by the human-biting mosquito, Ae. aegypti. It will be done through three specific objectives: (i) identify Ae. albopictus-adaptive mutations after serial cycling of the selvatic YFV-1D on Brazilian Ae. albopictus mosquitoes, (ii) evaluate their potential to be transmitted to a vertebrate host, and (iii) deepen the transmission of the experimentally selected viruses by field-collected mosquito populations.

The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections, including Influenza, HIV and Zika viruses, by inhibiting virus-cell fusion. We showed that IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses. We also reported that amino-terminal mutants of IFITM3 preventing ubiquitination or endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. Our aim is to analyze the RNA levels of the various members of the IFITM family, in various normal or pathological human or animal tissues.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by a biallelic mutation of the Ataxia Telangiectasia Mutated (ATM) gene. The ATM protein is involved in a very large number of cellular functions (over 700 substrates), mainly DNA repair, cell cycle regulation, mitochondrial and oxidative metabolism, as well as regulation of gene expression. The majority of hematological malignancies occurring in AT patients are non-Hodgkin's B lymphomas and Hodgkin's lymphomas. The association with the Epstein-Barr virus (EBV) is found in 50 to 100% of these cases according to the histological subtype. EBV is a widespread virus in human populations with a prevalence greater than 90% and is associated with the development of lymphomas in immunocompromised patients. We will study the transcriptome of EBV-infected cells in ATM patients, in order to determine the impact of ATM on EBV infection control.

Taking advantage of a murine model of controlled microbiota composed of 12 strains, we evaluated the activity of a cocktail of three virulent bacteriophages to target a murine Escherichia coli strain in the mammalian gut.

As a result of combined climate change and globalization (increased flow of travelers and goods), the distribution of the mosquito Aedes albopictus is expanding significantly outside tropical regions. Ae. albopictus has already established stable colonies in 20 European countries (https://ecdc.europa.eu/en/publications-data/aedes-albopictus-current-known-distribution-june-2018). Being the main mosquito that can be incriminated in the transmission of chikungunya, dengue, and zika viruses in Europe, Ae. albopictus were responsible for the first autochthonous cases in France and Italy. Accordingly, the European countries that are infested with Ae. albopictus are under the risk of arboviral diseases outbreak. The vector competence analysis of European Ae. albopictus is pivotal for evaluating the potential risk of diseases transmission. In this study, we analyzed the viral susceptibility of several European Ae. albopictus populations from Croatia, Greece, Montenegro, Italy, Switzerland, and also China (as a control), for chikungunya, dengue, and zika viruses. The results indicated that the European Ae. albopictus were able to transmit chikungunya and dengue viruses whereas they were not an efficient vector for zika virus.

A protein-protein interaction screen has been done beween viral proteins of influenza A viruses and a library of about 100 human factors invovled in RNA processing through RNA exonucleases activity. 20 factors were identified as interactors of viral proteins. To see if this targeting corresponds to specific RNA metabolism focntions of the cell, we analysed using Cytoscape the first neighbors of these factors in the human ORFeome. We then compared using the ClueGo pluging the functional enrichment of the factors targeted by viral factors and the functional enrichment of the non-interacting subset. We need now a statistical analysis of the results the functional enrichment comparison, which is required for further progress of the ongoing research project.

This project is integrated in the analysis of the gut ecosystem of children implicated in the AFRIBIOTA project, a translational project performed within a consortium of researchers and medical doctors from the Central African Republic, Madagascar, France and Canada (see https://research.pasteur.fr/fr/program_project/the-afribiota-project/). AFRIBIOTA aims at understanding the risk factors and pathophysiological changes underlying chronic child malnutrition as manifested through delayed growth. Within the project submitted, we aim at assessing a possible link between asymptomatic pathogen carriage, gut inflammation and stunted growth of children included in the study site in Antananarivo, Madagascar.

Bacteriophages infect bacteria. One bacteriophage can infect several strains. Around the world, many labs have performed spot tests to determine the host range of bacteriophages but this information is not accessible because there is no tool to process and store it. Developing such a tool will be useful for the phage community but also for the entire community of virologists.

We need an independent statistician to confirm that appropriate statistical tests were used to analyze the data in your manuscript for the final revision of our manuscript in Science Signaling (Manuscript Number: aar3993, \"LGP2 binds PACT to regulate RIG-I- and MDA5-mediated antiviral response\" ). Sincerely yours, Anastassia Komarova

Enteroviruses are known to recombine a lot during the replication cycle. A lot of viruses produce defective genomes, which are generated by the polymerase skipping several nucleotides and thus, forming internal deletions of the genome. We would like to look at this process more closely and come up with a model, which includes the role of the RNA virus genome itself, including its structure.

The chikungunya virus (CHIKV) is an emerging mosquito-borne virus which has widely spread around the world in the last two decades. The virus is transmitted between human hosts by Aedes mosquitoes, including Aedes albopictus which is now established in more than 51 departments in France. The transmissibility of CHIKV can be affected by a combination of both intrinsic and extrinsic factors. Since mosquitoes are poikilothermic insects, environmental temperature is of particular importance. Indeed, temperature can shape transmission of pathogens through direct effects on viral replication and/or indirect effects via the vector immune responses. The relation between temperature and virus transmission is very complex and remains poorly defined. The aim of the project is to better understand how temperature affects mosquito-virus interactions. To meet this objective, our studies will focus on (i) how CHIKV genetically evolves under low temperature pressure (in vitro and in vivo), (ii) mosquito molecular responses to a low temperature exposure, (iii) and ultimately, how mosquito microbiota is affected by a low temperature. Our study will help in identifying the determinants that facilitate CHIKV transmission by Ae. albopictus in temperate conditions.

We passaged EV71 in cell culture, followed by RNAseq analysis. The sequences were analysed for the presence of internal deletions within the viral genome. Such deletions can contribute to non-homologous and homologous recombination within Enteroviruses, which can have positive effects, such as the purging of deleterious mutations or to overcome host restrictions. On the other hand such deletions could result in the formation of defective interfering particles (DIs), which have the ability to interfere with wildtype virus infections and thus, reduce viral loads in ongoing infections. We aim to get a better understanding of these events during viral replication that can aid us to identify DIs and can help us explore these special forms of virus genome deletions as potential therapeutic agents.

We want to determine the function of a protein conserved among certain phages. Homology searches yield about a hundred hits that clearly share some motifs, however they remain 'putative', without an assigned function. Searching for known motifs doesn't give any hits neither. We would like to use a database of C3BI dedicated to viral proteins to find a possible clue about the protein's role.

The ViroScreen workflow is a bioinformatic pipeline dedicated the the analysis of NGS metagenomic data, especially in the field of virology (virome analysis). The aim of the project is to implement this bioinformatic workflow, developed with Corinne Maufrais (Bioinformatics and Biostatistics Hub), in Galaxy, to make it easly accessible to the scientific community, in easy-to-use way.

The transcriptome signature during rabies infection will be analyzed both in animal model (mouse) and in human, at the central nervous system level, to identify the main canonical pathways involved during the infection.

Viruses have evolved powerful countermeasures to evade host innate immunity, which produces immediate, but non-specific, immune response during infection. Among viruses possessing RNA genomes, the order of negative-single-strand viruses (Mononegavirales) encompasses many human and animal pathogens that cause severe disease, including measles virus, mumps virus and rabies virus. Rabies virus is known for its neurotropic retrograde progression from the site of transmission to brain parenchyma, and towards salivary glands, different organs linked through parasympathetic nervous system. In the cases of human infection, the exhibited symptoms such as hallucination, diplopia, hydrophobia, unsteadiness or paralysis all indicate that there is causality between rabies virus infection and dysfunction of neural activity. However, lack of pathological brain lesions observed at the point of autopsy or noncytolytic propagation devoid of apoptosis suggest that rabies virus possesses mechanisms to evade or delay immune responses and cell death at least for the duration of replication and transmission. Even though the details in molecular perspective of these discoveries are well laid out now, how these proteins work in coordination or if there are hidden components which connect them all together leading toward deterioration of neural cells on the benefit of virus is largely unclear. Moreover, considering the complexity of brain cell composition and how important the neighboring cells are to shape one neuron’s specialization and dependency onto others in homeostasis, which result in the astounding heterogeneity of gene expression, an integrated and holistic approach is mandatory to get a fully comprehensive view of the mechanisms involved. Consequently, we performed an RNASeq analysis in human interneuron cells derived from induced pluripotent stem cells and infected by two recombinant rabies viruses (Tha virus, isolated from a dog in Thailand and Th4M, a less pathogenic virus which is mutated on 4 different residues of the M gene; this virus can no longer escape the NF-KB pathway) in order to obtain transcriptome data by comparison with uninfected cells, and to have an overview of the temporal dynamics of the genes expression.

Rabies virus (RABV) is a causative agent of lethal neurological disease, a member of Lyssavirus genus, belonging to Rhabdoviridae family in the order Mononegavirales. It presents a public health threat in the world resulting in more than 59,000 human deaths every year around the world. RABV possesses negative strand RNA genome, 11.9 kb, encoding five viral proteins: Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M), Glycoprotein (G) and polymerase or Large protein (L). Structural modelling of L protein suggested that it contained different conserved domains: i) RdRp for RNA transcription and replication, ii) capping domain and iii) methyltransferase domain (MTase). The MTase domain is suggested to mediate the transfer of methyl molecules on mRNA cap structure and or at the ribose 2’OH of RNA residues. The MTase domain is assumed to be acquired for: 1) mRNA translation into viral proteins and, 2) protection of viral RNA from detection by host cytoplasmic sensors: RIG like receptors (RLR) such as RIG-I and MDA-5. However, the RABV MTase domain role in pathogenicity and immune evasion is not known. The objective of this work is to characterize the MTase domain and identify the critical residues involved in this function. To this aim, mutations of this domain are introduced by site directed mutagenesis, and recombinant viruses were recovered and characterized. We therefore evaluated the ability of MTase mutants to modify viral RNAs. We used RiboMethSeq analysis to identify eventual internal 2’O-methylated residues in RABV mRNAs {Ringeard et al., 2018}. This project was already started and developed in collaboration with biomics platform by Rachel Legendre (n°B885).

