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International audience; Viral haemorrhagic septicaemia virus (VHSV) is the cause of an important listed disease in European rainbow trout (Oncorhynchus mykiss) aquaculture and can be present in a wide range of fish species, including marine fish, which can act as viral reservoir. Recent studies revealed putative genetic virulence markers of VHSV to rainbow trout highlighting the roles of the nucleoprotein, phosphoprotein and non-virion protein. Using reverse genetics, we produced recombinant viruses by introducing parts of or the entire nucleoprotein from a high-virulent isolate VHSV into a low-virulent backbone. Furthermore, we also made recombinant viruses by introducing residue modifications in the nucleoprotein that seem to play a role in virulence. Rainbow trout challenged with these recombinant viruses (rVHSVs) by intraperitoneal injection (IP) developed clinical signs and showed lower survival when compared to the parental rVHSV whereas fish challenged by immersion did not show clinical signs except for the high-virulent control. The mutations did not influence the viral growth in cell culture. The recombinant viruses and parental recombinant were unable to replicate and show cytopathic effect in EPC cells whereas the high-virulent control was well adapted in all the fish cell lines tested. We showed evidence that corroborates with the hypothesis that the nucleoprotein has virulence motifs associated with VHSV virulence in rainbow trout.

International audience; Interferons are the first lines of defense against viral pathogen invasion during the early stages of infection. Their synthesis is tightly regulated to prevent excessive immune responses and possible deleterious effects on the host organism itself. The RIG-I-like receptor signaling cascade is one of the major pathways leading to the production of interferons. This pathway amplifies danger signals and mounts an appropriate innate response but also needs to be finely regulated to allow a rapid return to immune homeostasis. Recent advances have characterized different cellular factors involved in the control of the RIG-I pathway. This has been most extensively studied in mammalian species; however, some inconsistencies remain to be resolved. The IFN system is remarkably well conserved in vertebrates and teleost fish possess all functional orthologs of mammalian RIG-I-like receptors as well as most downstream signaling molecules. Orthologs of almost all mammalian regulatory components described to date exist in teleost fish, such as the widely used zebrafish, making fish attractive and powerful models to study in detail the regulation and evolution of the RIG-I pathway.

International audience; Viral hemorrhagic septicemia virus (VHSV) is a highly contagious virus leading to high mortality in a large panel of freshwater and marine fish species. VHSV isolates originating from marine fish show low pathogenicity in rainbow trout. The analysis of several nearly complete genome sequences from marine and freshwater isolates displaying varying levels of virulence in rainbow trout suggested that only a limited number of amino acid residues might be involved in regulating the level of virulence. Based on a recent analysis of 55 VHSV strains, which were entirely sequenced and phenotyped in vivo in rainbow trout, several amino acid changes putatively involved in virulence were identified. In the present study, these amino acid changes were introduced, alone or in combination, in a highly-virulent VHSV 23-75 genome backbone by reverse genetics. A total of 35 recombinant VHSV variants were recovered and characterized for virulence in trout by bath immersion. Results confirmed the important role of the NV protein (R116S) and highlighted a major contribution of the nucleoprotein N (K46G and A241E) in regulating virulence. Single amino acid changes in these two proteins drastically affect virus pathogenicity in rainbow trout. This is particularly intriguing for the N variant (K46G) which is unable to establish an active infection in the fins of infected trout, the main portal of entry of VHSV in this species, allowing further spread in its host. In addition, salmonid cell lines were selected to assess the kinetics of replication and cytopathic effect of recombinant VHSV and discriminate virulent and avirulent variants. In conclusion, three major virulence markers were identified in the NV and N proteins. These markers explain almost all phenotypes (92.7%) observed in trout for the 55 VHSV strains analyzed in the present study and herein used for the backward validation of virulence markers. The identification of VHSV specific virulence markers in this species is of importance both to predict the in vivo phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.

