T-cells and CD45-cells discovery in the central nervous system of healthy and nodavirus-infected teleost fish Dicentrarchuslabrax.

To achieve insights in antiviral immune defense of the central nervous system (CNS), we investigated T cells and CD45 cells in the marine fish model Dicentrarchus labrax infected with the CNS-tropic virus betanodavirus. By employing markers for pan-T cells (mAb DLT15) and CD45-cells (mAb DLT22) in immunofluorescence (IIF) of leukocytes from brain, we obtained 3,7 ± 2.3 % of T cells and 7.3 ± 3.2 % of CD45+ cells. Both IIF and immunoelectron microscopy confirmed a leukocyte/glial morphology for the immunoreactive cells. Quantitative immunohistochemistry (qIHC) of brain/eye sections showed 1.9 ± 0.8 % of T+ cells and 2 ± 0.9 % of CD45+ cells in the brain, and 3.6 ± 1.9 % and 4.1 ± 2.2 % in the eye, respectively. After in vivo RGNNV infection the number of T cells/CD45+ leukocytes in the brain increased to 8.3 ± 2.1 % and 11.6 ± 4.4 % (by IIF), and 26.1 ± 3.4 % and 45.6 ± 5.9 % (by qIHC), respectively. In the eye we counted after infection 8.5 ± 4.4 % of T cells and 10.2 ± 5.8 % of CD45 cells. Gene transcription analysis of brain mRNA revealed a strong increase of gene transcripts coding for: antiviral proteins Mx and ISG-12; T-cell related CD3ε/δ, TcRß, CD4, CD8α, CD45; and for immuno-modulatory cytokines TNFα, IL-2, IL-10. A RAG-1 gene product was also present and upregulated, suggesting somatic recombination in the fish brain. Similar transcription data were obtained in the eye, albeit with differences. Our findings provide first evidence for a recruitment and involvement of T cells and CD45+ leukocytes in the fish eye-brain axis during antiviral responses and suggest similarities in the CNS immune defense across evolutionary distant vertebrates.

Genomic characterization and transcription analysis of European sea bass (Dicentrarchus labrax) rtp3 genes.

Fish RTP3, belonging to the receptor-transporting protein family, display several functions, including a putative antiviral role as virus-responsive gene. In this work, we have identified and characterized two different European sea bass rtp3 genes. In addition, an in vivo transcription analysis in response to LPS, poly I:C and betanodavirus infection (RGNNV genotype) has been performed. The sequence analysis showed that European sea bass displays two rtp3 genes, X1 and X2, composed of two exons and a single intron (1007-bp and 888-bp long, respectively), located within the ORF sequence. The full-length cDNA is 1969 bp for rtp3 X1, and 1491 bp for rtp3 X2. Several ATTTA motifs have been found in the intron sequence of both genes, whereas rtp3 X1 also contains this motif in both untranslated regions. The transcription analyses revealed significant level of rtp3 X2 mRNA in brain and head kidney after LPS and poly I:C inoculation; however, the induction elicited by RGNNV infection was much higher, suggesting an essential role for this protein in controlling NNV infections.

The genetic variability and evolution of red-spotted grouper nervous necrosis virus quasispecies can be associated with its virulence.