Rabies virus (RABV) is a causative agent of lethal neurological disease. It presents a public health threat in the world resulting in more than 59,000 human deaths every year around the world. RABV possesses negative strand RNA genome, 11.9 kb, encoding five viral proteins: Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M), Glycoprotein (G) and polymerase or Large protein (L). Structural modelling of L protein suggested that L contained different conserved domains: i) RdRp for RNA transcription and replication, ii) capping domain and iii) methyltransferase domain (MTase) mediating the transfer of methyl molecule on mRNA cap structure. The detection of viral presence in a host cell is mediated by pattern recognition receptors (PRRs) that sense viral pathogen-associated molecular patterns (PAMPs). The cytosolic receptors include RIG-I-like receptors (RLRs). Three RLRs are known: RIG-I, MDA5 and LGP2. Upon ligand recognition, MDA5 and RIG-I activate signaling pathways leading to the production of pro-inflammatory cytokines, including type-I interferon (IFN), and the establishment of an antiviral state in the host. We have recently performed RLR siRNA knockdown experiments suggesting that upon RABV infection RIG-I is the major RLR implicated in RABV RNA sensing and IFN induction. However, the precise nucleic acid sequence of RABV RNA sensed by RLRs remains unknown. In order to determine RABV RNAs signatures recognized by RIG-I and MDA5, in collaboration with A. Komarova (IP), we have applied a novel ribonucleoproteomic approach that consists in affinity purification of tagged RLRs followed by extraction of associated RNA molecules. This approach has recently provided comparative views on viral RLR ligands in the context of infection with Measles, Chikungunya and Dengue viruses. We are now ready to perform NSG analysis of RIG-I- and MDA5- specific RNA ligands purified from RABV infected cells.

The RIG-I like receptors (RLRs) play a major role in sensing viral infection to initiate and modulate antiviral immunity. These are three RNA helicases entitled RIGI, MDA5 and LGP2 that interact with particular signatures of viral RNA molecules in cytoplasm of infected cell triggering a downstream signalling cascade resulting in the expression of type-I IFNs, proinflammatory cytokines and divers set of antiviral genes. We have recently generated stable cell lines expressing tagged RLRs. By applying affinity chromatography purification of RLR ribonucleoprotein complexes followed by RNA extraction, we observed that short-length RNAs were enriched on RLRs. We have previously sequenced small RNAs of RLRs (PF2 Pasteur) and now need to adjust our new bioinformatic analysis to these RNAseq data with the help of C3BI.

Because of the increasing biological data generated due to next-generation sequencing of the genetic material of organisms, storing and analyzing these data have become challenging both for molecular biologists and computer scientists. Here, we propose to design a system that attempts to solve this from building a secure repository up to designing algorithms that process sequences to produce biological insights, particularly mining for sequential patterns present in the sequences. Discovering sequence motifs/patterns has been essential to computational biologists as it is helpful in understanding protein function, structure, and evolution. Given the data generated from the project “A Study of Mosquitoes of Makiling Forest Reserve Areas with Characteristic Land Use and Survey of their Arbovirus Diversity through Vector-enabled Virome Sequencing” (funded by the Department of Science and Technology - Philippine Council of Health Research and Development; DOST-PCHRD) of Dr. Bautista, using the assembled contigs or even substantial portion of viruses’ full genome, we can take a look at the dynamics of genetics of viruses over time when analyzed alongside other publicly available sequences. This is a collaborative project between the University of the Philippines and Institut Pasteur in the context of this funded project that includes btoh partners. Our wish is for the Philippines bioinformatics engineer involved seeks guidance of the development their databse that may eventually be housed at Institut Pasteur.

The aim of this study is based on the analysis of the transcriptome during the infection with European bat lyssavirus type 1 (EBLV-1) in torpid bat. This study will be conducted using an in vitro model using another bat cell line from an European bat.

Hepatitis C virus (HCV) infection is a slow and asymptomatic progressive liver disease leading to metabolic disorders such as steatosis, an accumulation of lipid droplets (LDs) in hepatocytes. We have assessed the interplay between HCV variants recovered from chronically infected patients and LD content in infected hepatoma cells, through quantitative imaging approaches.

Modulation of infectious diseases transmission by climate conditions must be investigate in Sahel to prevent epidemics. Registration of data on flu transmission and on rainfalls+ temperature was conducted over the last ten years by the Flu national reference center in Niamey. Time series analysis must be conducted to investigate the correlation between the number of cases and climatic conditions.

Antibiotic resistance development is a public health liability and treatens treatment of bacterial infections. We are inetersed in genetic determinant of antibiotic resistance in Gram negative bacteria. We have identified V. cholerae mutants which are more tolerant to aminoglycosdies. Our aim is to identify cellular processes involved and characterize molecular mechanisms.

We propose to identify peptides derived from the S protein that can modulate the HLA-E restricted activity of NK cells. Through in-silico analyses, we have already identified in the S protein of SARS-Cov1, SARS-Cov2 and MERS-Cov regions coding for peptides with HLA-E binding motifs. We will perform functional and structural analyses of the binding of these peptides to MHC-E. We will study MHC-E restricted responses of NK cells in blood and tissues during SARS-Cov2 infection in non human primates. From longitudinally collected samples from blood and tissues before and after the SARS-CoV2 infection of the animals, we will analyse the phenotype and transcriptome profile of NK cells and compare to viral loads and disease progression of the animals.

Paleo(meta)genomics is an emerging and rapidly growing field where most is yet to be done. In most cases, it consists in the analysis of ancient DNA high-throughput sequencing data obtained from archaeological material or historical samples, and the goal is to retrieve and interpret the genomic information from species that from the past (microbial, eukaryotic, etc.) It combines tools borrowed from different fields, such as genomics, computational biology, microbial ecology, phylogenetics, population genetics, etc. However, at the moment there are no well-established tools for the analysis of this type of data. Hence, each lab must develop custom solutions, combining existing tools or developing new ones to meet the goals of their research programs. As a recently established lab, the microbial paleogenomics unit will spend the upcoming months setting up diverse pipelines and analysis tools for the different projects that will be developed in the coming years, many of which have been already used but need to be re-written in an understandable languaje and structure.

With an estimated 1031 particles on earth, bacteriophages are the most abundant genomic entities across all habitats and important drivers of microbial communities. Growing evidence suggest that they play roles in intestinal human microbiota homeostasis, and recent metagenomics studies on the viral fraction of this ecosystem have provided crucial information about their diversity and specificity. However, the bacterial hosts of this viral fraction, a necessary information to characterize further the balance of these ecosystems, remain poorly characterized. Here we unveil, using an enhanced metagenomic Hi-C approach, a large network of 6,651 host-phage relationships in the healthy human gut allowing to study in situ phage-host ratio. We notably found that half of these contigs appear to be sleeping prophages whereas ¼ exhibit a higher coverage than their associated MAG representing potentially active phages impacting the ecosystem. We also detect different candidate members of the crAss-like phage family as well as their bacterial hosts showing that these elusive phages infect different genus of Bacteroidetes. This work opens the door to single sample analysis and concomitant study of phages and bacteria in complex communities.

SARS-CoV-2 depends on the activation or inactivation of specific cellular pathways for efficient replication. The project screened small molecule compounds that affected epigenetic regulation of such pathway. The project aims to characterize these specific pathways, so identify specific steps important in the viral replication cycle and with this to describe the mode of action of these compounds.

HPV RNA-Seq, a novel and unique molecular test that allows detection of pre-cancerous lesions of the cervix from cervical samples.

Historically circulating in Africa, Rift Valley fever virus (RVFV) was firstly identified in Kenya in 1930, and responsible for dozens of outbreaks in Africa in the past decades. As an arbovirus, RVFV is mainly transmitted by mosquito vector, and needs to be altered between mammalian host and mosquito vector for maintaining the genome stability. In this project, we are analysing the mosquito vector competence for several RVFV variants obtained from different host cells, to better interpret the risk factors of transmission.

The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

We are working on the validation of protein interactions between SARS-CoV-2 proteins and human proteins that have been identified by Bio-ID. Our project is the validation of those interactions using our NanoLuciferase two-hybrid system.

Le projet a pour objectif de développer une page web pour présenter les travaux de modélisation de la pandémie de la COVID-19 au grand public.

Ndd expression in E.coli induces nuclear disruption where the nucleoid is swiftly relocated from a central position in the cell to a submembrane location. RNAseq analysis showed Ndd expression induces the differential expression of hundreds of E.coli genes. We would like to see if some of those genes are related to regulation networks of several nucleoid associated proteins.

Factors determining RABV tropism are largely unknown. Here, we are identifying the susceptibilities and influence of glial cells on RABV infection.

Using reverse genetics, we produced recombinant viruses within a prototypic HCV strain backbone, by substituting its core coding sequence by the equivalent sequences from from clinical isolates of different HCV genotypes. To verify that heterologous core sequences did not impair cross-talk with prototypic viral elements and could be used for comparative studies, we monitored the viral replication of our set of core intergenotypic viruses. For this, we quantified the amount of intracellular viral RNA, secreted genomes and infectious titers at different time points following infection of hepatoma cells with the recombinant viruses.

Describing viruses of invertebrate vectors of disease, provides better understanding of evolution and host range for various virus groups. In addition to filling the gaps in the current knowledge of the virosphere, viromes can serve as necessary contextual data for ongoing disease control measures and outbreak preparedness. This project aims at delivering a comprehensive analysis of RNA viromes from RNA sequencing data.

Advances in high-throughput sequencing may allow more rapid characterisation of viral diversity in samples. However, although there are many tools to perform taxonimuqye assignment of the reads obtained, there is no automated pipeline to obtain the different sequences by species and genus, and when the depth is sufficient to apply correction algorithms. In the end, obtaining these sequences will allow us to better understand the diversity and prevalence of each of the species highlighted in our samples.

