RESUMEN
Spring viremia of carp virus (SVCV) has a broad fish host spectrum and is responsible for a disease that generally affects juvenile fishes with a mortality rate of up to 90%. In the absence of treatments or vaccines against SVCV, the search for prophylactic or therapeutic solutions is thus relevant, particularly to identify solutions compatible with mass vaccination. In addition to being a threat to aquaculture and ecosystems, SVCV is a unique pathogen to study virus-host interactions in the zebrafish model. Establishing the first reverse genetics system for SVCV and the design of recombinant SVCV (rSVCV) expressing fluorescent or bioluminescent proteins adds a new dimension for the study of these interactions using innovative imaging techniques. The infection by bath immersion of zebrafish larvae with rSVCV expressing mCherry allows us to define the first SVCV replication sites and the host innate immune responses using different transgenic lines of zebrafish. The fins were found as the main initial sites of infection in both zebrafish and carp, its natural host. Hence, new insights into the physiopathology of SVCV infection have been described. We report that neutrophils are recruited at the sites of infection and persist up to the death of the animal leading to an uncontrolled inflammation correlated with the expression of the pro-inflammatory cytokine IL1ß. Tissue damage was observed at the site of initial replication, a likely consequence of virus-induced injury or the pro-inflammatory response. Interestingly, SVCV infection by bath immersion triggers a persistent pro-inflammatory response rather than activation of the antiviral IFN signaling pathway as observed following intravenous injection, highlighting the importance of the route of infection on the progression of pathogenicity. Thus, this model of zebrafish larvae infection by rSVCV offers new perspectives to study in detail virus-host interactions and to discover new prophylactic or therapeutic solutions.
Asunto(s)
Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Pez Cebra , Animales , Pez Cebra/virología , Rhabdoviridae/fisiología , Enfermedades de los Peces/virología , Infecciones por Rhabdoviridae/virología , Infecciones por Rhabdoviridae/inmunología , Carpas/virología , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Inmunidad Innata , ViremiaRESUMEN
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.
Asunto(s)
Septicemia Hemorrágica Viral , Nodaviridae , Novirhabdovirus , Vacunas , Animales , Nodaviridae/genética , Glicoproteínas , AntígenosRESUMEN
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.
Asunto(s)
Alphavirus/metabolismo , Antivirales/farmacología , Proteína 58 DEAD Box/metabolismo , Interferones/metabolismo , Salmonidae/virología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Alphavirus/genética , Infecciones por Alphavirus/virología , Animales , Línea Celular , Virus Chikungunya , Enfermedades de los Peces/virología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Inmunidad Innata , Interferón Tipo I/metabolismo , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
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.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Peces/genética , Peces/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras , Proteína 58 DEAD Box/genética , Peces/inmunología , Regulación de la Expresión Génica , Homeostasis , Inmunidad Innata , Interferones/biosíntesis , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismo , Unión Proteica , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
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.
Asunto(s)
Cyprinidae/genética , Cyprinidae/inmunología , Proteína 58 DEAD Box/genética , Enfermedades de los Peces/inmunología , Inmunidad Innata/genética , Interferones/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/fisiología , Animales , Línea Celular , Proteína 58 DEAD Box/metabolismo , Retroalimentación Fisiológica , Proteínas de Peces/genética , Proteínas de Peces/fisiología , Interferones/metabolismo , Novirhabdovirus/fisiología , Filogenia , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/veterinaria , Transducción de Señal , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genéticaRESUMEN
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.
Asunto(s)
Enfermedades de los Peces/metabolismo , Proteínas de Peces/metabolismo , Interferones/metabolismo , Novirhabdovirus/metabolismo , Oncorhynchus mykiss/metabolismo , Proteína Fosfatasa 2C/metabolismo , Infecciones por Rhabdoviridae/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Enfermedades de los Peces/genética , Enfermedades de los Peces/virología , Novirhabdovirus/genética , Oncorhynchus mykiss/virología , Infecciones por Rhabdoviridae/genética , Proteínas Virales/genéticaRESUMEN
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).
