Recombinant viral hemorrhagic septicemia virus with rearranged genomes as vaccine vectors to protect against lethal betanodavirus infection.

The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout (Oncorhynchus mykiss) and sole (Solea senegalensis). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture.

The C-Terminal Domain of Salmonid Alphavirus Nonstructural Protein 2 (nsP2) Is Essential and Sufficient To Block RIG-I Pathway Induction and Interferon-Mediated Antiviral Response.

Salmonid alphavirus (SAV) is an atypical alphavirus that has a considerable impact on salmon and trout farms. Unlike other alphaviruses, such as the chikungunya virus, SAV is transmitted without an arthropod vector, and it does not cause cell shutoff during infection. The mechanisms by which SAV escapes the host immune system remain unknown. By studying the role of SAV proteins on the RIG-I signaling cascade, the first line of defense of the immune system during infection, we demonstrated that nonstructural protein 2 (nsP2) effectively blocks the induction of type I interferon (IFN). This inhibition, independent of the protease activity carried by nsP2, occurs downstream of IRF3, which is the transcription factor allowing the activation of the IFN promoter and its expression. The inhibitory effect of nsP2 on the RIG-I pathway depends on the localization of nsP2 in the host cell nucleus, which is linked to two nuclear localization sequences (NLS) located in its C-terminal part. The C-terminal domain of nsP2 by itself is sufficient and necessary to block IFN induction. Mutation of the NLS of nsP2 is deleterious to the virus. Finally, nsP2 does not interact with IRF3, indicating that its action is possible through a targeted interaction within discrete areas of chromatin, as suggested by its punctate distribution observed in the nucleus. These results therefore demonstrate a major role for nsP2 in the control by SAV of the host cell's innate immune response. IMPORTANCE The global consumption of fish continues to rise, and the future demand cannot be met by capture fisheries alone due to limited stocks of wild fish. Aquaculture is currently the world's fastest-growing food production sector, with an annual growth rate of 6 to 8%. Recurrent outbreaks of SAV result in significant economic losses with serious environmental consequences for wild stocks. While the clinical and pathological signs of SAV infection are fairly well known, the molecular mechanisms involved are poorly described. In the present study, we focus on the nonstructural protein nsP2 and characterize a specific domain containing nuclear localization sequences that are critical for the inhibition of the host innate immune response mediated by the RIG-I pathway.

Deciphering the Fine-Tuning of the Retinoic Acid-Inducible Gene-I Pathway in Teleost Fish and Beyond.

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.

Zebrafish (Danio rerio) larvae as a model for real-time studies of propagating VHS virus infection, tissue tropism and neutrophil activity.

Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense single-stranded RNA virus that infects more than 140 different fish species. In this study, zebrafish larvae were employed as in vivo model organisms to investigate progression of disease, the correlation between propagation of the infection and irreversibility of disease, cell tropism and in situ neutrophil activity towards the VHSV-infected cells. A recombinant VHSV strain, encoding "tomato" fluorescence (rVHSV-Tomato), was used in zebrafish to be able to follow the progress of the infection in the live host in real-time. Two-day-old zebrafish larvae were injected into the yolk sac with the recombinant virus. The virus titre peaked 96 hr post-infection in zebrafish larvae kept at 18°C, and correlated with 33% mortality and high morbidity among the larvae. By utilizing the transgenic zebrafish line Tg(fli1:GFP)y1 with fluorescently tagged endothelial cells, we were able to demonstrate that the virus initially infected endothelial cells lining the blood vessels. By observing the rVHSV-Tomato infection in the neutrophil reporter zebrafish line Tg(MPX:eGFP)i114 , we inferred that only a subpopulation of the neutrophils responded to the virus infection. We conclude that the zebrafish larvae are suitable for real-time studies of VHS virus infections, allowing in vivo dissection of host-virus interactions at the whole organism level.

The Viral Hemorrhagic Septicemia Virus (VHSV) Markers of Virulence in Rainbow Trout (Oncorhynchus mykiss).