Nervous necrosis virus, NNV, is a neurotropic virus that causes viral nervous necrosis disease in a wide range of fish species, including European sea bass (Dicentrarchus labrax). NNV has a bisegmented (+) ssRNA genome consisting of RNA1, which encodes the RNA polymerase, and RNA2, encoding the capsid protein. The most prevalent NNV species in sea bass is red-spotted grouper nervous necrosis virus (RGNNV), causing high mortality in larvae and juveniles. Reverse genetics studies have associated amino acid 270 of the RGNNV capsid protein with RGNNV virulence in sea bass. NNV infection generates quasispecies and reassortants able to adapt to various selective pressures, such as host immune response or switching between host species. To better understand the variability of RGNNV populations and their association with RGNNV virulence, sea bass specimens were infected with two RGNNV recombinant viruses, a wild-type, rDl956, highly virulent to sea bass, and a single-mutant virus, Mut270Dl965, less virulent to this host. Both viral genome segments were quantified in brain by RT-qPCR, and genetic variability of whole-genome quasispecies was studied by Next Generation Sequencing (NGS). Copies of RNA1 and RNA2 in brains of fish infected with the low virulent virus were 1,000-fold lower than those in brains of fish infected with the virulent virus. In addition, differences between the two experimental groups in the Ts/Tv ratio, recombination frequency and genetic heterogeneity of the mutant spectra in the RNA2 segment were found. These results show that the entire quasispecies of a bisegmented RNA virus changes as a consequence of a single point mutation in the consensus sequence of one of its segments. Sea bream (Sparus aurata) is an asymptomatic carrier for RGNNV, thus rDl965 is considered a low-virulence isolate in this species. To assess whether the quasispecies characteristics of rDl965 were conserved in another host showing different susceptibility, juvenile sea bream were infected with rDl965 and analyzed as above described. Interestingly, both viral load and genetic variability of rDl965 in seabream were similar to those of Mut270Dl965 in sea bass. This result suggests that the genetic variability and evolution of RGNNV mutant spectra may be associated with its virulence.

Cytokine-like activity of European sea bass ISG15 protein on RGNNV-infected E-11 cells.

IFN-I generates an antiviral state by inducing the expression of numerous genes, called IFN-stimulated genes, ISGs, including ISG15, which is the only ISG with cytokine-like activity. In a previous study, we developed the Dl_ISG15_E11 cell line, which consisted of E11 cells able to express and secrete sea bass ISG15. The current study is a step forward, analysing the effect of secreted sea bass ISG15 on RGNNV replication in E11 cells, and looking into its immunomodulatory activity in order to corroborate its cytokine-like activity. The medium from ISG15-produccing cells compromised RGNNV replication, as it has been demonstrated both, by reduction in the viral genome synthesis and, specially, in the yield of infective viral particles. The implication of sea bass ISG15 in this protection has been demonstrated by ISG15 removal, which decreased the percentage of surviving cells upon viral infection, and by incubation of RGNNV-infected cells with a recombinant sea bass ISG15 protein, which resulted in almost full protection. Furthermore, the immunomodulatory activity of extracellular sea bass ISG15 has been demonstrated, which reaffirms a cytokine-like role for this protein.

Immune Response of Senegalese Sole against Betanodavirus Mutants with Modified Virulence.

Nervous necrosis virus (NNV), genus Betanodavirus, the etiological agent of the viral encephalopathy and retinopathy (VER), presents a genome with two positive-sense single-stranded RNA segments. Striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV), together with reassortants RGNNV/SJNNV, are the betanodaviruses predominantly isolated in Southern Europe. An RGNNV/SJNNV reassortant isolated from Senegalese sole (wt160) causes high mortalities in this fish species. This virus presents differences in the sequence of the 3' non-coding region (NCR) of both segments compared to RGNNV and SJNNV reference strains. Previously, it has been reported that the reversion of two of these differences (nucleotides 1408 and 1412) in the RNA2 3'NCR to the SJNNV-type (recombinant r1408-1412) resulted in a decrease in sole mortality. In the present study, we have applied an OpenArray® to analyse the involvement of sole immune response in the virulence of several recombinants: the r1408-1412 and two recombinants, developed in the present study, harbouring mutations at positions 3073 and 3093 of RNA1 3'NCR to revert them to RGNNV-type. According to the correlation values and to the number of expressed genes, the infection with the RNA2-mutant provoked the most different immune response compared to the immune response triggered after the infection with the rest of the viruses, and the exclusive and high upregulation of genes related to the complement system. The infection with the RNA1-mutants also provoked a decrease in mortality and their replication was delayed at least 24 h compared to the wt160 replication, which could provoke the lag observed in the immune response. Furthermore, the infection with the RNA1-mutants provoked the exclusive expression of pkr and the downregulation of il17rc.