Viral hemorrhagic septicemia virus (VHSV) is a highly contagious virus leading to high mortality in a large panel of freshwater and marine fish species. VHSV isolates originating from marine fish show low pathogenicity in rainbow trout. The analysis of several nearly complete genome sequences from marine and freshwater isolates displaying varying levels of virulence in rainbow trout suggested that only a limited number of amino acid residues might be involved in regulating the level of virulence. Based on a recent analysis of 55 VHSV strains, which were entirely sequenced and phenotyped in vivo in rainbow trout, several amino acid changes putatively involved in virulence were identified. In the present study, these amino acid changes were introduced, alone or in combination, in a highly-virulent VHSV 23–75 genome backbone by reverse genetics. A total of 35 recombinant VHSV variants were recovered and characterized for virulence in trout by bath immersion. Results confirmed the important role of the NV protein (R116S) and highlighted a major contribution of the nucleoprotein N (K46G and A241E) in regulating virulence. Single amino acid changes in these two proteins drastically affect virus pathogenicity in rainbow trout. This is particularly intriguing for the N variant (K46G) which is unable to establish an active infection in the fins of infected trout, the main portal of entry of VHSV in this species, allowing further spread in its host. In addition, salmonid cell lines were selected to assess the kinetics of replication and cytopathic effect of recombinant VHSV and discriminate virulent and avirulent variants. In conclusion, three major virulence markers were identified in the NV and N proteins. These markers explain almost all phenotypes (92.7%) observed in trout for the 55 VHSV strains analyzed in the present study and herein used for the backward validation of virulence markers. The identification of VHSV specific virulence markers in this species is of importance both to predict the in vivo phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.

International audience; Recently, we reported an emerging pathology named Brown Muscle Disease (BMD) affecting Asari clams inhabiting the most productive area for this species in France, the Arcachon Bay. The main macroscopic feature of the pathology relies on the atrophy of the posterior adductor muscle, affecting the ability of clams to burry. The research of the etiological agent of BMD privileged a viral infection. Contrary to healthy clams, infected animals are always found at the surface of the sediment and exhibit 30 nm virus-like particles in muscle, granulocytic and rectal cells. In order to get more insights on the etiology and impacts of the BMD on clams, we took advantage in the present study of next generation sequencing technologies. An RNA-Seq approach was used (i) to test whether viral RNA sequences can be specifically found in the transcriptome of diseased animals and (ii) to identify the genes that are differentially regulated between diseased and healthy clams. Contrary to healthy buried animals, in diseased clams one sequence showing extensive homologies with retroviridae-related genes was detected. Among the biological processes that were affected in diseased clams, the synaptic transmission process was the most represented. To deepen this result, a new sampling was carried out and the transcription level of genes involved in synaptic transmission was determined in healthy and diseased clams but also in clams with no visible sign of pathology but located at the surface of the sediment. Our findings suggest that muscle atrophy is a latter sign of the pathology and that nervous system could be instead a primary target of the BMD agent.

International audience; Interferon production is tightly regulated in order to prevent excessive immune responses. The RIG-I signaling pathway, which is one of the major pathways inducing the production of interferon, is therefore finely regulated through the participation of different molecules such as A20 (TNFAIP3). A20 is a negative key regulatory factor of the immune response. Although A20 has been identified and actively studied in mammals, nothing is known about its putative function in lower vertebrates. In this study, we sought to define the involvement of fish A20 orthologs in the regulation of RIG-I signaling. We showed that A20 completely blocked the activation of IFN and ISG promoters mediated by RIG-I. Furthermore, A20 expression in fish cells was sufficient to reverse the antiviral state induced by the expression of a constitutively active form of RIG-I, thus allowing the efficient replication of a fish rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We brought evidence that A20 interrupted RIG-I signaling at the level of TBK1 kinase, a critical point of convergence for many different pathways that activates important transcription factors involved in the expression of many cytokines. Finally, we showed that A20 expression was directly induced by the RIG-I pathway demonstrating that fish A20 acts as a negative feedback regulator of this key pathway for the establishment of an antiviral state.

International audience; Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full-length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G-gene to make the rIHNV-Gsvcv. The chimeric rIHNV-Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild-type SVCV infection was tested. The rIHNV-Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV-Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV-Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV-Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.

Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.

A recombinant sleeping disease virus (rSDV) was previously shown to be totally attenuated and provide long-term protection in trout (C. Moriette, M. Leberre, A. Lamoureux, T. L. Lai, M. Brémont, J. Virol. 80:4088–4098, 2006). Sequence comparison of the rSDV to wild-type genomes exhibited a number of nucleotide changes. In the current study, we demonstrate that the virulent phenotype of SDV was essentially associated with two amino acid changes, V8A and M136T, in the E2 glycoprotein, with the V8A change mostly being involved in the acquisition of the virulent phenotype.

