37 research outputs found

    Interest of reverse genetics applied to RNA viruses : Examples of a salmonid rhabdovirus and alphavirus

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    European and French rainbow trout farms are faced with viral infections which kill every year approximately one fifth of their production (i.e. 10,000 tons worth ?23m a year in France). Major viral diseases are caused by two coexisting rhabdoviruses: the viral hemorrhagic septicaemia virus (VHSV) and the infectious hematopoïetic necrosis virus (IHNV). The genome of rhabdoviruses, the best known being the rabies virus, consists of a single negative-strand RNA molecule of about 12 kb. These viral diseases are the most frequent, but the intensification of salmonid fish farms as well as the development of fish farming using new species have lead to the emergence of new diseases as well as an increase in the incidence of existing diseases. One of the more notable examples over the past few years is undoubtedly the emergence of salmonid sleeping disease, known since the ‘80s in most of the European countries and in North America. Although it used to be infrequent in French fish farms, it now affects 30-40 % of their production. The genome of the sleeping disease virus (SDV), a member of the alphavirus family, consists of a single positive-strand RNA molecule of about 12 kb. • Over the past few years, we have been focusing some of our research activity on the virus genetic engineering to obtain live vaccines. • Genetic engineering of RNA viruses is based on the availability of a DNA copy of the RNA genome. This approach is called “reverse genetics”. Reverse genetics on a positive-strand RNA virus was demonstrated for the first time on the poliovirus in the ‘80s. It took another fifteen years to establish a reverse genetics system for the recovery of a negative-strand RNA virus, the rabies virus. The synthesis of an intermediate cDNA, which is an exact copy of the viral RNA genome, helps manipulations such as gene deletions to study their role, or gene insertions to express heterologous genes. • Reverse genetics systems have now been established for IHNV and SDV, and have helped generate numerous recombinant viruses, of which some are currently being tested as vaccines in large-scale field trials.Les élevages piscicoles français et européens de truite arc-en-ciel sont confrontés à des infections virales qui tuent annuellement environ le cinquième de leur production (soit 10.000 tonnes pour la France, 23 millions d'Euros par an). Les principales viroses sont provoquées par deux rhabdovirus qui coexistent: le virus de la septicémie hémorragique virale, vSHV, et le virus de la nécrose hématopoïétique infectieuse, vNHI. Les rhabdovirus, dont le prototype le plus connu est le virus de la rage, ont un génome constitué d'une molécule d'ARN de polarité négative d'environ 12 kilobases (kb). Ces maladies virales sont les plus fréquentes mais l'intensification des élevages de salmonidés et le développement de l'élevage d'autres espèces entraînent l'apparition de pathologies nouvelles et, aussi, une augmentation de l'incidence des pathologies anciennes. L'un des exemples le plus notable de ces dernières années est certainement la maladie du sommeil des salmonidés, connue depuis les années 1980, présente dans la plupart des pays européens et sur le continent Nord américain. Jusqu'alors peu fréquente dans les élevages piscicoles français, elle affecte maintenant 30-40 % de ces élevages. Le virus responsable ou virus de la maladie du sommeil (VMS) appartient à la famille des alphavirus; son génome est une molécule d'ARN de polarité positive de 12 kb. Depuis quelques années, nous focalisons notre activité en partie sur le développement de stratégies vaccinales en manipulant le génome de ces virus, permettant à terme l'utilisation de vaccins vivants, La manipulation génétique des virus à ARN passe obligatoirement par l'aptitude à récupérer le virus à partir d'une copie ADN du génome ARN. C'est ce que l'on appelle « la génétique inverse». La première démonstration de génétique inverse pour un virus à ARN de polarité positive, le poliovirus, date des années 1980, et il a fallu attendre une quinzaine d'années pour qu'il en soit de même pour un virus à ARN de polarité négative, le virus de la rage. Le passage par un intermédiaire ADN artificiel, copie conforme du génome viral à ARN, offre la possibilité de manipuler aisément le génome et, par exemple, d'enlever des gènes pour étudier leur rôle ou bien d'en rajouter pour exprimer des gènes hétérologues. Des systèmes de génétique inverse pour le vNHI et le VMS ont été élaborés au laboratoire et ont permis de générer de nombreux virus recombinants dont certains sont en phase d'expérimentation à grande échelle comme outil vaccinal

    A Nuclear Localization of the Infectious Haematopoietic Necrosis Virus NV Protein Is Necessary for Optimal Viral Growth

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    The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the 32EGDL35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the 32EGDL35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of 32EGDL35 responsible for nuclear localization are important for the inhibitory activity of NV

    LE VIRUS RESPONSABLE DE LA MALADIE DU SOMMEIL CHEZ LES SALMONIDES (UN NOUVEAU TYPE D'ALPHAVIRUS)

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    ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

    Le virus de la maladie du sommeil des Salmonidés (mise au point d'un ADNc infectieux et obtention d'anticorps monoclonaux)

