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Keywords: Rainbow trout, Salmonids, Zebrafish, Virus and bacterial infections, VSHV, Fish viruses, Infection, Antiviral immunity, Interferons, ISG, Functional studies and antiviral activity, Genomics of virus-host interactions, Transcriptome, Imaging, TRIM Proteins, Adaptive Immunity, Ig and TCR Directories, Viral Infection Models, In Vivo Cellular and Inflammatory Response Monitoring.
Innate antiviral response in fish : functional approaches
The non-specific antiviral response induced by different viruses (rhabdobvirus, birnavirus) is studied in two "model" species of fish: rainbow trout, salmonid of aquaculture importance and zebrafish, cyprinid and model species in genomics, biology of development and immunology. Our work aims to characterize the innate immune response in fish and more specifically the IFN response. This knowledge makes it possible to consider: the development of diagnostic tools for infectious diseases in fish and have contributed in recent years to describe the evolution (conservation and / or diversification) of defense mechanisms antiviral through vertebrates.
The innate response is investigated by transcriptome analysis methods, and complemented by targeted functional approaches that combine the benefits of both model species. The mechanisms of action of viro-induced genes selected (ISG15, finTRIM, viperin) are then analyzed in vitro and in vivo. The evolution of the response: IFNs and viro-induced genes are studied by comparative genomics approaches.
The multigenic protein family "TRIM" or "RBCC" is a particular focus of the team, which has discovered a group of trIMs called "finTRIM" (fish novel TRIMs) very diverse (84 members) and specific species Pisces, ". The role of these proteins, particularly in antiviral responses, is characterized by combining various approaches in cellular models and zebrafish larvae. The analysis of the innate response against pathogenic bacteria (in particular F. psychrophilum) is conducted by transcriptome approaches in trout.
Imaging cellular and inflammatory responses in zebrafish
Zebrafish is a model of choice for the dynamic study of host-pathogen interactions in an entire organism. Its small size at the larval stage as well as its optical transparency allow to lead on this species, from dynamic imaging approaches to cellular resolutions. Relevant models (transgenic lines) have been developed to monitor in vivo, antiviral immune responses and inflammatory responses following different stimuli. The laboratory therefore has transgenic lines of interest expressing fluorescent proteins in different cellular sub-types (macrophages, neutrophils, neurons, epithelial cells, ...). Combined methods of real-time imaging and fixed tissue imaging are conducted on these lines at different stages of development.
The data is analyzed and quantified by image analysis software in collaboration with the platform MIMA2 (3D rendering) and Bioemergences (Tracking). Platform equipment (micromanipulation, microinjection and stereomicroscopes) can also lead to loss and gain of function experiments by microinjection of morpholino and RNA.
Analysis of vaccine-induced B and T responses in fish
The adaptive response induced by vaccines, and the basics of infection-induced protection, are studied in the team by analyzing the diversity of T-cell antigen and receptor antibodies in rainbow trout. In particular, high-throughput sequencing makes it possible to describe, at an unprecedented level of detail, the structure of the B and T repertoires after immunization or infection. These approaches explore the basics of vaccines-induced memory and protection in fish. Comparative analysis of these data with those available in mammals also provides clues to understanding the evolution of adaptive responses.
Main projects and collaborations
These approaches have been developed through numerous collaborations. The most prominent and sustainable are: the team of JP Levraud (Pasteur Institute), G Lutfalla (Univ Montpellier), Chris Secombes (Univ Aberdeen, UK) and G Wiegertjes (Univ Wageningen, NL) for the functional analysis of ISGs and IFNs; the team of JS Joly (CNRS), S. Saulnier (Imagine), N. Laguette (IGH, Montpellier), V. Beringue (INRA) and S. Bolte (Univ Paris 6) as well as all TEFOR network for zebrafish imaging projects; the teams of A Six (Univ Paris 6), T Mora and A Walshak (ENS), F Cazals (INRIA), S Fillatreau (INEM) and O Sunyer (Univ Penn, USA), and more recently O Evensen (Univ Oslo, N), Jorgensen J (Univ Tromsö, N) and A Krasnov (NOFIMA, N) for the analysis of immune repertoires; the B Verrier team (CNRS Lyon) for the RNA vaccination strategies.