Immunogene expression analysis in betanodavirus infected-Senegalese sole using an OpenArray® platform.

The transcriptomic response of Senegalese sole (Solea senegalensis) triggered by two betanodaviruses with different virulence to that fish species has been assessed using an OpenArray® platform based on TaqMan™ quantitative PCR. The transcription of 112 genes per sample has been evaluated at two sampling times in two organs (head kidney and eye/brain-pooled samples). Those genes were involved in several roles or pathways, such as viral recognition, regulation of type I (IFN-1)-dependent immune responses, JAK-STAT cascade, interferon stimulated genes, protein ubiquitination, virus responsive genes, complement system, inflammatory response, other immune system effectors, regulation of T-cell proliferation, and proteolysis and apoptosis. The highly virulent isolate, wSs160.3, a wild type reassortant containing a RGNNV-type RNA1 and a SJNNV-type RNA2 segments, induced the expression of a higher number of genes in both tested organs than the moderately virulent strain, a recombinant harbouring mutations in the protruding domain of the capsid protein. The number of differentially expressed genes was higher 2 days after the infection with the wild type isolate than at 3 days post-inoculation. The wild type isolate also elicited an exacerbated interferon 1 response, which, instead of protecting sole against the infection, increases the disease severity by the induction of apoptosis and inflammation-derived immunopathology, although inflammation seems to be modulated by the complement system. Furthermore, results derived from this study suggest a potential important role for some genes with high expression after infection with the highly virulent virus, such as rtp3, sacs and isg15. On the other hand, the infection with the mutant does not induce immune response, probably due to an altered recognition by the host, which is supported by a different viral recognition pathway, involving myd88 and tbkbp1.

Differential immunogene expression profile of European sea bass (Dicentrarchus labrax, L.) in response to highly and low virulent NNV.

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut247+270Dl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of Mut247+270Dl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection, and therefore, their functional activity could be analysed in future studies. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinants of RGNNV virulence to European sea bass.

Comparative analysis of marine and freshwater viral haemorrhagic septicaemia virus (VHSV) isolates antagonistic activity.

Viral Haemorrhagic Septicaemia Virus (VHSV) isolates virulent to marine fish species can replicate in freshwater species, although producing little or no mortality. Conversely, isolates from freshwater fish do not cause disease in marine species. An inverse relationship between VHSV virulence and host mx gene up-regulation has been described for several fish species, suggesting that differences between the antagonistic activity exerted by these isolates might be involved in the outcome of infections. In this study, the antagonistic activity against the type I interferon system of two representative marine and freshwater VHSV isolates has been characterised using RTG-2 cells stably transfected with the luciferase gene under the control of the Senegalese sole mx (ssmx) promoter, RTG pssmx-luc cells. Both isolates exerted a dose-dependent negative effect on the activation of ssmx promoter, showing a notably different minimal viral dose to exert the antagonism. In particular, an inverse relationship between the minimal MOI required and the viral virulence to sole has been recorded, which suggests this parameter as a possible in vivo VHSV virulence marker. Furthermore, the quantification of the endogenous inf I, mx1 and mx3 mRNA has demonstrated differences between both isolates in their antagonistic activity. Besides, a different nv RNA kinetics, which seems to depend on specific cellular factors, has been recorded for both isolates. This knowledge could contribute to the development of efficient tools to fight against viral infections in fish farming. For that purpose, the RTG pssmx-luc cells may be a suitable in vitro tool to identify the molecular mechanisms underlying VHSV-host interactions.

Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo.

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV-type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C-terminal side of the capsid. However, the concurrent mutation in the N-terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C-terminal and N-terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double- and triple-mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C-terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N-terminal side is also important for viral replication in vivo.

Differential antiviral activity of European sea bass interferon-stimulated 15 protein (ISG15) against RGNNV and SJNNV betanodaviruses.