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.

VHSV Single Amino Acid Polymorphisms (SAPs) Associated With Virulence in Rainbow Trout.

The Viral Hemorrhagic Septicemia Virus (VHSV) is an OIE notifiable pathogen widespread in the Northern Hemisphere that encompasses four genotypes and nine subtypes. In Europe, subtype Ia impairs predominantly the rainbow trout industry causing severe rates of mortality, while other VHSV genotypes and subtypes affect a number of marine and freshwater species, both farmed and wild. VHSV has repeatedly proved to be able to jump to rainbow trout from the marine reservoir, causing mortality episodes. The molecular mechanisms regulating VHSV virulence and host tropism are not fully understood, mainly due to the scarce availability of complete genome sequences and information on the virulence phenotype. With the scope of identifying in silico molecular markers for VHSV virulence, we generated an extensive dataset of 55 viral genomes and related mortality data obtained from rainbow trout experimental challenges. Using statistical association analyses that combined genetic and mortality data, we found 38 single amino acid polymorphisms scattered throughout the complete coding regions of the viral genome that were putatively involved in virulence of VHSV in trout. Specific amino acid signatures were recognized as being associated with either low or high virulence phenotypes. The phylogenetic analysis of VHSV coding regions supported the evolution toward greater virulence in rainbow trout within subtype Ia, and identified several other subtypes which may be prone to be virulent for this species. This study sheds light on the molecular basis for VHSV virulence, and provides an extensive list of putative virulence markers for their subsequent validation.

A20 (tnfaip3) is a negative feedback regulator of RIG-I-Mediated IFN induction in teleost.

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.

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination.

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.

A single amino acid change in the non-structural NV protein impacts the virulence phenotype of Viral hemorrhagic septicemia virus in trout.

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, rVHSVNVR116S, was generated by reverse genetics and used to infect juvenile trout. We showed that rVHSVNVR116S was highly attenuated and that surviving fish were almost completely protected from a challenge with the wild-type VHSV.

NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction.

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.

Complete Protection against Influenza Virus H1N1 Strain A/PR/8/34 Challenge in Mice Immunized with Non-Adjuvanted Novirhabdovirus Vaccines.

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°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.

Attenuated Infectious Hematopoietic Necrosis Virus with Rearranged Gene Order as Potential Vaccine.

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.

Expanding the tools for identifying mononuclear phagocyte subsets in swine: Reagents to porcine CD11c and XCR1.

Pig is a domestic species of major importance in the agro-economy and in biomedical research. Mononuclear phagocytes (MNP) are organized in subsets with specialized roles in the orchestration of the immune response and new tools are awaited to improve MNP subset identification in the pig. We cloned pig CD11c cDNA and generated a monoclonal antibody to pig CD11c which showed a pattern of expression by blood and skin MNP subsets similar to humans. We also developed a porcine XCL1-mCherry dimer which specifically reacted with the XCR1-expressing dendritic cell subset of the type 1 lineage in blood and skin. These original reagents will allow the efficient identification of pig MNP subsets to study their role in physiological and pathological processes and also to target these cells in novel intervention and vaccine strategies for veterinary applications and preclinical evaluations.

Efficient Co-Replication of Defective Novirhabdovirus.

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-ΔN-Red and rVHSV-ΔP-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 107 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-ΔN-Red and rVHSV-ΔP-Green are independent viral particles which could propagate only by simultaneously infecting the same cells.

Rainbow trout (Oncorhynchus mykiss) muscle satellite cells are targets of salmonid alphavirus infection.

Sleeping 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.

Fine mapping of a salmonid E2 alphavirus neutralizing epitope.