Before the WHO considered it as a public health emergency of international concern in February 2016, Zika virus (ZIKV, Flavivirus, Flaviviridae) was a neglected mosquito-borne virus. First identified in Uganda in a sylvatic cycle, ZIKV has caused in few months millions cases, emerging in the five continents (Latin America, the Caribbean, Southeast Asia/Pacific Ocean, Africa/Indian Ocean, European countries (Portugal, Spain, France, Switzerland, the Netherlands)). We have initiated the most comprehensive study on vector competence with almost 50 mosquito populations belonging to five main species (Aedes aegypti, Aedes albopictus, Aedes japonicus, Culex pipiens, Culex quinquefasciatus) infected with 3 different ZIKV and examined at 3 days post-infection (7, 14, and 21). The objective of the project will be to run a meta analysis on vector competence and to assess to which extent each mosquito species contributes to ZIKV transmission according to the geographical location and the viral genotype. It will help to improve our understanding of the vector status and adapt surveillance, prevention, and control of Zika.

Vaccines against COVID-19 have been developed for use as homologous two-dose regimens and several of them have demonstrated efficacy. There is limited data on the clinical and the immune response induced by heterologous vaccination regimens (HVR) using alternate vaccine modalities. CORSER-4 is a Viro-Immunology cohort promoted by the Institut Pasteur that will follow-up subjects from the time they receive a prime vaccine dose or a boosting dose. We determined subjects sub-groups of interest defined by the type of vaccine immunization they receive: (i) Heterologous two-dose vaccine regimen, the less frequently used at the moment, (ii) Homologous two-dose vaccine regimen, the more frequently used; (iii) One-dose vaccine regimen and (iv) Infectious-prime vaccine-boost immunization in COVID-19-experienced patients. The subject will be enrolled on the planed last boost injection visit and 5 Follow-up visits will be further conducted (Month1, M3, M6, M12 and M24). We will collect longitudinal clinical data and biological samples. We will explore Viral and Immune Response in sera, nasopharyngeal secretions and in saliva samples. We will assess the immune response obtained by various vaccination regimen against the reference strains and the variants of concern, and the durability of the humoral and cellular response. In addition, we will document any occurring acute infection episode with a supplemental unscheduled visit in order to detect an emerging variant viral strain. Altogether, the CORSER-4 project constitutes an opportunity to evaluate early anti-SARS-CoV-2 immunity in heterologous (and others) vaccine regimens and to estimate their efficacy and their sustainable (M24) protection against circulating virus strains (reference and variants).

We are studying a specific genomic signature and would like to evaluate how frequent is this signature in phage genomes.

The objective of this project is to study the virome present in specific bat colonies in China and their associated ectoparasites, in order to identify potential (re) emerging zoonotic viruses and to elucidate their transmission route, especially as putative arboviruses.

The objective of this project is to study the virome present in noctule bats from East Europe, in order to identify potential (re) emerging zoonotic viruses.

Measles virus (MeV) impairs the mitochondrial network. This leads to the fusion of mitochondria and simultaneous release of mitochondrial DNA (mtDNA). We have shown that released mtDNA is recognized as a danger signal, capable of stimulating signaling pathways and inducing the production of pro-inflammatory cytokines. The expression of human cytidine deaminase APOBEC3A is highly upregulated by interferon responses. This enzyme catalyzes the deamination of cytidine to uridine in single-stranded mitochondrial DNA substrates, resulting in the catabolism of edited DNA. Deep sequencing analyses of cytosolic mtDNA mutations revealed an APOBEC3A signature predominantly in the 5’TpCpG context. Using cell lines deprived of mtDNA, we have demonstrated the implication of mtDNA in the production of interferon and APOBEC3A expression during viral infection. We have shown that MeV induces mitochondrial network fragmentation and confirmed the implication of RNA polymerase III/RIG-I signaling in the capture of cytosolic mtDNA.

The recent propagation of resistance mechanisms to insecticides constitutes a major challenge in the fight against the transmission of vector-borne diseases. Even though alternatives are being explored in mosquitoes, there are few studies focusing on the role that these molecules could have or play in the transmission of arboviruses. Using an in cellulo model, this project aims to determine the impact that an insecticide selection can have on an infection caused by the chikungunya virus.

Yellow fever (YF) is a fatal hemorrhagic disease caused by an arbovirus, Yellow Fever Virus (YFV) transmitted by the Aedes aegypti mosquito vector. YFV is currently endemic in Africa (5 strains) and South America (2 strains), and although an effective vaccine is available, YFV remains a major public health issue. Native from Africa, YFV was transported to South America and the Caribbean during the slave trade (15th to 19th century) and caused devastating outbreaks. After two centuries of outbreaks, YF is no longer reported in the Caribbean thanks to the successful mosquito control program implemented at the beginning of the 20th century. The virus disappeared from the Caribbean and Martinique experienced its last outbreak in 1908. However, the relaxation of anti-vectorial control programs contributed to the reintroduction of Ae. aegypti in most Caribbean islands. We evaluated the vector competence to YFV of different populations of Ae. aegypti from Martinique and other mosquito populations of the Caribbean. Our results showed that Ae. aegypti from Martinique were competent to African as well as to American YFV genotypes with however differences of viral dissemination and transmission. In this project, we aim at determining the diversity of viral populations at the crossing of two anatomical barriers in mosquitoes: midgut and salivary glands. The virus should enter into the midgut epithelial cells, replicate and be released into the hemocel. The final step will be the infection of the salivary glands from where the virus is excreted with the saliva. At each step, a selection of viral populations operates with only a fraction of the viral population transmitted between different tissues within a same host. The virus diversification promoted by virus replication in mosquitoes depends on the mosquito species. Such vector-specific conditions may have great impacts on emergence processes.

Several arboviruses have emerged and/or reemerged in the New World in the past decades. While yellow fever and dengue are historical diseases which continue to cause deadly epidemics, Zika and chikungunya have recently invaded the South American continent, causing great concern. In Colombia, Aedes aegypti is the vector of most of human arboviruses. We collected Ae. aegypti eggs in Medellin in Colombia in 2020 and infected adults with dengue (DENV), chikungunya (CHIKV), yellow fever (YFV) and Zika virus (ZIKV). We show that Ae. aegypti Nor Oriental was more prone to become infected, to disseminate and transmit CHIKV and ZIKV than DENV and YFV. In this project, we aim at determining the diversity of viral populations at the crossing of two anatomical barriers in mosquitoes: midgut and salivary glands.

Since the advent of 3rd generation sequencing technologies, the application of long-read sequencing has had many applications in the study of infectious diseases. There is one area where these technological improvements can have a considerable impact on a better understanding of the disease, namely in virus-induced cancers. Indeed, in most cases, the viral genome integrates into the host genome and during this process may also induce alterations in the host genome at the site of integration (deletion, gene duplication, etc.). These modifications are difficult to observe with short read sequencing. However, the analysis of these long read data requires new tools to investigate them. Indeed, it is necessary to be able to identify chimeric long reads that contain both the viral and human genomes. But, in a second step, we must be able to precisely characterise the modifications that have been made in the viral genome (viral genomic region deleted, duplicated, etc.) as well as in the human genome (location, deletion or not at the insertion point, etc.). An additional difficulty, but also an advantage of long read sequencing, is that it should be easier to detect rarer and less frequent integration events. Although these could also be detected with previous technologies with short reads, it was more difficult to conclude due to the low number of reads that were detected in these situations. Therefore, it will be important to take into account when mapping these integration events that there will not necessarily be a single event, but several with varying frequencies. This investigation pipeline can be used to study several viruses whose genome integrates: the infectious team anaemia virus, the foamy retrovirus and the papillomaviruses.

FlavImmunity is an international project aiming to study the immune response developed after yellow fever vaccination.

According to the WHO, the Hepatitis B virus (HBV) infects chronically 295 million persons. After several decades, persistently infected patients become at risk to develop liver cirrhosis and/or liver cancer (hepatocellular carcinoma, HCC). Each year around one million persons die from these two complications directly triggered by HBV. In the course of infection, the HBV DNA genome accumulates mutations that become progressively dominant. Some of the recurrent mutations are suspected to play a significant role in the pathophysiology of liver disease by changing the biological activity of HBV-encoded genes or proteins. However, the virus is rarely acting alone to promote terminal liver diseases, and its deleterious activity is generally potentialized by other risk factors concomitant to viral presence. It is the case of alcohol or tobacco consumption, metabolism-associated fatty liver disease, herbal medicine containing aristolochic acid, or mycotoxin-contaminated food. Most of these agents are known to leave mutation fingerprints on the human genome. However, we do not currently know whether these mutagens contribute or not to the mutational burden observed on HBV DNA after decades of infection. We hypothesize that it could be the case. The characterization of the full mutation spectrum of the HBV genome including the application of tools initially proposed to analyze human mutations is the aim of this research program.

Persistent viral infections in mosquitoes require equilibrium between viral replication and efficient antiviral defenses. Arboviruses are able to counteract immune defenses and especially, the RNAi system. Production of DNA forms from several non-retroviral RNA viruses (vDNA) has been found to promote persistent infections and prevent lethal acute infections in mosquitoes and cell cultures. In vitro, vDNA was detected at the early stage of infection (24 hours post-infection for dengue virus). vDNA was suggested to be produced from defective viral genomes (DVGs) rather than from a full genome. NIRVs are non-retroviral integrated RNA virus sequences (NIRVS) present in host genomes. Most of the NIRVS found in Aedes mosquitoes and cell lines were from insect specific viruses (ISVs) belonging to Rhabdoviridae and Flaviviridae families. Among NIRVS originating from flaviviruses, most of them displayed similarities to sequences encoding non-structural proteins. These NIRVS are more often embedded in regions rich in retrotransposons and piRNA clusters. It is likely that according to their position in the mosquito genome, NIRVS functions (if any) would be different. We aim to characterize NIRVS production in in vitro models by establishing persistent infections. We selected five different viruses: two arboviruses causing important public health issues (chikungunya and dengue), two ISFs displaying high prevalence in Aedes mosquitoes (CFAV and KRV) and a rhabdovirus (vesicular stomatitis virus (VSV) which is also an arbovirus). Infections were performed in two different cell lines that are both RNAi-proficient and isolated from embryonic tissues: Ae. aegypti Aag2 and Ae. albopictus U4.4 cell lines. This experiment will help to characterize regions of the mosquito genome that are suitable for viral integration, but also parts of the viral genome that are more prone to integrate.