International audience; AbstractSleeping disease in rainbow trout is characterized by an abnormal swimming behaviour of the fish which stay on their side at the bottom of the tanks. This sign is due to extensive necrosis and atrophy of red skeletal muscle induced by the sleeping disease virus (SDV), also called salmonid alphavirus 2. Infections of humans with arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), are global causes of debilitating musculoskeletal diseases. The mechanisms by which the virus causes these pathologies are poorly understood due to the restrictive availability of animal models capable of reproducing the full spectrum of the disease. Nevertheless, it has been shown that CHIKV exhibits a particular tropism for muscle stem cells also known as satellite cells. Thus, SDV and its host constitute a relevant model to study in details the virus-induced muscle atrophy, the pathophysiological consequences of the infection of a particular cell-type in the skeletal muscle, and the regeneration of the muscle tissue in survivors together with the possible virus persistence. To study a putative SDV tropism for that particular cell type, we established an in vivo and ex vivo rainbow trout model of SDV-induced atrophy of the skeletal muscle. This experimental model allows reproducing the full panel of clinical signs observed during a natural infection since the transmission of the virus is arthropod-borne independent. The virus tropism in the muscle tissue was studied by immunohistochemistry together with the kinetics of the muscle atrophy, and the muscle regeneration post-infection was observed. In parallel, an ex vivo model of SDV infection of rainbow trout satellite cells was developed and virus replication and persistence in that particular cell type was followed up to 73 days post-infection. These results constitute the first observation of a specific SDV tropism for the muscle satellite cells.

The genome of infectious hematopoietic necrosis virus (IHNV), a salmonid novirhabdovirus, has been engineered to modify the gene order and to evaluate the impact on a possible attenuation of the virus in vitro and in vivo . By reverse genetics, eight recombinant IHNVs (rIHNVs), termed NxGy according to the respective positions of the nucleoprotein (N) and glycoprotein (G) genes along the genome, have been recovered. All rIHNVs have been fully characterized in vitro for their cytopathic effects, kinetics of replication, and profiles of viral gene transcription. These rIHNVs are stable through up to 10 passages in cell culture. Following bath immersion administration of the various rIHNVs to juvenile trout, some of the rIHNVs were clearly attenuated (N2G3, N2G4, N3G4, and N4G1). The position of the N gene seems to be one of the most critical features correlated to the level of viral attenuation. The induced immune response potential in fish was evaluated by enzyme-linked immunosorbent spot assay (ELISPOT) and seroneutralization assays. The recombinant virus N2G3 induced a strong antibody response in immunized fish and conferred 86% of protection against wild-type IHNV challenge in trout, thus representing a promising starting point for the development of a live attenuated vaccine candidate. IMPORTANCE In Europe, no vaccines are available against infectious hematopoietic necrosis virus (IHNV), one of the major economic threats in fish aquaculture. Live attenuated vaccines are conditioned by a sensible balance between attenuation and pathogenicity. Moreover, nonsegmented negative-strain RNA viruses (NNSV) are subject to a transcription gradient dictated by the order of the genes in their genomes. With the perspective of developing a vaccine against IHNV, we engineered various recombinant IHNVs with reordered genomes in order to artificially attenuate the virus. Our results validate the gene rearrangement approach as a potent and stable attenuation strategy for fish novirhabdovirus and open a new perspective for design of vaccines against other NNSV.

International audience; We report here the first full-length sequence of the eight ssRNA genome segments of the infectious salmon anemia virus (ISAV, Glesvaer/2/90 isolate), a salmonid orthomyxovirus-like. Comparison of ISAV genome sequence with those of others orthomyxovirus reveals low identity values, and a remarkable feature is the extremely long 59 end UTR of ISAV segments, which all contain an additional conserved motif of unknown function. In addition to the genome nucleotide sequence determination, specific mAbs have been produced through mice immunization with sucrose-purified ISAV. Four mAbs directed against the haemagglutininesterase glycoprotein, the nucleoprotein and free or actin-associated forms of the matrix protein have been characterized by (i) indirect fluorescent antibody test; (ii) virus neutralization; (iii) radioimmunoprecipitation and (iv) Western blot assays. These mAbs will potentially be useful for the development of new diagnostic tests, and the nucleotide sequences will help to establish a reverse genetics system for ISAV.

