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International audience; Salmonid alphavirus (SAV) is the etiological cause of pancreas disease (PD) in Atlantic salmon (Salmo salar). Several vaccines against SAV are in use, but PD still cause significant mortality and concern in European aquaculture, raising the need for optimal tools to monitor SAV immunity. To monitor and control the distribution of PD in Norway, all salmonid farms are regularly screened for SAV by RT-qPCR. While the direct detection of SAV is helpful in the early stages of infection, serological methods could bring additional information on acquired SAV immunity in the later stages. Traditionally, SAV antibodies are monitored in neutralization assays, but they are time-consuming and cumbersome, thus alternative assays are warranted. Enzyme-linked immunosorbent assays (ELISAs) have not yet been successfully used for anti-SAV antibody detection in aquaculture. We aimed to develop a bead-based immunoassay for SAV-specific antibodies. By using detergent-treated SAV particles as antigens, we detected SAV-specific antibodies in plasma collected from both a SAV challenge trial and a field outbreak of PD. Increased levels of SAV-specific antibodies were seen after most fish had become negative for viral RNA. The bead-based assay is time saving compared to virus neutralization assays, and suitable for non-lethal testing due to low sample size requirements. We conclude that the bead-based immunoassay for SAV antibody detection is a promising diagnostic tool to complement SAV screening in aquaculture.

B cell responses are a crucial part of the adaptive immune response to viral infection. Infection by salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) in Atlantic salmon (Salmo salar) and is a serious concern to the aquaculture industry. In this study, we have used intraperitoneal (IP) infection with SAV3 as a model to characterize local B cell responses in the peritoneal cavity (PerC) and systemic immune tissues (head kidney/spleen). Intraperitoneal administration of vaccines is common in Atlantic salmon and understanding more about the local PerC B cell response is fundamental. Intraperitoneal SAV3 infection clearly induced PerC B cell responses as assessed by increased frequency of IgM+ B cells and total IgM secreting cells (ASC). These PerC responses were prolonged up to nine weeks post-infection and positively correlated to the anti-SAV3 E2 and to neutralizing antibody responses in serum. For the systemic immune sites, virus-induced changes in B cell responses were more modest or decreased compared to controls in the same period. Collectively, data reported herein indicated that PerC could serve as a peripheral immunological site by providing a niche for prolonged maintenance of the ASC response in Atlantic salmon.

Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are involved in antiviral defense. Members of this protein family contain distinctive multiple structural motifs comprising tetratricopeptides that are tandemly arrayed or dispersed along the polypeptide. IFIT-encoding genes are upregulated by type I interferons (IFNs) and other stimuli. IFIT proteins inhibit virus replication by binding to and regulating the functions of cellular and viral RNA and proteins. In teleost fish, knowledge about genes and functions of IFITs is currently limited. In the present work, we describe an IFIT5 orthologue in Atlantic salmon (SsaIFIT5) with characteristic tetratricopeptide repeat motifs. We show here that the gene encoding SsaIFIT5 (SsaIfit5) was ubiquitously expressed in various salmon tissues, while bacterial and viral challenge of live fish and in vitro stimulation of cells with recombinant IFNs and pathogen mimics triggered its transcription. The profound expression in response to various immune stimulation could be ascribed to the identified IFN response elements and binding sites for various immune-relevant transcription factors in the putative promoter of the SsaIfit5 gene. Our results establish SsaIfit5 as an IFN-stimulated gene in A. salmon and strongly suggest a phylogenetically conserved role of the IFIT5 protein in antimicrobial responses in vertebrates.

The intraperitoneal route is favored for administration of inactivated and attenuated vaccines in Atlantic salmon. Nevertheless, the immune responses in the teleost peritoneal cavity (PerC) are still incompletely defined. In this study, we investigated the B cell responses after intraperitoneal Piscirickettsia salmonis (P. salmonis) challenge of Atlantic salmon, focusing on the local PerC response versus responses in the lymphatic organs: spleen and head kidney. We observed a major increase of leukocytes, total IgM antibody secreting cells (ASC), and P. salmonis-specific ASC in the PerC at 3- and 6-weeks post infection (wpi). The increase in ASC frequency was more prominent in the spleen and PerC compared to the head kidney during the observed 6 wpi. The serum antibody response included P. salmonis-specific antibodies and non-specific antibodies recognizing the non-related bacterial pathogen Yersinia ruckeri and the model antigen TNP-KLH. Finally, we present evidence that supports a putative role for the adipose tissue in the PerC immune response.