Voluntary organizations provide essential support to vulnerable populations and front-line health responders to the COVID-19 pandemic. The French Red Cross (FRC) is prominent among organizations offering health and support services in the current crisis. Comprised primarily of lay volunteers and some trained health workers, FRC volunteers in the Paris (France) region have faced challenges in adapting to pandemic conditions, working with sick and vulnerable populations, managing limited resources, and coping with high demand for their services. Existing studies focus on individual, social and organizational determinants of motivation, but attend less to contextual ones. Public health incertitude about the COVID-19 pandemic is an important feature of this pandemic. Whether and how uncertainty interacts with volunteer understandings and experiences of their work and organizational relations to contribute to Red Cross workonline social listening and surveys.

The chikungunya virus (CHIKV) is an emerging mosquito-borne virus which has widely spread around the world in the last two decades. The virus is transmitted between human hosts by Aedes mosquitoes, including Aedes albopictus which is now established in more than 51 departments in France. The transmissibility of CHIKV can be affected by a combination of both intrinsic and extrinsic factors. Since mosquitoes are poikilothermic insects, environmental temperature is of particular importance. Indeed, temperature can shape transmission of pathogens through direct effects on viral replication and/or indirect effects via the vector immune responses. The relation between temperature and virus transmission is very complex and remains poorly defined. The aim of the project is to better understand how temperature affects mosquito-virus interactions. To meet this objective, our studies will focus on (i) how CHIKV genetically evolves under low temperature pressure (in vitro and in vivo), (ii) mosquito molecular responses to a low temperature exposure, (iii) and ultimately, how mosquito microbiota is affected by a low temperature. Our study will help in identifying the determinants that facilitate CHIKV transmission by Ae. albopictus in temperate conditions.

Because of the increasing biological data generated due to next-generation sequencing of the genetic material of organisms, storing and analyzing these data have become challenging both for molecular biologists and computer scientists. Here, we propose to design a system that attempts to solve this from building a secure repository up to designing algorithms that process sequences to produce biological insights, particularly mining for sequential patterns present in the sequences. Discovering sequence motifs/patterns has been essential to computational biologists as it is helpful in understanding protein function, structure, and evolution. Given the data generated from the project “A Study of Mosquitoes of Makiling Forest Reserve Areas with Characteristic Land Use and Survey of their Arbovirus Diversity through Vector-enabled Virome Sequencing” (funded by the Department of Science and Technology - Philippine Council of Health Research and Development; DOST-PCHRD) of Dr. Bautista, using the assembled contigs or even substantial portion of viruses’ full genome, we can take a look at the dynamics of genetics of viruses over time when analyzed alongside other publicly available sequences. This is a collaborative project between the University of the Philippines and Institut Pasteur in the context of this funded project that includes btoh partners. Our wish is for the Philippines bioinformatics engineer involved seeks guidance of the development their databse that may eventually be housed at Institut Pasteur.

Several arboviruses have emerged and/or reemerged in the New World in the past decades. While yellow fever and dengue are historical diseases which continue to cause deadly epidemics, Zika and chikungunya have recently invaded the South American continent, causing great concern. In Colombia, Aedes aegypti is the vector of most of human arboviruses. We collected Ae. aegypti eggs in Medellin in Colombia in 2020 and infected adults with dengue (DENV), chikungunya (CHIKV), yellow fever (YFV) and Zika virus (ZIKV). We show that Ae. aegypti Nor Oriental was more prone to become infected, to disseminate and transmit CHIKV and ZIKV than DENV and YFV. In this project, we aim at determining the diversity of viral populations at the crossing of two anatomical barriers in mosquitoes: midgut and salivary glands.

As a result of combined climate change and globalization (increased flow of travelers and goods), the distribution of the mosquito Aedes albopictus is expanding significantly outside tropical regions. Ae. albopictus has already established stable colonies in 20 European countries (https://ecdc.europa.eu/en/publications-data/aedes-albopictus-current-known-distribution-june-2018). Being the main mosquito that can be incriminated in the transmission of chikungunya, dengue, and zika viruses in Europe, Ae. albopictus were responsible for the first autochthonous cases in France and Italy. Accordingly, the European countries that are infested with Ae. albopictus are under the risk of arboviral diseases outbreak. The vector competence analysis of European Ae. albopictus is pivotal for evaluating the potential risk of diseases transmission. In this study, we analyzed the viral susceptibility of several European Ae. albopictus populations from Croatia, Greece, Montenegro, Italy, Switzerland, and also China (as a control), for chikungunya, dengue, and zika viruses. The results indicated that the European Ae. albopictus were able to transmit chikungunya and dengue viruses whereas they were not an efficient vector for zika virus.

The recent propagation of resistance mechanisms to insecticides constitutes a major challenge in the fight against the transmission of vector-borne diseases. Even though alternatives are being explored in mosquitoes, there are few studies focusing on the role that these molecules could have or play in the transmission of arboviruses. Using an in cellulo model, this project aims to determine the impact that an insecticide selection can have on an infection caused by the chikungunya virus.

Persistent viral infections in mosquitoes require equilibrium between viral replication and efficient antiviral defenses. Arboviruses are able to counteract immune defenses and especially, the RNAi system. Production of DNA forms from several non-retroviral RNA viruses (vDNA) has been found to promote persistent infections and prevent lethal acute infections in mosquitoes and cell cultures. In vitro, vDNA was detected at the early stage of infection (24 hours post-infection for dengue virus). vDNA was suggested to be produced from defective viral genomes (DVGs) rather than from a full genome. NIRVs are non-retroviral integrated RNA virus sequences (NIRVS) present in host genomes. Most of the NIRVS found in Aedes mosquitoes and cell lines were from insect specific viruses (ISVs) belonging to Rhabdoviridae and Flaviviridae families. Among NIRVS originating from flaviviruses, most of them displayed similarities to sequences encoding non-structural proteins. These NIRVS are more often embedded in regions rich in retrotransposons and piRNA clusters. It is likely that according to their position in the mosquito genome, NIRVS functions (if any) would be different. We aim to characterize NIRVS production in in vitro models by establishing persistent infections. We selected five different viruses: two arboviruses causing important public health issues (chikungunya and dengue), two ISFs displaying high prevalence in Aedes mosquitoes (CFAV and KRV) and a rhabdovirus (vesicular stomatitis virus (VSV) which is also an arbovirus). Infections were performed in two different cell lines that are both RNAi-proficient and isolated from embryonic tissues: Ae. aegypti Aag2 and Ae. albopictus U4.4 cell lines. This experiment will help to characterize regions of the mosquito genome that are suitable for viral integration, but also parts of the viral genome that are more prone to integrate.

Dengue is an arthropod-borne viral disease caused by dengue virus (DENV), which is transmitted by Aedes mosquitoes. Recent studies estimate that 100 million cases occur annually worldwide, making dengue the most prevalent mosquito-borne viral disease of human beings. DENV transmission results from interactions between humans, mosquitoes, viruses and their environment. If the human immune response against DENV is well studied, there are knowledge gaps in understanding how mosquitoes and DENV interact with each other. Moreover, it was shown that virus transmission does not only depend on the viral strain and mosquito population but also on the specific interaction between the two partners, named genotype-by-genotype (GxG) interaction. One aim of the team is to highlight factors at the mosquito and virus levels that regulate vector competence, i.e. the ability for a mosquito to acquire viral infection and subsequently transmit the virus to a new host. To this aim, we use field-derived Aedes aegypti populations that we challenge in the lab with virus isolates from DENV infected patients. This allows us to refine our understanding of the basic biology of virus transmission by mosquitoes.

Dengue prevention relies primarily on controlling populations of the main mosquito vector, Aedes aegypti, which is failing in many parts of the world because of the lack of sustained commitment of resources and ineffective implementation. Novel entomological approaches to dengue control are being developed that aim at replacing or suppressing mosquito vector populations. Insufficient genomic resources for Ae. aegypti, however, have until now impeded progress in both basic and applied research on this medically important mosquito species. The only available reference genome for Ae. aegypti is a draft that consists of over 4,800 unassembled fragments with incomplete annotation. Moreover, the inbred Ae. aegypti laboratory strain that was sequenced does not universally represent the considerable genetic and ecological diversity of the species worldwide. The large size of the genome and its high content in repeat-rich sequences of transposable elements was a major difficulty to assemble the Ae. aegypti genome sequence. In the present project, we aim to overcome this difficulty using a novel strategy for genome sequencing and assembly. The ultimate goal is to produce several, fully assembled, well-annotated, new Ae. aegypti reference genomes from epidemiologically relevant populations. The expected outcome is a genome reference panel including a catalog of species-wide genetic variation that will significantly improve genomic resources for Ae. aegypti research and help address a broad range of biological questions related to Ae. aegypti vectorial capacity and dengue virus transmission.

We would like to uncover associations between transcriptomic features and dengue infection outcome. In order to do so, we want to take advantage not only of our data but also of all publicly available data. A main challenge is to translate the measurements across different transcriptomic technologies into a summary expression level per gene. For this C3BI project we would like to concentrate on the problem of mapping probe IDs into a common identifier for all experiments.

As a result of combined climate change and globalization (increased flow of travelers and goods), the distribution of the mosquito Aedes albopictus is expanding significantly outside tropical regions. Ae. albopictus has already established stable colonies in 20 European countries (https://ecdc.europa.eu/en/publications-data/aedes-albopictus-current-known-distribution-june-2018). Being the main mosquito that can be incriminated in the transmission of chikungunya, dengue, and zika viruses in Europe, Ae. albopictus were responsible for the first autochthonous cases in France and Italy. Accordingly, the European countries that are infested with Ae. albopictus are under the risk of arboviral diseases outbreak. The vector competence analysis of European Ae. albopictus is pivotal for evaluating the potential risk of diseases transmission. In this study, we analyzed the viral susceptibility of several European Ae. albopictus populations from Croatia, Greece, Montenegro, Italy, Switzerland, and also China (as a control), for chikungunya, dengue, and zika viruses. The results indicated that the European Ae. albopictus were able to transmit chikungunya and dengue viruses whereas they were not an efficient vector for zika virus.