International audience; BACKGROUND: Chronic HCV infection is associated with the development of hepatic fibrosis. The direct role of HCV in the fibrogenic process is unknown. Specifically, whether HCV is able to infect hepatic stellate cells (HSCs) is debated. OBJECTIVE: To assess whether human HSCs are susceptible to HCV infection. DESIGN: We combined a set of original HCV models, including the infectious genotype 2a JFH1 model (HCVcc), retroviral pseudoparticles expressing the folded HCV genotype 1b envelope glycoproteins (HCVpp) and a subgenomic genotype 1b HCV replicon, and two relevant cellular models, primary human HSCs from different patients and the LX-2 cell line, to assess whether HCV can infect/replicate in HSCs. RESULTS: In contrast with the hepatocyte cell line Huh-7, neither infectious HCVcc nor HCVpp infected primary human HSCs or LX-2 cells. The cellular expression of host cellular factors required for HCV entry was high in Huh-7 cells but low in HSCs and LX-2 cells, with the exception of CD81. Finally, replication of a genotype 2a full-length RNA genome and a genotype 1b subgenomic replicon was impaired in primary human HSCs and LX-2 cells, which expressed low levels of cellular factors known to play a key role in the HCV life-cycle, suggesting that human HSCs are not permissive for HCV replication. CONCLUSIONS: Human HSCs are refractory to HCV infection. Both HCV entry and replication are deficient in these cells, regardless of the HCV genotype and origin of the cells. Thus, HCV infection of HSCs does not play a role in liver fibrosis. These results do not rule out a direct role of HCV infection of hepatocytes in the fibrogenic process.

International audience; We previously reported that betanodavirus reassortant strains [redspotted grouper nervous necrosis virus/striped jack nervous necrosis virus (SJNNV)] isolated from Senegalese sole (Solea senegalensis) exhibited a modified SJNNV capsid amino acid sequence, with changes at aa 247 and 270. In the current study, we investigated the possible role of both residues as putative virulence determinants. Three recombinant viruses harbouring site-specific mutations in the capsid protein sequence, rSs160.03(247) (S247A), rSs160.03(270) (S270N) and rSs160.03(247+270) (S247A/S270N), were generated using a reverse genetics system. These recombinant viruses were studied in cell culture and in vivo in the natural fish host. The three mutant viruses were shown to be infectious and able to replicate in E-11 cells, reaching final titres similar to the WT virus, although with a somewhat slower kinetics of replication. When the effect of the amino acid substitutions on virus pathogenicity was evaluated in Senegalese sole, typical clinical signs of betanodavirus infection were observed in all groups. However, fish mortality induced by all three mutant viruses was clearly affected. Roughly 40% of the fish survived in these three groups in contrast with the WT virus which killed 100% of the fish. These data demonstrated that aa 247 and 270 play a major role in betanodavirus virulence although when both mutated aa 247 and 270 are present, corresponding recombinant virus was not further attenuated.

International audience; The first salmonid alphaviruses, Salmon pancreas disease virus (SPDV) and Sleeping disease virus (SDV), were discovered and molecularly characterized more than 15 years ago, and since then few studies have really focused on vaccine development. However, these viruses are now becoming a real threat for farmed fish and wild fish populations. Pancreas disease has recently been listed as a notifiable disease in Norway, and there is renewed interest in developing strategies to protect fish from these pathogens. In this chapter we will present an update on what is known of the molecular biology of these viruses, the diagnostics tools that have been developed, the more recent epidemiological studies, and the existing and possible future vaccines to fight against these emerging viruses.

West Nile Virus (WNV) is a zoonotic mosquito-transmitted flavivirus that can infect and cause disease in mammals including humans. Our study aimed at developing a WNV vectored vaccine based on a fish Novirhabdovirus, the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20uC and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed in our laboratory for VHSV allowing the addition of genes in the viral genome and the recovery of the respective recombinant viruses (rVHSV). In this study, we have generated rVHSV vectors bearing the complete WNV envelope gene (EWNV) (rVHSV-EWNV) or fragments encoding E subdomains (either domain III alone or domain III fused to domain II) (rVHSV-DIIIWNV and rVHSV-DII-DIIIWNV, respectively) in the VHSV genome between the N and P cistrons. With the objective to enhance the targeting of the EWNV protein or EWNV-derived domains to the surface of VHSV virions, Novirhadovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EWNV at its amino and carboxy termini, respectively. By Western-blot analysis, electron microscopy observations or inoculation experiments in mice, we demonstrated that both the EWNV and the DIIIWNV could be expressed at the viral surface of rVHSV upon addition of SPG. Every constructs expressing EWNV fused to SPG protected 40 to 50% of BALB/cJ mice against WNV lethal challenge and specifically rVHSV-SPGEWNV induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EWNV-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly incorporated in the virion and expressed at the viral surface. In this study we demonstrated that a heterologous glycoprotein and non membrane-anchored protein, can be efficiently expressed at the surface of rVHSV making this approach attractive to develop new vaccines against various pathogens.