In this study, we aimed to characterize the epitope recognized by the neutralizing 17H23 mAb directed against the E2 glycoprotein of most of salmonid alphavirus (SAV) subtypes and widely used in several laboratories to routinely diagnose SAV. We hypothesized that the 17H23 epitope was located in the major domain B, previously identified in the E2 of mammalian alphaviruses as the domain recognized by most of the E2 neutralizing mAbs. Indeed, the SAV E2 domain B counterpart is contained in the protein domain previously characterized as being recognized by mAb 17H23. Thus, to precisely characterize the 17H23 epitope, we developed an alanine scanning mutagenesis approach coupled with the generation of the respective recombinant SAV (rSAV) by using the available infectious cDNA. Ten mutant rSAVs termed A-J from E2 aa 223-236 were produced and characterized in vitro using indirect immunofluorescence assays on virus-infected cells with mAbs 17H23, 51B8 (another non-neutralizing anti-E2 mAb) and 19F3 directed against the non-structural protein nsp1. Two of the mutant rSAVs (G and H) escaped neutralization by mAb 17H23. In addition, we showed that when juvenile trout were infected by bath immersion with the rSAV mutants, some of them were either totally (D, E and G) or partially (H) attenuated. Together, the data from the in vitro and in vivo experiments indicated that the putative 17H23 amino acid sequence epitope comprised the short amino acid sequence (227)FTSDS(231).

In vitro and in vivo characterization of molecular determinants of virulence in reassortant betanodavirus.

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, rSs160.03247 (S247A), rSs160.03270 (S270N) and rSs160.03247+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.

A recombinant novirhabdovirus presenting at the surface the E Glycoprotein from West Nile Virus (WNV) is immunogenic and provides partial protection against lethal WNV challenge in BALB/c mice.

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 20°C 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.

High virulence differences among phylogenetically distinct isolates of the fish rhabdovirus viral hemorrhagic septicaemia virus are not explained by variability of the surface glycoprotein G or the non-virion protein Nv.

Viral hemorrhagic septicaemia virus (VHSV) is an important viral pathogen in European rainbow trout farming. Isolates from wild marine fish and freshwater trout farms show highly different virulence profiles: isolates from marine fish species cause little or no mortality in rainbow trout following experimental waterborne challenge, whilst challenge with rainbow trout isolates results in high levels of mortality. Phylogenetic analyses have revealed that the highly virulent trout-derived isolates from freshwater farms have evolved from VHSV isolates from marine fish host species over the past 60 years. Recent isolates from rainbow trout reared in marine zones show intermediate virulence. The present study aimed to identify molecular virulence markers that could be used to classify VHSV isolates according to their ability to cause disease in rainbow trout. By a reverse genetics approach using a VHSV-related novirhabdovirus [infectious hematopoietic necrosis virus (IHNV)], four chimaeric IHNV-VHSV recombinant viruses were generated. These chimaeric viruses included substitution of the IHNV glyco- (G) or non-structural (Nv) protein with their counterparts from either a trout-derived or a marine VHSV strain. Comparative challenge experiments in rainbow trout fingerlings revealed similar levels of survival induced by the recombinant (r)IHNV-VHSV chimaeric viruses regardless of whether the G or Nv genes originated from VHSV isolated from a marine fish species or from rainbow trout. Interestingly, recombinant IHNV gained higher virulence following substitution of the G gene with those of the VHSV strains, whilst the opposite was the case following substitution of the Nv genes.

Lack of correlation between the resistances to two rhabdovirus infections in rainbow trout.

The Viral Hemorrhagic Septicemia Virus (VHSV) and the Infectious Hematopoietic Necrosis Virus (IHNV) are two rhabdoviruses responsible for serious outbreaks in salmonid farms. To date, little is known about the variability of host response to these viruses. Using gynogenetic clonal lines of rainbow trout exhibiting a wide range of resistance to viral infections, we showed that there was no correlation between the resistance to VHSV and IHNV. We also confirmed the importance of fish weight for its susceptibility to IHNV infection. Finally, using a chimeric recombinant IHNV expressing the VHSV glycoprotein, we showed that the glycoprotein plays a key role in the virulence and in the level of resistance observed in different genetic backgrounds. Taken together, our results provide new prospects for a better understanding of host responses to rhabdovirus infections in salmonids.