Persistent viral infections in mosquitoes require equilibrium between viral replication and efficient antiviral defenses. Arboviruses are able to counteract immune defenses and especially, the RNAi system. Production of DNA forms from several non-retroviral RNA viruses (vDNA) has been found to promote persistent infections and prevent lethal acute infections in mosquitoes and cell cultures. In vitro, vDNA was detected at the early stage of infection (24 hours post-infection for dengue virus). vDNA was suggested to be produced from defective viral genomes (DVGs) rather than from a full genome. NIRVs are non-retroviral integrated RNA virus sequences (NIRVS) present in host genomes. Most of the NIRVS found in Aedes mosquitoes and cell lines were from insect specific viruses (ISVs) belonging to Rhabdoviridae and Flaviviridae families. Among NIRVS originating from flaviviruses, most of them displayed similarities to sequences encoding non-structural proteins. These NIRVS are more often embedded in regions rich in retrotransposons and piRNA clusters. It is likely that according to their position in the mosquito genome, NIRVS functions (if any) would be different. We aim to characterize NIRVS production in in vitro models by establishing persistent infections. We selected five different viruses: two arboviruses causing important public health issues (chikungunya and dengue), two ISFs displaying high prevalence in Aedes mosquitoes (CFAV and KRV) and a rhabdovirus (vesicular stomatitis virus (VSV) which is also an arbovirus). Infections were performed in two different cell lines that are both RNAi-proficient and isolated from embryonic tissues: Ae. aegypti Aag2 and Ae. albopictus U4.4 cell lines. This experiment will help to characterize regions of the mosquito genome that are suitable for viral integration, but also parts of the viral genome that are more prone to integrate.

The 2013-2015 Ebola virus disease epidemic is the largest outbreak so far described with 27 305 cases and 11 169 deaths. The virus spread by human to human contact throughout Western Africa and never before has a variant been transmitted for such a sustained period of time. Ebola virus are RNA virus so as other RNA viruses they could accumulate mutations during evolution. Therefore it is an emergency to monitor viral changes and adaptation within and between individuals in order to help researchers to better understand susceptibility to Ebola infections, to guide research on therapeutic targets and to ensure accurate diagnosis. New technologies can provide information about pathogen’s evolution and in our lab we have access to an Ion PGMTM sequencer. Thanks to the national reference center for viral hemorrhagic fever (VHF) we have at our disposal a large number of samples collected from Ebola infected patients especially from Guinea. We have developed an Amplicon approach using sixteen couples of specific primers for Ebola viruses and a RNA sequencing method based on randomly primed cDNA synthesis to product our libraries. Ion PGMTM Hi-Q sequencing kit will be used to sequence up to 400 bp inserts loaded onto 316v2TM or 318v2TM chip. Through high depth sequencing we would like to follow up the profiling of intra and inter host viral quasispecies at different time of the epidemic in the geographic area of the Ebola Treatment Centre in Macenta. Thanks to the activities of national reference center for VHF and the Biomics Pole one aim of the project is also to occasionally compare viral quasispecies and consensus sequences between patients who get uncommon symptoms from those who get classical illness and to study intra host quasispecies in different biological fluids (cerebrospinal fluid, sperm, urine) to see if there are differences between persistent species and viral quasispecies found during symptomatic step.

Blood cells were collected in the field from patients admited in the Ebola treatment center of Macenta (Guinea). After red cells lysis, blood cells were frozen and RNA was extracted after the shipment of samples in France. Despite poor RNA quality, Affimetrix chips have been performed and transcriptomic data are available. The aim of this study is to compare the transcriptomic data from patients who survived and those who died and also to perform a kinetic analysis of the infection in the two groups of patients.  Samples from febrile patients who were not infected with Ebola virus have been used as negative controls.

Samples were collected in the field from infected patients admited in Ebola treatment Center of Macenta (ETC Guinea). Deep sequencing was performed in order to look for majority and minority variants. The aim of this study is to compare quasispecies of patients who survived and those who died and also to get detailed description of EBOV evolution in an ETC to identify phylogenetic clusters or transmission chain.

Poliomyelitis has been a major public health concern and currently, efforts are being made towards eradicating wild poliovirus type 2 (WPV2). A global switch from trivalent oral poliovirus vaccine (tOPV) to bivalent oral poliovirus vaccine (bOPV without PV2) is planned to take place this year in countries, like Madagascar, where epidemics of type 2 pathogenic circulating vaccine-derived polioviruses (cVDPVs) occur when low polio vaccine coverage allows the circulation and genetic drift of vaccine attenuated PV2 that can thus become pathogenic. In an attempt to monitor the presence and eventually absence of wild poliovirus 2, environmental and human surveillance will be conducted before, during, and after the switch in two regions in Madagascar. PV2, in particular cVDPVs and other enteroviruses will be genetically characterized. These data will support monitoring efforts and confirm the elimination of type 2 PV strains that have been implicated in outbreaks in this country.

Notre objectif est d'étudier comment la recombinaison génétique génère de la diversité au sein des écosystèmes d'entérovirus.

Enteroviruses are known to recombine a lot during the replication cycle. A lot of viruses produce defective genomes, which are generated by the polymerase skipping several nucleotides and thus, forming internal deletions of the genome. We would like to look at this process more closely and come up with a model, which includes the role of the RNA virus genome itself, including its structure.

We passaged EV71 in cell culture, followed by RNAseq analysis. The sequences were analysed for the presence of internal deletions within the viral genome. Such deletions can contribute to non-homologous and homologous recombination within Enteroviruses, which can have positive effects, such as the purging of deleterious mutations or to overcome host restrictions. On the other hand such deletions could result in the formation of defective interfering particles (DIs), which have the ability to interfere with wildtype virus infections and thus, reduce viral loads in ongoing infections. We aim to get a better understanding of these events during viral replication that can aid us to identify DIs and can help us explore these special forms of virus genome deletions as potential therapeutic agents.

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by a biallelic mutation of the Ataxia Telangiectasia Mutated (ATM) gene. The ATM protein is involved in a very large number of cellular functions (over 700 substrates), mainly DNA repair, cell cycle regulation, mitochondrial and oxidative metabolism, as well as regulation of gene expression. The majority of hematological malignancies occurring in AT patients are non-Hodgkin's B lymphomas and Hodgkin's lymphomas. The association with the Epstein-Barr virus (EBV) is found in 50 to 100% of these cases according to the histological subtype. EBV is a widespread virus in human populations with a prevalence greater than 90% and is associated with the development of lymphomas in immunocompromised patients. We will study the transcriptome of EBV-infected cells in ATM patients, in order to determine the impact of ATM on EBV infection control.

Despite effective prevention against HBV infection, 300 million people worldwide are chronic HBV carriers, of whom 25% will die of liver cirrhosis or hepatocellular carcinoma (HCC). Current treatments for chronic hepatitis B (CHB) are inefficient to completely clear the virus and liver cancer is a lethal disease, thus representing an area of highly unmet medical need. Viral persistence is due to the maintenance, in the nuclei of infected cells, of the viral nuclear DNA : the cccDNA that is not targeted by the antiviral treatment and to the impairment of both the innate and adaptive immune responses that accompanies CHB infection. Viral replication depends on a balance between factors that benefit and those that restrict viral infection. However little is known about cellular factors that repress HBV replication. Using quantitative temporal viromics approach developed by P. Lenher in Cambridge we have performed a quantitative analysis of temporal changes in host and viral proteins in primary human hepatocytes through the course of HBV infection. We will in particular search for cellular factors involved in virus restriction and uncover the mechanism of viral escape.

According to the WHO, the Hepatitis B virus (HBV) infects chronically 295 million persons. After several decades, persistently infected patients become at risk to develop liver cirrhosis and/or liver cancer (hepatocellular carcinoma, HCC). Each year around one million persons die from these two complications directly triggered by HBV. In the course of infection, the HBV DNA genome accumulates mutations that become progressively dominant. Some of the recurrent mutations are suspected to play a significant role in the pathophysiology of liver disease by changing the biological activity of HBV-encoded genes or proteins. However, the virus is rarely acting alone to promote terminal liver diseases, and its deleterious activity is generally potentialized by other risk factors concomitant to viral presence. It is the case of alcohol or tobacco consumption, metabolism-associated fatty liver disease, herbal medicine containing aristolochic acid, or mycotoxin-contaminated food. Most of these agents are known to leave mutation fingerprints on the human genome. However, we do not currently know whether these mutagens contribute or not to the mutational burden observed on HBV DNA after decades of infection. We hypothesize that it could be the case. The characterization of the full mutation spectrum of the HBV genome including the application of tools initially proposed to analyze human mutations is the aim of this research program.

Hepatitis C virus (HCV) infection is a slow and asymptomatic progressive liver disease leading to metabolic disorders such as steatosis, an accumulation of lipid droplets (LDs) in hepatocytes. We have assessed the interplay between HCV variants recovered from chronically infected patients and LD content in infected hepatoma cells, through quantitative imaging approaches.

Using reverse genetics, we produced recombinant viruses within a prototypic HCV strain backbone, by substituting its core coding sequence by the equivalent sequences from from clinical isolates of different HCV genotypes. To verify that heterologous core sequences did not impair cross-talk with prototypic viral elements and could be used for comparative studies, we monitored the viral replication of our set of core intergenotypic viruses. For this, we quantified the amount of intracellular viral RNA, secreted genomes and infectious titers at different time points following infection of hepatoma cells with the recombinant viruses.

HIV-1 replication requires the integration of the viral genome into the cell genome. A viral-encoded enzyme, integrase (IN), performs this critical step of infection and is a promising target for anti-viral therapeutics. If the catalytic properties of INs are well characterized, the mechanisms responsible for their site selectivity are still under investigation. Several cellular proteins, such as the LEDFGF/p75 transcription regulator, the RNA polymerase II machinery, nuclear pore proteins and specific modified histones have been proposed to be involved in IN selectivity at a genomic level. In addition, structural parameters of the target DNA helix (curvature, flexibility and topology) are proposed to regulate IN selectivity at a local level. Our team is studying the role and molecular mechanisms associated with these various parameters (Botbol et al., 2008; Lesbats et al., 2011; Morchikh et al., 2013; Benleulmi et al., 2015; Naughtin et al.,). This project aims to define the role of cellular DNA topology during HIV-1 integration. We will first compare already mapped integration sites and superhelicity profiles and search for possible correlations between these two parameters. We will then modify topoisomerases activity in infected cells and study the consequences on viral replication and integration. Finally, we will study in vitro, the direct effects on integration of two parameters of DNA topology, the twist and writhe of the DNA helix. This project relies on complementary in vivo, in vitro and in silico approaches. Bio-informatics tools are crucial for the correlative and statistical analyses of integration sites and superhelicity maps.