Asunto(s)
Infecciones por Alphavirus/inmunología , Alphavirus/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/química , Enfermedades de los Peces/inmunología , Proteínas del Envoltorio Viral/inmunología , Alphavirus/genética , Infecciones por Alphavirus/genética , Infecciones por Alphavirus/virología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/química , Línea Celular , Mapeo Epitopo , Epítopos/inmunología , Enfermedades de los Peces/genética , Enfermedades de los Peces/virología , Expresión Génica , Interacciones Huésped-Patógeno , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Oncorhynchus mykiss/virología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
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.
Asunto(s)
Alphavirus/genética , Alphavirus/patogenicidad , Sustitución de Aminoácidos , Glicoproteínas/genética , Mutación Missense , Proteínas Virales/genética , Factores de Virulencia/genética , Animales , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Enfermedades de los Peces/virología , Glicoproteínas/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Análisis de Supervivencia , Trucha , Proteínas Virales/metabolismo , Virulencia , Factores de Virulencia/metabolismoRESUMEN
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).
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ARN Helicasas DEAD-box/metabolismo , Peces/metabolismo , Interferones/biosíntesis , Proteínas de la Membrana/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Virus ADN/inmunología , Retículo Endoplásmico/metabolismo , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Peces/genética , Peces/inmunología , Expresión Génica , Regulación de la Expresión Génica , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutación , Filogenia , Unión Proteica , Transporte de Proteínas , Virus ARN/inmunología , Alineación de Secuencia , Virosis/genética , Virosis/inmunología , Virosis/virologíaRESUMEN
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 5' 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 haemagglutinin-esterase 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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Genoma Viral , Isavirus/genética , Isavirus/inmunología , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Neutralización , ARN Viral/genética , Ensayo de Radioinmunoprecipitación , Análisis de Secuencia de ADN , Proteínas Virales/genéticaRESUMEN
The genome sequence of a hypervirulent novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) French strain 23-75, was determined. Compared to the genome of the prototype Fil3 strain, a number of substitutions, deletions, and insertions were observed. Following the establishment of a plasmid-based minigenome replication assay, recombinant VHSV (rVHSV) was successfully recovered. rVHSV exhibits wild-type-like growth properties in vitro as well as in vivo in rainbow trout. The dispensable role of NV for the novirhabdovirus replication was confirmed by generating rVHSV-DeltaNV, in which the NV gene was deleted. This deletion mutant was shown to be as debilitated as that previously described for infectious hematopoietic necrosis virus (IHNV), a distantly related novirhabdovirus (S. Biacchesi, M. I. Thoulouze, M. Bearzotti, Y. X. Yu, and M. Bremont, J. Virol. 74:11247-11253, 2000). Recombinant VHSV and IHNV expressing tdTomato and GFP(max) reporter genes, respectively, were generated, demonstrating the potential of these rhabdoviruses to serve as viral vectors. Interestingly, rIHNV-GFP(max) could be recovered using the replicative complex proteins of either virus, whereas rVHSV-Tomato could be recovered only by using its own replicative complex, reflecting that the genome signal sequences of VHSV are relatively distant from those of IHNV and do not allow their cross-recognition. Moreover, the use of heterologous protein combinations underlined the importance of strong protein-protein interactions for the formation of a functional ribonucleoprotein complex. The rIHNV-GFP(max) and rVHSV-Tomato viruses were used to simultaneously coinfect cell monolayers. It was observed that up to 74% of the cell monolayer was coinfected by both viruses, demonstrating that a limited interference phenomenon exists during the early stage of primary infection, and it was not mediated by a cellular antiviral protein or by some of the viral proteins.