Novirhabdoviruses like Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) are fish-infecting Rhabdoviruses belonging to the Mononegavirales order. By reverse genetics, we previously showed that a recombinant VHSV expressing the West Nile Virus (WNV) E glycoprotein could serve as a vaccine platform against WNV. In the current study, we aimed to evaluate the potential of the Novirhabdovirus platform as a vaccine against influenza virus. Recombinant Novirhabdoviruses, rVHSV-HA and rIHNV-HA, expressing at the viral surface the hemagglutinin HA ectodomain were generated and used to immunized mice. We showed that mice immunized with either, rVHSV-HA or rIHNV-HA, elicited a strong neutralizing antibody response against influenza virus. A complete protection was conferred to the immunized mice when challenged with a lethal dose of influenza H1N1 A/PR/8/34 virus. Furthermore we showed that although acting as inert antigen in mice, since naturally inactivated over 20 degrees C, mice immunized with rVHSV-HA or rIHNV-HA in the absence of adjuvant were also completely protected from a lethal challenge. Novirhabdoviruses platform are of particular interest as vaccines for mammals since they are cost effective to produce, relatively easy to generate and very effective to protect immunized animals.

International audience

International audience; We have generated defective Viral Hemorrhagic Septicemia Viruses (VHSV) which express either the green fluorescent protein (GFP) or a far-red fluorescent protein (mKate) by replacing the genes encoding the nucleoprotein N or the polymerase-associated P protein. To recover viable defective viruses, rVHSV-Delta N-Red and rVHSV-Delta P-Green, fish cells were co-transfected with both deleted cDNA VHSV genomes, together with plasmids expressing N, P and L of the RNA-dependent RNA polymerase. After one passage of the transfected cell supernatant, red and green cell foci were observed. Viral titer reached 10(7) PFU/mL after three passages. Infected cells were always red and green with the very rare event of single red or green cell foci appearing. To clarify our understanding of how such defective viruses could be so efficiently propagated, we investigated whether (i) a recombination event between both defective genomes had occurred, (ii) whether both genomes were co-encapsidated in a single viral particle, and (iii) whether both defective viruses were always replicated together through a complementation phenomenon or even as conglomerate. To address these hypotheses, genome and viral particles have been fully characterized and, thus, allowing us to conclude that rVHSV-Delta N-Red and rVHSV-Delta P-Green are independent viral particles which could propagate only by simultaneously infecting the same cells.

International audience; Viral infections are detected in most cases by the host innate immune system through pattern-recognition receptors (PRR), the sensors for pathogen-associated molecular patterns (PAMPs), which induce the production of cytokines, such as type I interferons (IFN). Recent identification in mammalian and teleost fish of cytoplasmic viral RNA sensors, RIG-I-like receptors (RLRs), and their mitochondrial adaptor: the mitochondrial antiviral signaling (MAVS) protein, also called IPS-1, highlight their important role in the induction of IFN at the early stage of a virus infection. More recently, an endoplasmic reticulum (ER) adaptor: the stimulator of interferon genes (STING) protein, also called MITA, ERIS and MPYS, has been shown to play a pivotal role in response to both non-self-cytosolic RNA and dsDNA. In this study, we cloned STING cDNAs from zebrafish and showed that it was an ortholog to mammalian STING. We demonstrated that overexpression of this ER protein in fish cells led to a constitutive induction of IFN and interferon-stimulated genes (ISGs). STING-overexpressing cells were almost fully protected against RNA virus infection with a strong inhibition of both DNA and RNA virus replication. In addition, we found that together with MAVS, STING was an important player in the RIG-I IFN-inducing pathway. This report provides the demonstration that teleost fish possess a functional RLR pathway in which MAVS and STING are downstream signaling molecules of RIG-I. The Sequences presented in this article have been submitted to GenBank under accession numbers: Zebrafish STING (HE856619); EPC STING (HE856620); EPC IRF3 (HE856621); EPC IFN promoter (HE856618).