HIV-1 replication requires the integration of the viral genome into the cell genome. A viral-encoded enzyme, integrase (IN), performs this critical step of infection and is a promising target for anti-viral therapeutics. If the catalytic properties of INs are well characterized, the mechanisms responsible for their site selectivity are still under investigation. Several cellular proteins, such as the LEDFGF/p75 transcription co-activator, the RNA polymerase II machinery, nuclear pore proteins and specific modified histones have been proposed to be involved in IN selectivity at a genomic level but the underlying molecular mechanisms remain to be demonstrated. In addition, structural parameters of the target DNA helix (curvature, flexibility, topology) are proposed to regulate IN selectivity at a local level. Our aims are to study the role of these different parameters of IN selectivity, using both in vitro and in vivo approaches. In vitro, we will map integration sites on various target DNA substrates (naked DNA or chromatin, minicircles, plasmids with different topologies, transcribed templates) and will test the effect of purified proteins suspected to regulate IN selectivity. In vivo, integration sites will be mapped in cells depleted of these suspected regulators or in cells incubated with drugs targeting enzymes involved in transcription, DNA topology or histone modifications. Integration sites will be mapped using published or “home-made” protocols and the sites will be compared with DNA structural parameters, nucleosome positions, histone modifications or transcriptional parameters (published maps). Bio-informatics tools are crucial for these correlative and statistical analyses of integration sites. Our project relies on complementary in vivo, in vitro and in silico approaches. It should establish molecular and mechanistic rules of HIV-1 integration selectivity that could serve in the development of new antiviral strategies and of safer gene therapy vectors.

Controlling virus replication by generating a strong CD8+ T cell response against HIV is one of the major goals in the development of an effective HIV vaccine candidate. Indeed, during the chronic phase of infection, HIV-specific CD8+ T cells were shown to have impaired functionality and fail to control viral replication. Understanding the profile of CD8+ T cells able to efficiently tackle HIV is therefore much needed. HIV controllers (HIC) are individuals who control viremia without antiretroviral therapy. CD8+ T cells seems to play a major role in their HIV control. We hypothesized that HIV-specific CD8+ T cells associated with control of infection bear particular transcriptional signature (cell cycle, survival, activation, cytolytic function…) when compared to HIV-specific CD8+ T cells not associated with control of infection sharing the same memory phenotype.

The objective of this study is to evaluate how the different programming of distinct CD4+ T cell subsets affected their susceptibility to HIV infection and the survival of infected cells. Dr. Saez-Cirion's Team evaluates how the metabolic state of CD4⁺ T cells and the regulation of cellular factors shape the distribution of the HIV reservoir in distinct CD4⁺ T cell subset. This project uses new technologies and reagents to analyze cells from uninfected donors, and from HIV patients in acute infection, chronic infection and HIV remission.

Les mutations de résistance aux traitements apparaissent sous l’effet de la sélection. Ces mutations sont transmises, les patients pouvant être infectés par des souches déjà résistantes à certains traitements. Ces mutations posent des problèmes considérables en limitant l’arsenal des traitements disponibles, pour les individus comme pour la population sur le long cours. Dans le cas du VIH, nous avons travaillé sur la transmission de ces mutations au sein de la population anglaise, et montré que la majorité de celles-ci étaient transmises par des patients naïfs ignorant leur infection (Mourad et al. AIDS 2015). La méthode employée était souvent ad-hoc et n’utilisait qu’une partie des données disponibles. L’objectif de ce projet est de mettre en place des modèles rigoureux et des méthodes efficaces d’estimation des paramètres (taux de transmission suivant les caractéristiques du donneur, taux de réversion, etc.).

We would like to be able to use IgBlast on the Galaxy platform. We are studying B cells in adaptive immune response, and are particularly interested in the antibodies termed as broadly neutralizing antibodies (bNAbs). By definition, these antibodies can neutralize most known HIV-1 strains, and are produced by rare infected individuals several years post-infection. We are currently investigating the bNabs immunoglobulin repertoire by focusing our NGS (454 pyrosequencing) analysis on  immunoglobulin sequences (V-domains) from HIV-infected patients who developed bNAbs. As immunoglobulin sequences result from the combinatorial rearrangement of 3 gene segments : V , (D) and J gene segments, we need a specific tool to analyze these sequences. Indentifying the germline genes which are involved in the rearrangment is an essential step. Two main tools are being widely used to analyze Immunoglobulins: IMGT and IgBlast. IgBlast has several advantages; it is based on BLAST (it is then possible for the user to build his own database), open source, can use protein or nucleotide sequences as input, and most of all, IgBlast is already installed on the Institut Pasteur's cluster as well as the germline genes database. As it would be very convenient for us to use the bic cluster and galaxy platform to run our analyzes, we would be grateful if IgBlast could be implemented in the Pasteur Galaxy Platform. In this regard, we are of course fully disposed to help in any ways. We also believe that it would be very useful to people working on immunoglobulin sequences in the immunology department by building specific pipelines. Thank you very much.

Integration of the viral reverse-transcribed genome into the genome of infected cells is an essential step of retroviral replication and is performed by a viral-encoded enzyme, named integrase (IN). In the case of HIV-1, IN is a new and efficient anti-viral target. The selectivity of this enzyme for its cellular genomic sites is also a major parameter of HIV replication and is regulated by several cellular parameters. One of them is chromatin, and different levels of this nucleoprotein complex are involved in the regulation of IN selectivity. Using in vitro integration assays, established by our team and collaborators, we have studied this regulation at two levels of chromatin architecture: large poly-nucleosome templates (Botbol et al., 2008; Lesbats et al., 2011; Benleulmi et al., 2015; Naughtin et al., 2015) or nucleosome-induced DNA curvature mimicked by DNA minicircles (Pasi et al., 2016). Our present project is to study IN selectivity into mononucleosomes (MN). These MNs will be used as target substrates of integration and the role of MN structure, histone modifications and IN cofactors will be studied. Results obtained in vitro, will be confronted to structural data obtained by molecular modeling and to integration sites observed in infected cells. This project will benefit from our expertise in integration in chromatin templates and a previous collaboration with the C3BI on the analysis of integration sites (Pasi, M., Mornico, D., S. Volant, S., et al., 2016). This project is funded by the ANRS.

We previously showed that humanized immune system (HIS) mice generated in Balb/c Rag2^-/-γ_c^-/- SIRP^NOD (BRGS) recipients are susceptible to HIV-1 infection (X4 and R5 isolates) and maintain circulating HIV-1 in the plasma, resulting in a dramatic depletion of human CD4⁺ T cells. We also characterized features of HIV physiopathology in this model. Human thymocyte subsets developing in the thymus of HIS mice appear phenotypically normal, but in the periphery the T cell repertoire is restricted compared with that of human peripheral blood T cells. This negatively impacts on the ability of HIS mice to generate antigen-specific human immune responses when mice are vaccinated with protein antigens or following infection with lymphotropic viruses such as HIV. One likely explanation for these functional deficiencies involves the fact that human T cells are selected intrathymically by mouse MHC molecules and that naïve T cells in peripheral lymphoid organs interact primarily with mouse DC (as human DC development in HIS mice is limited). As a first line of improvement, we recently generated a novel mouse model by crossing our BRGS mice with the HLA-A*02-HHD class I transgenic mice and the HLA-DRB1*15 class II transgenic mice, resulting in BRGS-A2DR2 mice. Following intra-hepatic injection of these mice with MHC-matched CD34⁺ stem cells we observed increased engraftment, with faster kinetics. Moreover BRGS-A2DR2 HIS mice have an increased T cell development leading to a more equilibrated B/T and CD4/CD8 phenotype. We showed that BRGS-A2DR2 HIS mice were able to sustain replication of HIV R5 virus as the BRGS hosts. Viremia was similar in a first phase and then lower in a second phase in BRGS-A2DR2 compared to BRGS HIS mice, which could be a consequence of a better quality of the immune response. However, the viremia reached a similar plateau in the last phase. We propose to study the impact of the immune res

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants underlying this response, we characterized the TCR repertoire directed at the most immunodominant CD4 epitope in HIV-1 capsid, Gag293. HIV Controllers from the ANRS CO21 CODEX cohort showed a highly skewed TCR repertoire characterized by a predominance of the TRAV24 and TRBV2 variable gene families. Controllers shared public clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control (Benati D. et al., J Clin Invest 2016).   We propose to test the generality of these findings by characterizing the TCRs specific for a series of immunodominant HIV Gag and Env epitopes, and comparing the frequencies of public clonotypes in groups of HIV Controllers and treated patients. We will then assay the functions of the most prevalent public clonotypes through lentivector-based TCR transfer, and correlate the panel of T cell functions to TCR affinity and frequency.

HIV infects and depletes CD4+ T cells, leading to a progressive loss of adaptive immune responses and ultimately to AIDS. In addition, HIV preferentially targets HIV-specific CD4+ T cells, resulting in an attrition of the very cells that should orchestrate the antiviral immune response. Due to this preferential depletion, HIV-specific CD4+ T cells are few and remain incompletely characterized. The emergence of single cell technologies opens the opportunity for an in depth analysis of the rare specific CD4+ T cells that persist in the circulation of chronically infected patients. We have sorted single CD4+ T cells specific for the most immunodominant epitope in HIV-1 capsid, using an optimized MHC class II tetramer labeling protocol. We now propose to analyze these single cells by multiplexed real time PCR in a microfluidics device, to define their transcriptional profile. We will analyze the differentiation status of HIV-specific CD4+ T cells in rare patients who naturally control HIV infection and in progressor patients who control HIV replication due to antiretroviral therapy. This project will help define the parameters of an efficient T cell response against HIV, and may provide insights into the type of responses that should be induced by candidate HIV vaccines.

Molecular markers of cervix lesions linked to HPV infections.

HPV RNA-Seq, a novel and unique molecular test that allows detection of pre-cancerous lesions of the cervix from cervical samples.