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    Le Virus de la Maladie du Sommeil (VMS) a été caractérisé au laboratoire comme étant le premier alphavirus aquatique appartenant donc à la famille des Togaviridae. Au cours de ce travail de thèse nous avons poursuivi 3 objectifs : (i) élaboration d'un ADNc infectieux, (ii) génération d'un éventail d'anticorps monoclonaux contre les protéines non structurales et structurales du VMS, et cartographie de certains de ces anticorps, (iii) développement de méthodes diagnostiques pour le VMS.(i) Le génome du VMS est un ARN positif de 12 kb environ, entièrement séquencé. Il a été converti en ADNc et cloné dans un vecteur d'expression eucaryote sous le contrôle de différentes séquences promotrices : soit SP6 (dérivée du phage SP6), soit T7 (dérivée du phage T7), soit du CMV (promoteur précoce du cytomégalovirus). En premier lieu, des minigénomes dans lesquels un gène rapporteur (luciférase, GFP) remplace la région codante pour les protéines structurales ont été construits. Leur expression a été testée et mise au point en transfectant à des cellules de poisson, soit l'ARN synthétisé in vitro à partir du promoteur SP6, soit l'ADN plasmidique porteur des promoteurs CMV ou T7. De cette façon, nous avons déterminé les conditions nécessaires à la réplication du génome viral et à sa détection. Le VMS ne peut être exprimé lorsqu'il est fusionné à la séquence promotrice qui le précède, ce qui constitue une autre particularité de cet alphavirus atypique. Dans le système d'expression CMV/T7, le remplacement du gène rapporteur par la région codant pour les protéines structurales du VMS conduit à la production de particules virales réplicatives identiques au virus sauvage lorsque l'ADNc est transfecté à des cellules de poisson.Sleeping Disease Virus (SDV) has been characterised in our laboratory as being the first aquatic alphavirus belonging to the Togaviridae family. During this work, we pursue three objectives : (i) elaboration of an infectious cDNA, (ii) generation of a panel of monoclonal antibodies directed against structural and non structural viral proteins, (iii) development of an RT-PCR diagnostic method.(i) The SDV genome is a positive single strand of RNA of approximately 12 kb which sequence is determined. It has been converted in a full lenght cDNA and cloned in an eukaryotic expression vector under the control of several different promoters : either the SP6 promoter (derived from SP6 phage), or the T7 promoter (derived from T7 phage), or the CMV promoter (early promoter of cytomegalovirus). First, replicons expressing a reporter gene (luciferase, GFP) instead of the structural genes have been constructed. Expression of these replicons has been tested through transfection of fish cells using either RNA transcribed from SP6 promoter, or plasmid DNA carrying CMV or T7 promoters. It was not possible to express SDV genome when it is directly fused to the promoter. In the CMV/T7 expression system, replacement of reporter gene by the structural genes allowed the recovery of infectious viral particles when cDNA was transfected into fish cells. The virulence of this recombinant virus has been studied in vivo on juvenile rainbow trouts. We show that this virus is greatly attenuated and induce a long lasting protection.Otherwise, this system has been tested for development of a gene vector : a second transcriptional unit coding to the GFP protein has been inserted either upstream, or downstream of the structural genes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

    Interaction of the attenuated recombinant rIHNV-Gvhsv GFP virus with macrophages from rainbow trout (Oncorhynchus mykiss)

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    7 figurasOne of the most important threats to the salmonid aquaculture industry is infection caused by novirhabdoviruses such as infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV). Using reverse genetics, an avirulent recombinant rIHNV-Gvhsv GFP strain was generated, which was able to replicate as effectively as wild type IHNV in a fish cell line and in macrophages. Although this recombinant virus induced protective responses against IHNV and VHSV, the response did not involve the production of antibodies or modulate the expression of some antiviral genes. To determine the immune mechanisms underlying the protection conferred by the rIHNV-Gvhsv GFP virus, different immune parameters (NO production, respiratory burst activity and the induction of apoptosis) were assessed in the macrophage population. The results obtained in the present work may indicate that the Nv protein could be important in the modulation of NO and ROS production. rIHNV-Gvhsv GFP did not appear to have a clear effect on nitric oxide production or apoptosis. However, an increased respiratory burst activity (with levels induced by the recombinant virus significantly higher than the levels induced by the wild type virus), suggests a stimulation of the macrophage population, which could be related to the protection against virulent viruses.This work was supported by the projects FAIRCT 98-4398 from the European Union and BIO 2000-0906 from the Spanish Ministerio de Ciencia y Tecnología. European structural funds were used for confocal microscopy. Fondo Europeo de Desarrollo Regional (FEDER) y Ministerio de Ciencia e Innovación. A. Romero acknowledges the CSIC for the I3P fellowship.Peer reviewe

    Recombinant infectious hematopoietic necrosis viruses induce protection for rainbow trout Oncorhynchus mykiss

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    13 páginas, 8 figuras, 1 tablaInfectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicaemia virus (VHSV) are rhabdoviruses that infect salmonids, producing serious economic losses. Two recombinant IHN viruses were generated by reverse genetics. For one (rIHNV GFP) the IHNV NV gene was replaced with the green fluorescent protein (GFP) gene. In the other (rIHNV-Gvhsv GFP) the G gene was also exchanged for that of VHSV. No mortalities, external signs or histological lesions were observed in experimental infections conducted with the recombinant viruses. Neither the rIHNV GFP nor rIHNV-Gvhsv GFP was detected by RT-PCR in any of the examined tissues from experimentally infected fish. In order to assess their potential as vaccines against the wild type viruses, rainbow trout were vaccinated with the recombinant viruses by intraperitoneal injection and challenged 30 d later with virulent IHNV or VHSV. The GFP viruses provided protection against both wild type viruses. None of the recombinant viruses induced antibody production, and the expression of interferon (IFNαβ) and interferon induced genes such as Mx protein and ISG-15 was not different to that of controls. The rIHNV-Gvhsv GFP did not inhibit cellular apoptosis as it was observed in an IHNV inoculated fish cell line. These studies suggest that the recombinant rIHNV-Gvhsv GFP is a promising candidate as a live recombinant vaccine and also provides a good model to further study viral pathogenicity and the molecular basis of protection against these viral infectionsThis work was supported by the projects FAIRCT 98-4398 from the European Union, BIO 2000-0906 and BIO 2001-2324-C02-01 from the Spanish Ministerio de Ciencia y TecnologíaPeer reviewe
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