Viral hemorrhagic septicemia virus (VHSV) is a member of the Novirhabdovirus genus in the Rhabdoviridae family. VHSV is considered by many countries and international organizations to be one of the most important viral pathogens of finfish (38). During recent years, VHSV has been isolated from at least 50 different species from marine and freshwater fish and is present throughout the northern hemisphere (45). The transmission of the virus from fish to fish occurs directly through the water or by contact between infected and healthy individuals. VHSV is thought to enter the body through the gills or possibly through wounds on the skin. However, we recently showed that fins may represent the main portal of entry for the novirhabdoviruses (25). The virus usually causes severe hemorrhages in the skin, muscles, eyes, kidney, and liver, with mortality rates as high as 90%. As for all members of the Rhabdoviridae family, the VHSV genome consists of a negative-sense single-stranded RNA molecule of about 11 kb encoding five structural proteins: N, the nucleoprotein; P, a polymerase-associated protein; M, the matrix protein; G, the unique viral surface glycoprotein; and L, the large RNA-dependent RNA polymerase. In addition, like the other members of the Novirhabdovirus genus, such as infectious hematopoietic necrosis virus (IHNV), the VHSV genome encodes a small nonstructural NV protein, which has been shown to be dispensable for IHNV replication in cell culture and is involved in virus-induced pathogenicity in rainbow trout (8, 50). The sequence analysis of the glycoprotein (G) and nucleoprotein (N) genes of VHSV has shown that VHSV isolates can be divided into four genotypes that generally correlate with geographic location rather than the host species (4, 19, 47, 49). Isolates belonging to VHSV genotypes I, II, and III are present in continental Europe, the north Atlantic Ocean, the Baltic Sea, the North Sea, and waters around Scotland. Genotype IV consists of isolates from the marine environment in North America. Recently, viral hemorrhagic septicemia has become an emerging disease of freshwater fish in the Great Lakes region of North America (2, 54). Thus, it is quite obvious that VHSV is becoming a worldwide and very-broad-host-range fish virus and that the development of efficient vaccines is needed. Reverse genetics, allowing the introduction of targeted modifications into the viral genome and the production of attenuated live vaccine, may help to fight this rapidly spreading and emerging virus. It is routinely observed in farm trouts exposed to viral diseases that VHSV and IHNV coexist (26). By developing experimental coinfections by VHSV and IHNV in rainbow trout, Brudeseth et al. studied the pathogenesis and virus distribution (10). They found that both viruses established an infection and raised similar virus titers in kidneys, but the distribution of IHNV was more restricted in internal organs during the acute stage of the infection and was not detected in the brain. However, it generally is admitted that infection by one virus renders host cells resistant to a superinfecting virus. Superinfection exclusion, also known as homologous interference, is the phenomenon in which a cell infected with one type of virus or transfected with a viral replicon becomes resistant to a secondary infection with the same virus, whereas infection with unrelated viruses normally is unaffected (40, 51). Superinfection exclusion has been observed in a broad range of viruses, including vaccinia virus (14, 18), human immunodeficiency virus (HIV) (36, 37), vesicular stomatitis virus (VSV) (32, 43, 53), Borna disease virus (BDV) (24), measles virus (34), Sindbis virus (28), Semliki Forest virus (44), rubella virus (15), hepatitis C virus (HCV) (40, 51), and bovine viral diarrhea virus (BVDV) (31). Mechanisms of exclusion are diverse and have not been determined in all cases, but mechanisms described so far are caused by competition among different viruses for critical replicative pathways (for example, the use of the same receptors for the entry) or depend on the direct interaction of products of the primary infection with the secondary infecting virus. For example, the superinfection exclusion of VSV was found to be caused by a combination of three distinct effects on endocytosis by VSV-infected cells: (i) a decreased rate of the formation of endocytic vesicles, (ii) a decreased rate of the internalization of receptor-bound ligands, and (iii) a competition with newly synthesized virus for the occupancy of coated pits (43). In contrast, the cytoplasmic accumulation of BDV nucleocapsid components appeared to prevent subsequent infection through a blockage of the polymerase activity of incoming viruses (24). Superinfection exclusion by BVDV was the result of dual mechanisms that were mediated by the structural protein E2, which blocks the entry of a homologous second virus, and by a blockage at the level of replication dependent on the level of primary viral RNA replication but not influenced by the expression of viral structural proteins, as observed for BDV (31). HIV employs its early gene product Nef to efficiently interfere with superinfection at the virus entry step by downregulating cell surface receptors (36). Finally, vaccinia virus expresses in newly infected cells two surface proteins that mark cells as infected and induce the repulsion of superinfecting viruses (18). In the present study, we described a reverse-genetics system for VHSV allowing the generation of a wild-type-like recombinant VHSV and a recombinant virus expressing a red fluorescent protein (Tomato). The system is based on the French strain 23/75 of VHSV, which is a hypervirulent and devastating strain for farmed rainbow trout belonging to genotype I (serotype III) and was isolated in France in 1975 from a brown trout (16, 23). Thus, this system provides a suitable starting point for identifying potential virulence determinants, as demonstrated by the deletion of the NV gene, and for developing attenuated derivatives as candidate vaccines. Using the available reverse-genetics system elaborated with IHNV, a recombinant IHNV expressing a green fluorescent protein (GFP) also was produced (8), and it was of interest to study whether a superinfection exclusion phenomenon could be observed between both VHSV and IHNV, whose cohabitation has been recorded often. We showed that up to 74% of a cell monolayer could be simultaneously infected by the viruses, demonstrating a limited viral interference between salmonid novirhabdoviruses and that, based on previous data, chimeric or pseudotyped viruses could be generated (6).

