Expertise

Hub members Have many expertise, covering most of the fields in bioinformatics and biostatistics. You'll find below a non-exhaustive list of these expertise

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Searched keyword : Virus

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Thomas BIGOT

Group : GIPhy - Embedded : Biology of Infection

I joined the C3BI Hub in 2016 after a curriculum widely dedicated to Bioinformatics studies, and more precisely to Phylogeny and Evolution, topics of my PhD thesis. At Institut Pasteur, I am involved in projects dealing with sequences homology : alignments, hmm profiles, making homologous family databases, kmers signatures. I am also a developer (Python / C++) with a solid interest in optimization as well as in developing usable tools for final user such as automated pipeline for metagenomics sequence analysis. I’m currently embedded in Marc Eloit’s team (80% of my work time). My main task in this team is to develop strategies to identify, in their metagenomics samples, new pathogens, or new combination pathogen / symptoms. The rest of my time, I manage small projects and participate to the Hub life. I am currently experimenting with functional programming (for now, using Python) and its applicability to bioinformatics issues.


Keywords
AlgorithmicsScientific computingSofware development and engineeringParallel computingGraph theory and analysis
Organisms
BacteriaFungiVirus
Projects (10)

Julien GUGLIELMINI


After a PhD in Microbiology on bacterial toxin-antitoxin systems at the Free University of Brussels, I joined the Institut Pasteur for a 3 years postdoc in Eduardo Rocha’s lab. During this period, I performed comparative genomics and pylogenetic analysis on bacterial conjugation and type IV secretion systems. Then, I worked 2 years in Olivier Tenaillon’s team on the modelling and evolution of organismal complexity. I joined the HUB in 2015, and I am involved in phylogenetic and comparative genomics projects.


Keywords
GenomicsPhylogeneticsSequence analysisGenome analysisGeneticsEvolutionPopulation genetics
Organisms
ArchaeaBacteriaVirus
Projects (12)

Related projects (161)

Regulation of HIV replication by cellular DNA topology

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.



Project status : Declined

Evolutionary relationships between giant viruses and eukaryotes

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).



Project status : Closed

viral evolution around Ebola Treatment Centre in Macenta and according to disease outcomes

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.



Project status : Closed

Identification of new cellular parameters involved in HIV-1 integration selectivity

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.



Project status : Closed

A reference panel of dengue vector genomes

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.



Project status : Closed

Identification of new or unexpected pathogens, including viruses, bacteria, fungi and parasites associated with acute or progressive diseases

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.



Project status : In Progress

IgBlast on Galaxy

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.



Project status : Closed

Regulation of HIV-1 integration selectivity by chromatin

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.



Project status : In Progress

Utilize mouse models to study infection by HIV-1

We previously showed that humanized immune system (HIS) mice generated in Balb/c Rag2-/-γc-/- 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



Project status : Closed

Single cell analysis of HIV-specific CD4+ T cell differentiation



Project status : Closed

Study of the early pathogenesis during Lassa fever in cynomolgus monkeys and its correlation with the outcome

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.



Project status : In Progress

The resurgence of a neglected disease, Yellow fever: from jungle to urban environments

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.



Project status : Closed

An Aedes albopictus-driven epidemiological prediction for arboviral diseases outbreak in Europe



Project status : Closed

Modulation of cellular pathways involved in neuropathology of rabies 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.



Project status : Closed

Identification of internal methylations of RABV mRNAs using RiboMeth sequencing approach

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).



Project status : In Progress

Determination of RABV RNAs signatures recognized by RLRs

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.



Project status : Awaiting Publication

Building a database with a pattern mining system designed for virus nucleic acid data

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.



Project status : Declined

Phages - bacteria interactions network of the healthy human gut



Project status : Closed

Global BioID-based SARS-CoV-2 proteins proximal interactome unveils novel ties between viral polypeptides and host factors involved in multiple COVID19-associated mechanisms

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.



Project status : In Progress

CORSER-4 Cohort Study: Assessment of the humoral immune response to COVID-19 vaccination in each subpopulation defined by type of vaccination regimen, at 1-3-6-12-24 months

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).



Project status : In Progress

Genetic diversity of yellow fever virus populations in mosquitoes

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.



Project status : Pending

Better characterization of long chimeric virus-human reads after target enrichment on integrated viral genomes.

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.



Project status : New

Deciphering mutation signatures on hepatitis B virus genome



Project status : New

Detection of newly produced NIRVS in mosquito persistently infected cells

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.



Project status : Pending

Building a database with a pattern mining system designed for virus nucleic acid data

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.



Project status : Declined

An Aedes albopictus-driven epidemiological prediction for arboviral diseases outbreak in Europe



Project status : Closed

Detection of newly produced NIRVS in mosquito persistently infected cells

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.



Project status : Pending

A reference panel of dengue vector genomes

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.



Project status : Closed

An Aedes albopictus-driven epidemiological prediction for arboviral diseases outbreak in Europe



Project status : Closed

Detection of newly produced NIRVS in mosquito persistently infected cells

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.



Project status : Pending

viral evolution around Ebola Treatment Centre in Macenta and according to disease outcomes

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.



Project status : Closed

Deciphering mutation signatures on hepatitis B virus genome



Project status : New

Regulation of HIV replication by cellular DNA topology

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.



Project status : Declined

Identification of new cellular parameters involved in HIV-1 integration selectivity

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.



Project status : Closed

IgBlast on Galaxy

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.



Project status : Closed

Regulation of HIV-1 integration selectivity by chromatin

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.



Project status : In Progress

Utilize mouse models to study infection by HIV-1

We previously showed that humanized immune system (HIS) mice generated in Balb/c Rag2-/-γc-/- 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



Project status : Closed

Single cell analysis of HIV-specific CD4+ T cell differentiation



Project status : Closed

Study of the early pathogenesis during Lassa fever in cynomolgus monkeys and its correlation with the outcome

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.



Project status : In Progress

Modulation of cellular pathways involved in neuropathology of rabies 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.



Project status : Closed

Identification of internal methylations of RABV mRNAs using RiboMeth sequencing approach

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).



Project status : In Progress

Determination of RABV RNAs signatures recognized by RLRs

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.



Project status : Awaiting Publication

CORSER-4 Cohort Study: Assessment of the humoral immune response to COVID-19 vaccination in each subpopulation defined by type of vaccination regimen, at 1-3-6-12-24 months

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).



Project status : In Progress

The resurgence of a neglected disease, Yellow fever: from jungle to urban environments

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.



Project status : Closed

Genetic diversity of yellow fever virus populations in mosquitoes

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.



Project status : Pending