Analyses fonctionnel des protéines humains de type ubiquitin qui interagir avec les virales protéines de la grippe

L'Analyses phylogénétique par séquence protéique, temporal et géographique des échantillons grippales avant et après l’épidémie de 2009.

analyses et classification des échantillons grippales.

Collect statistics on variants found in different strains of influenza virus. Determine variant effects to the protein sequence.

A protein-protein interaction screen has been done beween viral proteins of influenza A viruses and a library of about 100 human factors invovled in RNA processing through RNA exonucleases activity. 20 factors were identified as interactors of viral proteins. To see if this targeting corresponds to specific RNA metabolism focntions of the cell, we analysed using Cytoscape the first neighbors of these factors in the human ORFeome. We then compared using the ClueGo pluging the functional enrichment of the factors targeted by viral factors and the functional enrichment of the non-interacting subset. We need now a statistical analysis of the results the functional enrichment comparison, which is required for further progress of the ongoing research project.

Modulation of infectious diseases transmission by climate conditions must be investigate in Sahel to prevent epidemics. Registration of data on flu transmission and on rainfalls+ temperature was conducted over the last ten years by the Flu national reference center in Niamey. Time series analysis must be conducted to investigate the correlation between the number of cases and climatic conditions.

Lassa virus (LASV) is an arenavirus causing hemorrhagic fever in human. 300 000 to 500 000 cases of LASV infection are reported every year in western Africa, including 5 000 to 6 000 deaths. LASV is highly pathogenic, and no vaccine or treatment is available in endemic areas. LASV pathogenesis mechanisms are not well documented, and further investigations are needed to understand viral and immunological factors involved during infection. As previously shown by studies conducted on patients and non-human primates infected by LASV, T-cell response and type I interferon (IFN-I) are important for an effective response to LASV infection. Among dendritic cells (DC), myeloid DC can induce T-cell activation and plasmacytoid DC are specialized in IFN-I response. DC are also the first target of LASV during the infection of a new host, and studies on in vitro differentiated DC suggest a role of DC in T-cell response to LASV infection. Therefore, plasmacytoid and myeloid DC could be important for an efficient response to LASV infection.

The aim of this project is to implement, at the level of the research units, a bioinformatics workflow dedicated to the analysis of the microbiome, and of the virome in particular, based on Next Generation Sequencing (NGS) data (Illumina technology). This workflow will be applied in different contexts, from the identification of nucleotide sequences belonging to a novel or emerging pathogen (mainly viruses) presente in a clinical sample (such as serum or cerebrospinal fluid), to the determination of the whole sequence genome of isolated and uncharacterized viruses (such as bacteriophages of Leptospira).  

Because of their increasing incidence, dramatic severity, lack of treatment or vaccine, complicated diagnosis, misreading of the pathogenesis, and need for a maximum containment, Viral Hemorrhagic Fevers (VHF) constitute a major public health problem. There is therefore an urgent need to further study VHF to understand the pathogenesis of the severe disease and the host responses involved in their control or in the dramatic damages. Among VHF, Lassa fever (LF) is probably the most worrying one because of its endemicity and the large number of cases. LF is caused by the Old-World arenavirus Lassa virus (LASV). It is endemic to West Africa and is responsible for 300,000 cases and 5,000 to 6,000 deaths each year. We propose here to study the pathogenesis of VHF by using LF in cynomolgus monkeys as a paradigm, with a particular emphasis on the very early events. The viral tropism, pathophysiological mechanisms, and immune responses will be studied during the course of infection, including the incubation period. Powerful approaches will be used to (1) identify early biological markers of infection, to be able to confirm infection and isolate patients; (2) determine the viral tropism and dynamics during the course of infection to understand the natural history of virus into its host. (3) characterize the early pathogenic events that lead to the severe hemorrhagic syndrome to fully understand the pathophysiogenesis of VHF and identify new therapeutic targets. (4) identify the immune responses involved in the control of infection or in the fatal outcome, to reveal the involvement of immunopathological mechanisms and help to design a vaccine approach. This ambitious and unprecedented project will allow to develop therapeutic and prophylactic approaches but also to identify early biological markers of infection and improve the early diagnosis to optimize the management of outbreaks in the field and increase the survival rate in patients.

We need an independent statistician to confirm that appropriate statistical tests were used to analyze the data in your manuscript for the final revision of our manuscript in Science Signaling (Manuscript Number: aar3993, \"LGP2 binds PACT to regulate RIG-I- and MDA5-mediated antiviral response\" ). Sincerely yours, Anastassia Komarova

The RIG-I like receptors (RLRs) play a major role in sensing viral infection to initiate and modulate antiviral immunity. These are three RNA helicases entitled RIGI, MDA5 and LGP2 that interact with particular signatures of viral RNA molecules in cytoplasm of infected cell triggering a downstream signalling cascade resulting in the expression of type-I IFNs, proinflammatory cytokines and divers set of antiviral genes. We have recently generated stable cell lines expressing tagged RLRs. By applying affinity chromatography purification of RLR ribonucleoprotein complexes followed by RNA extraction, we observed that short-length RNAs were enriched on RLRs. We have previously sequenced small RNAs of RLRs (PF2 Pasteur) and now need to adjust our new bioinformatic analysis to these RNAseq data with the help of C3BI.

Measles virus (MeV) impairs the mitochondrial network. This leads to the fusion of mitochondria and simultaneous release of mitochondrial DNA (mtDNA). We have shown that released mtDNA is recognized as a danger signal, capable of stimulating signaling pathways and inducing the production of pro-inflammatory cytokines. The expression of human cytidine deaminase APOBEC3A is highly upregulated by interferon responses. This enzyme catalyzes the deamination of cytidine to uridine in single-stranded mitochondrial DNA substrates, resulting in the catabolism of edited DNA. Deep sequencing analyses of cytosolic mtDNA mutations revealed an APOBEC3A signature predominantly in the 5’TpCpG context. Using cell lines deprived of mtDNA, we have demonstrated the implication of mtDNA in the production of interferon and APOBEC3A expression during viral infection. We have shown that MeV induces mitochondrial network fragmentation and confirmed the implication of RNA polymerase III/RIG-I signaling in the capture of cytosolic mtDNA.

Poliomyelitis has been a major public health concern and currently, efforts are being made towards eradicating wild poliovirus type 2 (WPV2). A global switch from trivalent oral poliovirus vaccine (tOPV) to bivalent oral poliovirus vaccine (bOPV without PV2) is planned to take place this year in countries, like Madagascar, where epidemics of type 2 pathogenic circulating vaccine-derived polioviruses (cVDPVs) occur when low polio vaccine coverage allows the circulation and genetic drift of vaccine attenuated PV2 that can thus become pathogenic. In an attempt to monitor the presence and eventually absence of wild poliovirus 2, environmental and human surveillance will be conducted before, during, and after the switch in two regions in Madagascar. PV2, in particular cVDPVs and other enteroviruses will be genetically characterized. These data will support monitoring efforts and confirm the elimination of type 2 PV strains that have been implicated in outbreaks in this country.

The transcriptome signature during rabies infection will be analyzed both in animal model (mouse) and in human, at the central nervous system level, to identify the main canonical pathways involved during the infection.

Viruses have evolved powerful countermeasures to evade host innate immunity, which produces immediate, but non-specific, immune response during infection. Among viruses possessing RNA genomes, the order of negative-single-strand viruses (Mononegavirales) encompasses many human and animal pathogens that cause severe disease, including measles virus, mumps virus and rabies virus. Rabies virus is known for its neurotropic retrograde progression from the site of transmission to brain parenchyma, and towards salivary glands, different organs linked through parasympathetic nervous system. In the cases of human infection, the exhibited symptoms such as hallucination, diplopia, hydrophobia, unsteadiness or paralysis all indicate that there is causality between rabies virus infection and dysfunction of neural activity. However, lack of pathological brain lesions observed at the point of autopsy or noncytolytic propagation devoid of apoptosis suggest that rabies virus possesses mechanisms to evade or delay immune responses and cell death at least for the duration of replication and transmission. Even though the details in molecular perspective of these discoveries are well laid out now, how these proteins work in coordination or if there are hidden components which connect them all together leading toward deterioration of neural cells on the benefit of virus is largely unclear. Moreover, considering the complexity of brain cell composition and how important the neighboring cells are to shape one neuron’s specialization and dependency onto others in homeostasis, which result in the astounding heterogeneity of gene expression, an integrated and holistic approach is mandatory to get a fully comprehensive view of the mechanisms involved. Consequently, we performed an RNASeq analysis in human interneuron cells derived from induced pluripotent stem cells and infected by two recombinant rabies viruses (Tha virus, isolated from a dog in Thailand and Th4M, a less pathogenic virus which is mutated on 4 different residues of the M gene; this virus can no longer escape the NF-KB pathway) in order to obtain transcriptome data by comparison with uninfected cells, and to have an overview of the temporal dynamics of the genes expression.

Rabies virus (RABV) is a causative agent of lethal neurological disease, a member of Lyssavirus genus, belonging to Rhabdoviridae family in the order Mononegavirales. It presents a public health threat in the world resulting in more than 59,000 human deaths every year around the world. RABV possesses negative strand RNA genome, 11.9 kb, encoding five viral proteins: Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M), Glycoprotein (G) and polymerase or Large protein (L). Structural modelling of L protein suggested that it contained different conserved domains: i) RdRp for RNA transcription and replication, ii) capping domain and iii) methyltransferase domain (MTase). The MTase domain is suggested to mediate the transfer of methyl molecules on mRNA cap structure and or at the ribose 2’OH of RNA residues. The MTase domain is assumed to be acquired for: 1) mRNA translation into viral proteins and, 2) protection of viral RNA from detection by host cytoplasmic sensors: RIG like receptors (RLR) such as RIG-I and MDA-5. However, the RABV MTase domain role in pathogenicity and immune evasion is not known. The objective of this work is to characterize the MTase domain and identify the critical residues involved in this function. To this aim, mutations of this domain are introduced by site directed mutagenesis, and recombinant viruses were recovered and characterized. We therefore evaluated the ability of MTase mutants to modify viral RNAs. We used RiboMethSeq analysis to identify eventual internal 2’O-methylated residues in RABV mRNAs {Ringeard et al., 2018}. This project was already started and developed in collaboration with biomics platform by Rachel Legendre (n°B885).