Source at https://doi.org/10.3389/fimmu.2019.00037. Fish immunology research is at a pivotal point with the increasing availability of functional immunoassays and major advances in omics approaches. However, studies on fish B cells and their distinct subsets remain a challenge due to the limited availability of differentially expressed surface markers. To address this constraint, cell surface proteome of Atlantic salmon IgM+ B cells were analyzed by mass spectrometry and compared to surface proteins detected from two adherent salmon head kidney cell lines, ASK and SSP-9. Out of 21 cluster of differentiation (CD) molecules identified on salmon IgM+ B cells, CD22 and CD79A were shortlisted as potential markers based on the reported B cell-specific surface expression of their mammalian homologs. Subsequent RT-qPCR analyses of flow cytometry-sorted subpopulations from head kidney leukocytes confirmed that both cd22 and cd79a genes were highly expressed in IgM+ lymphoid cells but were observed in barely detectable levels in IgM− non-lymphoid suspension and adherent cells. Similarly, significantly high cd22 and cd79a mRNA levels were observed in IgM+ or IgT+ lymphoid cells from the spleen and peritoneal cavity, but not in their corresponding IgM− IgT− non-lymphoid fractions. This suggests that the B cell restrictive expression of CD22 and CD79A extend down to the transcription level, which was consistent across different lymphoid compartments and immunoglobulin isotypes, thus strongly supporting the potential of CD22 and CD79A as pan-B cell markers for salmon. In addition, this study provides novel information on the salmon B cell surface protein repertoire, as well as insights on B cell evolution. Further investigation of the identified salmon CD molecules, including development of immunological tools for detection, will help advance our understanding of the dynamics of salmon B cell responses such as during infection, vaccination, or immunostimulation.

Viral diseases represent one of the major threats for salmonid aquaculture. Survival from viral infections are highly dependent on host innate antiviral immune defense, where interferons are of crucial importance. Neutralizing antibodies and T cell effector mechanisms mediate long-term antiviral protection. Despite an immune cell repertoire comparable to higher vertebrates, farmed fish often fail to mount optimal antiviral protection. In the quest to multiply and spread, viruses utilize a variety of strategies to evade or escape the host immune system. Understanding the specific interplay between viruses and host immunity at depth is crucial for developing successful vaccination and treatment strategies in mammals. However, this knowledge base is still limited for pathogenic fish viruses. Here, we have focused on five RNA viruses with major impact on salmonid aquaculture: Salmonid alphavirus, Infectious salmon anemia virus, Infectious pancreatic necrosis virus, Piscine orthoreovirus and Piscine myocarditis virus. This review explore the protective immune responses that salmonids mount to these viruses and the existing knowledge on how the viruses counteract and/or bypass the immune response, including their IFN antagonizing effects and their mechanisms to establish persisting infections.

Fish immunology research is at a pivotal point with the increasing availability of functional immunoassays and major advances in omics approaches. However, studies on fish B cells and their distinct subsets remain a challenge due to the limited availability of differentially expressed surface markers. To address this constraint, cell surface proteome of Atlantic salmon IgM+ B cells were analyzed by mass spectrometry and compared to surface proteins detected from two adherent salmon head kidney cell lines, ASK and SSP-9. Out of 21 cluster of differentiation (CD) molecules identified on salmon IgM+ B cells, CD22 and CD79A were shortlisted as potential markers based on the reported B cell-specific surface expression of their mammalian homologs. Subsequent RT-qPCR analyses of flow cytometry-sorted subpopulations from head kidney leukocytes confirmed that both cd22 and cd79a genes were highly expressed in IgM+ lymphoid cells but were observed in barely detectable levels in IgM− non-lymphoid suspension and adherent cells. Similarly, significantly high cd22 and cd79a mRNA levels were observed in IgM+ or IgT+ lymphoid cells from the spleen and peritoneal cavity, but not in their corresponding IgM− IgT− non-lymphoid fractions. This suggests that the B cell restrictive expression of CD22 and CD79A extend down to the transcription level, which was consistent across different lymphoid compartments and immunoglobulin isotypes, thus strongly supporting the potential of CD22 and CD79A as pan-B cell markers for salmon. In addition, this study provides novel information on the salmon B cell surface protein repertoire, as well as insights on B cell evolution. Further investigation of the identified salmon CD molecules, including development of immunological tools for detection, will help advance our understanding of the dynamics of salmon B cell responses such as during infection, vaccination, or immunostimulation.