ISG15 is an antiviral protein acting intracellularly, by conjugation to viral or cellular proteins, or extracellularly, as cytokine. In this work, an in vitro system, consisting of E-11 cells over-expressing European sea bass ISG15 (Dl_ISG15_E11 cells), has been developed to evaluate the European sea bass ISG15 protein activity against RGNNV and SJNNV isolates. Regarding RGNNV, RNA2 copy number and viral titres were similar in E-11 and Dl_ISG15_E11 cells, and the cellular survival analyses demonstrated that Dl_ISG15_E11 cells were not protected from this virus. In contrast, ISG15 compromises SJNNV replication, since a reduction of the SJNNV genome synthesis has been recorded. The ISG15 anti-SJNNV activity was confirmed by viral titration and survival assays. In addition, a role of the intracellular ISG15 in modulating the transcription of endogenous genes has being recorded, with tlr3 gene being knocked out and e3 gene being up-regulated in RGNNV-inoculated Dl_ISG15_E11 cells. Sea bass ISG15 has also been detected extracellularly, and its activity has been evaluated by co-culture. The survival rate of RGNNV-inoculated E-11 cells increased from 25% to 46% when they were co-cultured with ISG15-producing cells. Similarly, the survival rate of SJNNV-inoculated E-11 cells increased from 27% to 51% in co-culture with ISG15-producing cells. To our knowledge, this is the first description of a differential antiviral activity of an ISG15 protein against two betanodavirus species, and the first evaluation of the cytokine-like activity of a fish ISG15 protein on non-immune cells.

Transcriptomic Profiles of Senegalese Sole Infected With Nervous Necrosis Virus Reassortants Presenting Different Degree of Virulence.

Betanodaviruses [nervous necrosis virus (NNV)] are the causative agent of the viral encephalopathy and retinopathy, a disease that affects cultured Senegalese sole (Solea senegalensis). NNV reassortants, combining genomic segments from redspotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV) genotypes, have been previously isolated from several fish species. The wild-type reassortant wSs160.03, isolated from Senegalese sole, has been proven to be more virulent to sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine) in the wSs160.03 capsid protein have allowed us to obtain a mutant reassortant (rSs160.03247+270), which provokes a 40% mortality decrease. In this study, the RNA-Seq technology has been used to comparatively analyze Senegalese sole transcriptomes in two organs (head kidney and eye/brain) after infection with wild-type and mutant strains. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wild-type isolate (with higher virulence), whereas 393 genes were differentially expressed in animals infected with the mutant strain (37.9% decrease in the number of DEGs). To study the biological functions of detected DEGs involved in NNV infection, a gene ontology (GO) enrichment analysis was performed. Different GO profiles were obtained in the following subclasses: (i) biological process; (ii) cellular component; and (iii) molecular function, for each viral strain tested. Immune response and proteolysis have been the predominant biological process after the infection with the wild-type isolate, whereas the infection with the mutant strain induces proteolysis in head kidney and inhibition of vasculogenesis in nervous tissue. Regarding the immune response, genes coding for proteins acting as mediators of type I IFN expression (DHX58, IRF3, IRF7) and IFN-stimulated genes (ISG15, Mx, PKR, Gig1, ISG12, IFI44, IFIT-1, to name a few) were upregulated in animals infected with the wild-type isolate, whereas no-differential expression of these genes was observed in samples inoculated with the mutant strain. The different transcriptomic profiles obtained could help to better understand the NNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment.

Immuno-related gene transcription and antibody response in nodavirus (RGNNV and SJNNV)-infected European sea bass (Dicentrarchus labrax L.).

The immune response of European sea bass to RGNNV and SJNNV infections has been evaluated by quantifying the transcription of some genes involved in the IFN I system, as well as in the inflammatory and adaptive immune mechanisms. The transcription of IFN-I, ISG-12, ISG-15 and MxA genes has been analyzed in brain and head kidney, showing that RGNNV genotype induces a more intense response of the IFN I system than SJNNV in both organs. In addition, the results obtained indicate the importance of the inflammatory response in nodavirus pathogenesis, with the transcription of IL-8 and TNF-α significantly higher in brain than in head kidney, being RGNNV the strongest inductor. An important difference between the immune response induced by both genotypes refers to the IgM titre in sera, which was higher in SJNNV-inoculated fish. The acquired response is also important locally, since TR-γ transcription is higher in brain than in head kidney (especially in the RGNNV-inoculated group). To our knowledge, this is the first study addressing the sea bass anti-SJNNV immune response.