Rabies virus (RABV) is a causative agent of lethal neurological disease. It presents a public health threat in the world resulting in more than 59,000 human deaths every year around the world. RABV possesses negative strand RNA genome, 11.9 kb, encoding five viral proteins: Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M), Glycoprotein (G) and polymerase or Large protein (L). Structural modelling of L protein suggested that L contained different conserved domains: i) RdRp for RNA transcription and replication, ii) capping domain and iii) methyltransferase domain (MTase) mediating the transfer of methyl molecule on mRNA cap structure. The detection of viral presence in a host cell is mediated by pattern recognition receptors (PRRs) that sense viral pathogen-associated molecular patterns (PAMPs). The cytosolic receptors include RIG-I-like receptors (RLRs). Three RLRs are known: RIG-I, MDA5 and LGP2. Upon ligand recognition, MDA5 and RIG-I activate signaling pathways leading to the production of pro-inflammatory cytokines, including type-I interferon (IFN), and the establishment of an antiviral state in the host. We have recently performed RLR siRNA knockdown experiments suggesting that upon RABV infection RIG-I is the major RLR implicated in RABV RNA sensing and IFN induction. However, the precise nucleic acid sequence of RABV RNA sensed by RLRs remains unknown. In order to determine RABV RNAs signatures recognized by RIG-I and MDA5, in collaboration with A. Komarova (IP), we have applied a novel ribonucleoproteomic approach that consists in affinity purification of tagged RLRs followed by extraction of associated RNA molecules. This approach has recently provided comparative views on viral RLR ligands in the context of infection with Measles, Chikungunya and Dengue viruses. We are now ready to perform NSG analysis of RIG-I- and MDA5- specific RNA ligands purified from RABV infected cells.

Factors determining RABV tropism are largely unknown. Here, we are identifying the susceptibilities and influence of glial cells on RABV infection.

Screening for NSm function in infected mammalian cells by proteomic analysis.

In order to study a differential regulation of RNA and protein expression level during Rift valley fever virus (RVFV) infection in a murine model, we want to compare transcriptomic and proteomic analysis.  

Historically circulating in Africa, Rift Valley fever virus (RVFV) was firstly identified in Kenya in 1930, and responsible for dozens of outbreaks in Africa in the past decades. As an arbovirus, RVFV is mainly transmitted by mosquito vector, and needs to be altered between mammalian host and mosquito vector for maintaining the genome stability. In this project, we are analysing the mosquito vector competence for several RVFV variants obtained from different host cells, to better interpret the risk factors of transmission.

This project is integrated in the analysis of the gut ecosystem of children implicated in the AFRIBIOTA project, a translational project performed within a consortium of researchers and medical doctors from the Central African Republic, Madagascar, France and Canada (see https://research.pasteur.fr/fr/program_project/the-afribiota-project/). AFRIBIOTA aims at understanding the risk factors and pathophysiological changes underlying chronic child malnutrition as manifested through delayed growth. Within the project submitted, we aim at assessing a possible link between asymptomatic pathogen carriage, gut inflammation and stunted growth of children included in the study site in Antananarivo, Madagascar.

We propose to identify peptides derived from the S protein that can modulate the HLA-E restricted activity of NK cells. Through in-silico analyses, we have already identified in the S protein of SARS-Cov1, SARS-Cov2 and MERS-Cov regions coding for peptides with HLA-E binding motifs. We will perform functional and structural analyses of the binding of these peptides to MHC-E. We will study MHC-E restricted responses of NK cells in blood and tissues during SARS-Cov2 infection in non human primates. From longitudinally collected samples from blood and tissues before and after the SARS-CoV2 infection of the animals, we will analyse the phenotype and transcriptome profile of NK cells and compare to viral loads and disease progression of the animals.

SARS-CoV-2 depends on the activation or inactivation of specific cellular pathways for efficient replication. The project screened small molecule compounds that affected epigenetic regulation of such pathway. The project aims to characterize these specific pathways, so identify specific steps important in the viral replication cycle and with this to describe the mode of action of these compounds.

Vaccines against COVID-19 have been developed for use as homologous two-dose regimens and several of them have demonstrated efficacy. There is limited data on the clinical and the immune response induced by heterologous vaccination regimens (HVR) using alternate vaccine modalities. CORSER-4 is a Viro-Immunology cohort promoted by the Institut Pasteur that will follow-up subjects from the time they receive a prime vaccine dose or a boosting dose. We determined subjects sub-groups of interest defined by the type of vaccine immunization they receive: (i) Heterologous two-dose vaccine regimen, the less frequently used at the moment, (ii) Homologous two-dose vaccine regimen, the more frequently used; (iii) One-dose vaccine regimen and (iv) Infectious-prime vaccine-boost immunization in COVID-19-experienced patients. The subject will be enrolled on the planed last boost injection visit and 5 Follow-up visits will be further conducted (Month1, M3, M6, M12 and M24). We will collect longitudinal clinical data and biological samples. We will explore Viral and Immune Response in sera, nasopharyngeal secretions and in saliva samples. We will assess the immune response obtained by various vaccination regimen against the reference strains and the variants of concern, and the durability of the humoral and cellular response. In addition, we will document any occurring acute infection episode with a supplemental unscheduled visit in order to detect an emerging variant viral strain. Altogether, the CORSER-4 project constitutes an opportunity to evaluate early anti-SARS-CoV-2 immunity in heterologous (and others) vaccine regimens and to estimate their efficacy and their sustainable (M24) protection against circulating virus strains (reference and variants).

Voluntary organizations provide essential support to vulnerable populations and front-line health responders to the COVID-19 pandemic. The French Red Cross (FRC) is prominent among organizations offering health and support services in the current crisis. Comprised primarily of lay volunteers and some trained health workers, FRC volunteers in the Paris (France) region have faced challenges in adapting to pandemic conditions, working with sick and vulnerable populations, managing limited resources, and coping with high demand for their services. Existing studies focus on individual, social and organizational determinants of motivation, but attend less to contextual ones. Public health incertitude about the COVID-19 pandemic is an important feature of this pandemic. Whether and how uncertainty interacts with volunteer understandings and experiences of their work and organizational relations to contribute to Red Cross workonline social listening and surveys.

Analysis of the immune response to yellow fever 17D vaccination

We have identified a candidate gene associated with increased resistance to a pathogen. This gene is poorly annotated in public databases. To get insight into its function we are focusing on genes that are co-expressed with this candidate gene in various datasets, including microarray collection on various mouse tissues or in a given tissue across inbred strains of mice. Indeed, coexpression is one of the central idea in gene expression analysis. The 'Guilt by association' principle states that gene coexpression might indicate shared regulatory mechanisms and roles in related biological processes. We have established lists of genes that are co-expressed in various tissues, and in collection of individuals with different genotypes. We are willing to get graphical representation giving a compact overview of genes that are coexpressed with our candidate gene.

Yellow fever virus (YFV), a Flavivirus transmitted by mosquitoes causes a severe hemorrhagic fever in humans. Despite the availability of a safe and effective vaccine (17D), YFV is still a public health problem in tropical Africa and South America. In the Americas, the massive campaign of mosquito control during the first half of the 20th century led to the eradication of Aedes aegypti from most American countries, and as a consequence, urban outbreaks of YF were no longer observed. However, the relaxation of vector control led to the reinfestation of urban areas by Ae. aegypti and the subsequent establishment of the Asian tiger mosquito Aedes albopictus. In Brazil, while human cases are sporadically detected in the Amazonian basin where sylvatic YFV strains circulate between non-human primates and arboreal canopy-dwelling mosquitoes (Haemagogus sp.), they are increasingly reported outside the jungle moving towards the Atlantic coast, the most populated area. In the absence of routine immunization programs, YF may come back in the American towns as it was in the past. The causes leading to the current YF resurgence are multifactorial. From a mosquito vector viewpoint, changes in vector densities, distribution, vector competence or vector as a site of selection for epidemic YFV strains, can be regarded as critical factors. Our project aims to address the contribution of the invasive mosquito Ae. albopictus as a missing link to allow a selvatic YF strain (1D) to become adapted for a transmission in urban areas by the human-biting mosquito, Ae. aegypti. It will be done through three specific objectives: (i) identify Ae. albopictus-adaptive mutations after serial cycling of the selvatic YFV-1D on Brazilian Ae. albopictus mosquitoes, (ii) evaluate their potential to be transmitted to a vertebrate host, and (iii) deepen the transmission of the experimentally selected viruses by field-collected mosquito populations.

Yellow fever (YF) is a fatal hemorrhagic disease caused by an arbovirus, Yellow Fever Virus (YFV) transmitted by the Aedes aegypti mosquito vector. YFV is currently endemic in Africa (5 strains) and South America (2 strains), and although an effective vaccine is available, YFV remains a major public health issue. Native from Africa, YFV was transported to South America and the Caribbean during the slave trade (15th to 19th century) and caused devastating outbreaks. After two centuries of outbreaks, YF is no longer reported in the Caribbean thanks to the successful mosquito control program implemented at the beginning of the 20th century. The virus disappeared from the Caribbean and Martinique experienced its last outbreak in 1908. However, the relaxation of anti-vectorial control programs contributed to the reintroduction of Ae. aegypti in most Caribbean islands. We evaluated the vector competence to YFV of different populations of Ae. aegypti from Martinique and other mosquito populations of the Caribbean. Our results showed that Ae. aegypti from Martinique were competent to African as well as to American YFV genotypes with however differences of viral dissemination and transmission. In this project, we aim at determining the diversity of viral populations at the crossing of two anatomical barriers in mosquitoes: midgut and salivary glands. The virus should enter into the midgut epithelial cells, replicate and be released into the hemocel. The final step will be the infection of the salivary glands from where the virus is excreted with the saliva. At each step, a selection of viral populations operates with only a fraction of the viral population transmitted between different tissues within a same host. The virus diversification promoted by virus replication in mosquitoes depends on the mosquito species. Such vector-specific conditions may have great impacts on emergence processes.

FlavImmunity is an international project aiming to study the immune response developed after yellow fever vaccination.