National Research Agency:Project Zebraflam (2011-2013). The objective of this project was to monitor in real time the spread of a viral infection in an entire vertebrate organism, and simultaneously observe the development of the host response, at a resolution where the individual cells are identifiable. The potential role of these molecules in the modulation of the inflammatory response can thus be established. The different components of the host response - leukocyte attraction, phagocytosis, granuloma formation, cytokine production - will be monitored by real-time microscopy. In order to complete the microscopic studies, a global analysis of the transcriptome variations induced during the early phase of a viral infection was performed to determine the role of the response to IFNs, TNfa and IL-10.
National Research Agency:FishRNAVax Project (2016-). The objective of this project is to test the effectiveness of different biodegradable nanosystems for the administration of vaccines to fish, particularly by mucosal route (immersion for example). The distribution of nanoparticles is analyzed in zebrafish by imaging, and the immune response is studied in this model or by analysis of Igs in rainbow trout. An innovative strategy of RNA vaccines is developed.
European Projects Lifecycle PCRD7 2009-2013 & "Targetfish" PCRD7 (2012-2017):The group's contribution to these two projects was the analysis of the structure of the B and T directories of rainbow trout by massive sequencing after different vaccinations according to multiple routes and protocols. The origin of the public Ig responses induced by vaccination against Viral Haemorrhagic Septicemia has been analyzed in detail. The diversity of TCRs expressed by T lymphocytes with different phenotypes defined by CD4 and CD8 receptors has been characterized, giving indications on the functions of these cells.
European Project Veterinary Biotechnology Research Facility Network (2017): This project aims to strengthen the cooperation and accessibility of European infectious disease platforms, to better know, control and prevent diseases of farm animals. This project integrates in particular the unit housing fish in Jouy-en-Josas and brings together 30 partners from 14 countries. In addition to the reception and support activities for access to infectious disease infrastructures spread across the nine European partner countries, VetBioNet is developing research activities that concern the team, the zebrafish model.
Fish for Pharma ITN PCRD7 (2012-2016):The participation of the team in this consortium, associated with the JP levraud group at the Pasteur Institute, has led to the development of work on the functions of finTRIMs and ISGs, and establish a model of zebrafish infection by the neurotropic Sinbis virus, identifying the pathway into the brain.
Tefor "Infrastructures for the future" 2013-2018: The TEFOR infrastructure (Transgenesis for Functional Studies in model ORgansimes) (www.tefor.net) is a distributed infrastructure that supports research on two main model species: the zebrafish and Drosophila. Its main objectives are to provide transgenesis, mutagenesis or phenotyping services and to generate new collections of transgenic lines presented in 3D image databases. The VIM unit, a partner in this project, is in charge of phenotyping zebrafish lines of interest for the study of the immune system. The phenotyping platform attached to this unit, is equipped with two microinjection stations and a two-photon microscope located in the pathogenic zone of the experimental fish facilities of IERP in Jouy-en-Josas. In recent years, the laboratory has contributed to the development of viral infection models in zebrafish (Novirhabdovirus, Alphavirus, ...) and to the production of new transgenic lines of interest for the study of the anti-viral immune response. . The phenotyping approaches currently carried out at the platform are based on the development of innovative immunolabeling techniques (IHC in toto, transparency) as well as the monitoring of the dynamics of immune and inflammatory responses by real-time microscopy (post-infection or immunization). Technological developments as well as image analysis are carried out in close collaboration with the other infrastructure partners (TeforCoreFacility & CASBAH Team: Comparative Analysis of Stem cells, Brain Anatomy and Homeostasis (JS Joly), Bioemergences (N Peyrieras), CNRS Gif on Yvette, Amagen platform INRA / CNRS (F Sohm) and LPGP-INRA (V Thermes), INRA Rennes).
Service / collaboration contract with L'Oréal company 2014-2015-2016