Identification of an interferon-stimulated gene, isg15, involved in host immune defense against viral infections in gilthead seabream (Sparus aurata L.).

Interferons (IFNs) play a key role in the innate immunity of vertebrates against viral infections by inducing hundreds of IFN-stimulated genes (ISGs), such as isg15. Isg15 is an ubiquitin-like protein, which can conjugate cellular and viral proteins in a process called ISGylation, although it can also act as a cytokine-like protein. Gilthead seabream (Sparus aurata L.) is an important asymptomatic carrier of viral haemorrhagic septicaemia virus (VHSV) and nodavirus, representing a threat to other co-cultivated susceptible species. In order to better understand virus-host interactions in this fish species, this study addresses the identification and molecular characterization of seabream isg15 (sb-isg15). In addition, the modulation of transcript levels of sb-isg15 was analysed in SAF-1 cells and seabream acidophilic granulocytes (AGs) stimulated in vitro with different pathogen-associated molecular patterns (PAMPs) or inoculated with VHSV and striped jack nervous necrosis virus (SJNNV). The full-length cDNA of sb-isg15 gene, encoding a predicted protein of 155 amino acids, was identified and seen to share the same characteristics as other fish and mammalian isg15 genes. Here we report the clear induction of sb-isg15 transcript levels in SAF-1 cells and AGs stimulated with toll-like receptor (TLR) ligands, such as polyinosinic:polycytidylic acid (poly I:C) or genomic DNA from Vibrio anguillarum (VaDNA), respectively. Furthermore, VHSV and SJNNV inoculation induced a significant degree of sb-isg15 transcription in SAF-1 cells and AGs. However, the relative levels of viral RNA transcription showed that SJNNV replication seems to be more efficient than VHSV in both in vitro systems. Interestingly, sb-isg15 transcript induction elicited by VaDNA was reduced in VHSV- and SJNNV-inoculated AGs, suggesting an interference prompted by the viruses against the type I IFN system. Taken together, these findings support the use of seabream AGs as a valuable experimental system to study virus-host interactions, in which sb-isg15 seems to play an important role.

Molecular characterization and expression analyses of the Solea senegalensis interferon-stimulated gene 15 (isg15) following NNV infections.

Interferons are essential in fish resistance to viral infections. They induce interferon-stimulated genes, such as isg15. In this study, the Senegalese sole isg15 gene (ssisg15) has been characterized. As other isg15, ssisg15 contains a 402-bp intron sited in the 5'-UTR, and the full length cDNA is 1492-bp, including a 480-bp ORF. The expression analyses revealed basal levels of isg15 transcripts, and a clear induction after poly I:C injection, that reached maximum values in brain, head kidney and gills. The ssisg15 induction patterns were similar in RGNNV- and SJNNV-inoculated fish, whereas the reassortant (RG/SJ) isolate, which has higher replication fitness, triggered delayed but higher transcript levels. Furthermore, RG/SJ infection after poly I:C treatment reduced the induction of ssisg15 transcripts, suggesting an antagonistic mechanism against interferon type I system, that might allow an efficient viral replication at the initial steps of the infective process.

Genetic characterization and transcription analyses of the European sea bass (Dicentrarchus labrax) isg15 gene.