This research was originally published in The Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology; International audience; Chronic hepatitis C virus (HCV) infection is associated with altered lipid metabolism and hepatocellular steatosis. Virus-induced steatosis is a cytopathic effect of HCV replication. The goal of this study was to examine the mechanisms underlying HCV-induced lipid metabolic defects in a transgenic mouse model expressing the full HCV protein repertoire at levels corresponding to natural human infection. In this model, expression of the HCV full-length open reading frame was associated with hepatocellular steatosis and reduced plasma triglyceride levels. Triglyceride secretion was impaired, whereas lipogenesis was activated. Increased lipogenic enzyme transcription was observed, resulting from maturational activation and nuclear translocation of sterol regulatory element-binding protein 1c (SREBP1c). However, endoplasmic reticulum (ER) stress markers were expressed at similar levels in both HCV transgenic mice and their wild type counterparts, suggesting that SREBP1c proteolytic cleavage in the presence of HCV proteins was independent of ER stress. In conclusion, transgenic mice expressing the HCV full-length polyprotein at low levels have decreased plasma triglyceride levels and develop hepatocellular steatosis in the same way as HCV-infected patients. In these mice, SREBP1c activation by one or several HCV proteins induces de novo triglyceride synthesis via the lipogenic pathway, in a manner independent of ER stress, whereas triglyceride secretion is simultaneously reduced.

International audience; Abdominal pain is common in the general population and, in patients with irritable bowel syndrome, is attributed to visceral hypersensitivity. We found that oral administration of specific Lactobacillus strains induced the expression of mu-opioid and cannabinoid receptors in intestinal epithelial cells, and mediated analgesic functions in the gut-similar to the effects of morphine. These results suggest that the microbiology of the intestinal tract influences our visceral perception, and suggest new approaches for the treament of abdominal pain and irritable bowel syndrome.

International audience

International audience; Novirhabdoviruses like the Viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses infecting fish. In the current study, RNA genomes of different VHSV field isolates classified as high, medium or low virulent phenotypes have been sequenced by next generation sequencing and compared. Various amino acid changes, depending on the VHSV phenotype, have been identified in all the VHSV proteins. As a starting point, we focused our study on the non-virion (NV) non-structural protein in which an arginine residue (R116) is present in all the virulent isolates and replaced by a serine/asparagine residue S/N116 in the attenuated isolates. A recombinant virus derived from a virulent VHSV strain in which the NV R116 residue has been replaced by a serine, rVHSV(NVR116S), was generated by reverse genetics and used to infect juvenile trout. We showed that rVHSV(NvR116s) was highly attenuated and that surviving fish were almost completely protected from a challenge with the wild-type VHSV.