Asunto(s)
Virus de la Necrosis Hematopoyética Infecciosa/fisiología , Novirhabdovirus/fisiología , Infecciones por Rhabdoviridae/virología , Interferencia Viral/fisiología , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , ADN Viral/genética , Genes Reporteros , Genoma Viral , Proteínas Fluorescentes Verdes/genética , Técnicas In Vitro , Virus de la Necrosis Hematopoyética Infecciosa/genética , Virus de la Necrosis Hematopoyética Infecciosa/patogenicidad , Datos de Secuencia Molecular , Mutación , Novirhabdovirus/genética , Novirhabdovirus/patogenicidad , Oncorhynchus mykiss , Plásmidos/genética , Recombinación Genética , Interferencia Viral/genética , Virulencia/genética , Virulencia/fisiología , Replicación ViralRESUMEN
Viral infection triggers host innate immune responses through cellular sensor molecules which activate multiple signaling cascades that induce the production of interferons (IFN) and other cytokines. The recent identification of mammalian cytoplasmic viral RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and their mitochondrial adaptor, the mitochondrial antiviral signaling protein (MAVS), also called IPS-1, VISA, and Cardif, highlights the significance of these molecules in the induction of IFN. Teleost fish also possess a strong IFN system, but nothing is known concerning the RLRs and their downstream adaptor. In this study, we cloned MAVS cDNAs from several fish species (including salmon and zebrafish) and showed that they were orthologs of mammalian MAVS. We demonstrated that overexpression of these mitochondrial proteins in fish cells led to a constitutive induction of IFN and IFN-stimulated genes (ISGs). MAVS-overexpressing cells were almost fully protected against RNA virus infection, with a strong inhibition of both DNA and RNA virus replication (1,000- and 10,000-fold decreases, respectively). Analyses of MAVS deletion mutants showed that both the N-terminal CARD-like and C-terminal transmembrane domains, but not the central proline-rich region, were indispensable for MAVS signaling function. In addition, we cloned the cDNAs encoding a RIG-I-like molecule from salmonid and cyprinid cell lines. Like the case with MAVS, overexpression of RIG-I CARDs in fish cells led to a strong induction of both IFN and ISGs, conferring on fish cells full protection against RNA virus infection. This report provides the first demonstration that teleost fish possess a functional RLR pathway in which MAVS may play a central role in the induction of the innate immune response.
Asunto(s)
Virus ADN/fisiología , Enfermedades de los Peces/virología , Proteínas de Peces/inmunología , Peces/inmunología , Proteínas Mitocondriales/inmunología , Virus ARN/fisiología , Transducción de Señal , Virosis/veterinaria , Secuencia de Aminoácidos , Animales , Línea Celular , Enfermedades de los Peces/inmunología , Proteínas de Peces/química , Proteínas de Peces/genética , Peces/clasificación , Peces/genética , Peces/virología , Inmunidad Innata , Interferones/inmunología , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Virosis/inmunología , Virosis/virologíaRESUMEN
Sleeping disease virus (SDV) is a member of the new Salmonid alphavirus genus within the Togaviridae family. The single-stranded RNA genome of SDV is 11,894 nucleotides long, excluding the 3' poly(A) tail. A full-length cDNA has been generated; the cDNA was fused to a hammerhead ribozyme sequence at the 5' end and inserted into a transcription plasmid (pcDNA3) backbone, yielding pSDV. By transfection of pSDV into fish cells, recombinant SDV (rSDV) was successfully recovered. Demonstration of the recovery of rSDV was provided by immunofluorescence assay on rSDV-infected cells and by the presence of a genetic tag, a BlpI restriction enzyme site, introduced into the rSDV RNA genome. SDV infectious cDNA was used for two kinds of experiments (i) to evaluate the impact of various targeted mutations in nsP2 on viral replication and (ii) to study the virulence of rSDV in trout. For the latter aspect, when juvenile trout were infected by immersion in a water bath with the wild-type virus-like rSDV, no deaths or signs of disease appeared in fish, although they were readily infected. In contrast, cumulative mortality reached 80% in fish infected with the wild-type SDV (wtSDV). When rSDV-infected fish were challenged with wtSDV 3 and 5 months postinfection, a long-lasting protection was demonstrated. Interestingly, a variant rSDV (rSDV14) adapted to grow at a higher temperature, 14 degrees C instead of 10 degrees C, was shown to become pathogenic for trout. Comparison of the nucleotide sequences of wtSDV, rSDV, and rSDV14 genomes evidenced several amino acid changes, and some changes may be linked to the pathogenicity of SDV in trout.