Fish interferons are cytokines involved in its resistance to viral infections by inducing the transcription of several interferon-induced genes, such as isg15. The aim of the present study was the genetic characterization of the European sea bass isg15 gene, describing the regulatory motifs found in its sequence. In addition, an in vivo analysis of transcription in response to betanodavirus (RGNNV genotype) and poly I:C has been performed. The analysis of the resulting sequences showed that sea bass isg15 gene is composed of two exons and a single 276-bp intron located at the 5'-UTR region. The full length cDNA is 1143-bp, including a 102-bp 5'-UTR region, a 474-bp ORF, and a 291-bp 3'-UTR region. Several mRNA-regulatory elements, including three unusual ATTTA instability motifs in the intron, and four ATTTA motifs along with a cytoplasmic polyadenylation element in the 3'-UTR region, have been found in this sequence. The in vivo analyses revealed a similar kinetics and level of transcription in fish brain and head kidney after poly I:C inoculation; however, the induction caused by RGNNV started earlier in brain, where the upregulation of isg15 gene transcription was high. The present study contributes to further characterize the European sea bass IFN I response against RGNNV infections.

SJNNV down-regulates RGNNV replication in European sea bass by the induction of the type I interferon system.

European sea bass is highly susceptible to the betanodavirus RGNNV genotype, although the SJNNV genotype has also been detected in this fish species. The coexistence of both genotypes may affect the replication of both viruses by viral interaction or by stimulation of the host antiviral defense system in which the IFN I system plays a key role. IFN I triggers the transcription of interferon-stimulated genes, including Mx genes, whose expression has been used as a reporter of IFN I activity. The present study evaluated the effect of a primary exposure to an SJNNV isolate on a subsequent RGNNV infection and analyzed the role of the IFN I system in controlling VNNV infections in sea bass using different in vivo approaches. VNNV infection and Mx transcription were comparatively evaluated after single infections, superinfection (SJ+RG) and co-infection (poly I:C+RG). The single RGNNV infection resulted in a 24% survival rate, whereas the previous SJNNV or poly I:C inoculation increased the survival rate up to 96 and 100%, respectively. RGNNV replication in superinfection was reduced compared with RGNNV replication after a single inoculation. Mx transcription analysis shows differential induction of the IFN I system by both isolates. SJNNV was a potent Mx inducer, whereas RGNNV induced lower Mx transcription and did not interfere with the IFN I system triggered by SJNNV or poly I:C. This study demonstrates that an antiviral state exists after SJNNV and poly I:C injection, suggesting that the IFN I system plays an important role against VNNV infections in sea bass.

Role of the IFN I system against the VHSV infection in juvenile Senegalese sole (Solea senegalensis).

Senegalese sole is susceptible to marine VHSV isolates but is not affected by freshwater isolates, which may indicate differences regarding virus-host immune system interaction. IFN I induces an antiviral state in fish, stimulating the expression of genes encoding antiviral proteins (ISG). In this study, the stimulation of the Senegalese sole IFN I by VHSV infections has been evaluated by the relative quantification of the transcription of several ISG (Mx, Isg15 and Pkr) after inoculation with marine (pathogenic) and freshwater (non-pathogenic) VHSV isolates. Compared to marine VHSV, lower levels of RNA of the freshwater VHSV induced transcription of ISG to similar levels, with the Isg15 showing the highest fold induction. The protective role of the IFN I system was evaluated in poly I:C-inoculated animals subsequently challenged with VHSV isolates. The cumulative mortality caused by the marine isolate in the control group was 68%, whereas in the poly I:C-stimulated group was 5%. The freshwater VHSV isolate did not cause any mortality. Furthermore, viral RNA fold change and viral titers were lower in animals from the poly I:C + VHSV groups than in the controls. The implication of the IFN I system in the protection observed was confirmed by the transcription of the ISG in animals from the poly I:C + VHSV groups. However, the marine VHSV isolate exerts a negative effect on the ISG transcription at 3 and 6 h post-inoculation (hpi), which is not observed for the freshwater isolate. This difference might be partly responsible for the virulence shown by the marine isolate.

Differential response of the Senegalese sole (Solea senegalensis) Mx promoter to viral infections in two salmonid cell lines.

Mx proteins are main effectors of the antiviral innate immune defence mediated by type I interferon (IFN I). The IFN I response is under a complex regulation; hence, one of the key issues in understanding virus-host interaction is the knowledge of the regulatory mechanisms governing this response. With this purpose, in this study Chinook salmon embryo cells (CHSE-214) and rainbow trout gonad cells (RTG-2) were transiently transfected with a vector containing the luciferase reporter gene under the control of the Senegalese sole Mx promoter. These transfected cells were infected with infectious pancreatic necrosis virus (IPNV), viral haemorrhagic septicaemia virus (VHSV) and epizootic haematopoietic necrosis virus (EHNV) at different doses in order to study the luciferase fold induction in response to viral infections. Transfected CHSE-214 cells infected with EHNV showed significant induction of the luciferase reporter gene, compared to control non-infected cells, at different times post infection (p.i.). The maximum expression was recorded at 24h p.i. in cells inoculated with 5 × 10(2)TCID50/mL (2.17 folds compared to control cells). In these cells, the infection with IPNV and VHSV did not result in the luciferase expression at any time and doses tested. In transfected RTG-2 cells, VHSV stimulated luciferase expression, obtaining a maximum activity at 48 h p.i. in cells infected with 5 × 10(2)TCID50/mL (2.9 folds compared to control cells), whereas RTG-2 cells infected with IPNV and EHNV did not show significant luciferase activity at any time point. The different induction of the Senegalese sole Mx promoter in CHSE-214 and RTG-2 cells after infection with the same viruses indicates that cell-specific factors are significantly involved in the IFN-signalling response, and, probably, on the success of the strategies of these viruses to escape the IFN mechanisms. The use of these two different cellular systems might be an interesting approach to identify such cellular factors.

Structural and functional characterization of the Senegalese sole (Solea senegalensis) Mx promoter.

Mx proteins are one of the most studied interferon-stimulated genes (ISGs). The antiviral activity against different fish viruses has been demonstrated for diverse fish Mx proteins, including the Senegalese sole (Solea senegalensis) Mx protein (SsMx). The aim of the current study is to characterize the structure and functional activity of the SsMx promoter. Several polyclonal cell populations expressing the luciferase reporter gene under the control of the SsMx promoter have been used to determine the ability of this promoter to drive the expression of the luciferase gene after poly I:C stimulation. In addition, the implication of each interferon-stimulated response element (ISRE) in the activation of the promoter has also been analysed. The genomic structure of the Senegalese sole and Japanese flounder Mx promoters (containing three ISREs) differs from the rest of the fish Mx promoters described to date. The ISRE1, the one closest to the start codon, is the main ISRE involved in the SsMx promoter activity, whereas ISRE2 and ISRE3 show a minor additive effect on this activity. Another feature differing SsMx promoter from the rest of the fish Mx promoters is the presence of a 24-bp GC island close to the ATG codon, including one Sp1 binding site, which may constitute the transcriptional start site. Furthermore, the SsMx promoter contains a gamma interferon activation site (GAS) element.

Antiviral specificity of the Solea senegalensis Mx protein constitutively expressed in CHSE-214 cells.

Interferons play a key role in fish resistance to viral infections by inducing the expression of antiviral proteins, such as Mx. The aim of the present study was to test the antiviral activity of the Senegalese sole Mx protein (SsMx) against RNA and DNA viruses pathogenic to fish, i.e. the infectious pancreatic necrosis virus (IPNV, dsRNA), the viral haemorrhagic septicaemia virus (VHSV, ssRNA), and the European sheatfish virus (ESV, dsDNA), using a CHSE-214 cell clone expressing this antiviral protein. A strong inhibition of IPNV and VHSV replication was recorded in SsMx-expressing cells, as has been shown by the virus yield reduction and the decrease in the synthesis of the viral RNA encoding the polyprotein (for IPNV) and the nucleoprotein (for VHSV). The titres of these viruses replicating on SsMx-expressing cells were 100 times lower than those recorded on non-transfected cells. In contrast, SsMx did not inhibit ESV replication since no significant differences were observed regarding the virus yield or the major capsid protein gene transcription in transfected and non-transfected cells.