While TLR-activated pathways are key regulators of B cell responses in mammals, their impact on teleost B cells are scarcely addressed. Here, the potential of Atlantic salmon B cells to respond to TLR ligands was shown by demonstrating a constitutive expression of nucleic-acid sensing TLRs in magnetic sorted IgM+ cells. Of the two receptors recognizing CpG in teleosts, tlr9 was the dominating receptor with over ten-fold higher expression than tlr21. Upon CpG-stimulation, IgM secretion increased for head kidney (HK) and splenic IgM+ cells, while blood B cells were marginally affected. The results suggest that CpG directly affects salmon B cells to differentiate into antibody secreting cells (ASCs). IgM secretion was also detected in the non-treated controls, again with the highest levels in the HK derived population, signifying that persisting ASCs are present in this tissue. In all tissues, the IgM+ cells expressed high MHCII levels, suggesting antigen-presenting functions. Upon CpG-treatment the co-stimulatory molecules cd83 and cd40 were upregulated, while cd86 was down-regulated under the same conditions. Finally, ifna1 was upregulated upon CpG-stimulation in all tissues, while a restricted upregulation was evident for ifnb, proposing that salmon IgM+ B cells exhibit a type I IFN-response.

This is the submitted manuscript version of the following article Iliev, D., Strandskog, G., Nepal, A., Aspar, A., Olsen, R., Jørgensen, J., ... Mironova, R. (2018). Stimulation of exosome release by extracellular DNA is conserved across multiple cell types. The FEBS Journal, 285(16), 3114-3133. https://doi.org/10.1111/febs.14601. Published version available at https://doi.org/10.1111/febs.14601. Exosomes are distinguished from other types of extracellular vesicles by their small and relatively uniform size (30-100 nm) and their composition which reflects their endo-lysosomal origin. Involvement of these extracellular organelles in intercellular communication and their implication in pathological conditions has fuelled intensive research on mammalian exosomes; however, currently, very little is known about exosomes in lower vertebrates. Here we show that, in primary cultures of head kidney leukocytes from Atlantic salmon (Salmo salar), phosphorothioate CpG oligodeoxynucleotides induce secretion of vesicles with characteristics very similar to these of mammalian exosomes. Further experiments revealed that the oligonucleotide-induced exosome secretion did not depend on the CpG motifs but it relied on the phosphorothioate modification of the internucleotide linkage. Exosome secretion was also induced by genomic bacterial and eukaryotic DNA in toll-like receptor 9-negative piscine and human cell lines demonstrating that this is a phylogenetically conserved phenomenon which does not depend on activation of immune signaling pathways. In addition to exosomes, stimulation with phosphorothioate oligonucleotides and genomic DNA induced secretion of LC3B-II, an autophagosome marker, which was associated with vesicles of diverse size and morphology, possibly derived from autophagosome-related intracellular compartments. Overall, this work reveals a previously unrecognized biological activity of phosphorothioate ODNs and genomic DNA – their capacity to induce secretion of exosomes and other types of extracellular vesicles. This finding might help shed light on the side effects of therapeutic phosphorothioate oligodeoxynucleotides and the biological activity of extracellular genomic DNA which is often upregulated in pathological conditions.

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Pancreas disease (PD) and heart and skeletal muscle inflammation (HSMI) are viral diseases associated with SAV (salmonid alphavirus) and PRV (piscine reovirus), which induce systemic infections and pathologies in cardiac and skeletal muscle tissue of farmed Atlantic salmon (Salmo salar L), resulting in severe morbidity and mortality. While general features of the clinical symptoms and pathogenesis of salmonid viral diseases are relatively well studied, much less is known about molecular mechanisms associated with immunity and disease-specific changes. In this study, transcriptomic analyses of heart tissue from PD and HSMI challenged Atlantic salmon were done, focusing on the mature phases of both diseases at respectively 28-35 and 42-77 days post infection. A large number of immune genes was activated in both trials with prevalence of genes associated with early innate antiviral responses, their expression levels being slightly higher in PD challenged fish. Activation of the IFN axis was in parallel with inflammatory changes that involved diverse humoral and cellular factors. Adaptive immune response genes were more pronounced in fish with HSMI, as suggested by increased expression of a large number of genes associated with differentiation and maturation of B lymphocytes and cytotoxic T cells. A similar down-regulation of non-immune genes such as myofiber and mitochondrial proteins between diseases was most likely reflecting myocardial pathology. A suite of genes important for cardiac function including B-type natriuretic peptide and four neuropeptides displayed differential expression between PD and HSMI. Comparison of results revealed common and distinct features and added to the understanding of both diseases at their mature phases with typical clinical pictures. A number of genes that showed disease-specific changes can be of interest for diagnostics.

Infectious pancreatic necrosis virus (IPNV) is a prevalent pathogen in fish worldwide. The virus causes substantial mortality in Atlantic salmon juveniles and smolts when transferred to sea water and persistent infection in surviving fish after disease outbreaks. Here, we have investigated the occurrence of the virus as well as the innate immune marker Mx in the head kidney (HK) of Atlantic salmon throughout an experimental challenge covering both a fresh and a seawater phase. The fish were challenged with a high (HV) and low virulence (LV) IPNV. Both isolates caused mortality due to reactivation of the virus after transfer to sea water. In the freshwater phase, higher levels of virus transcripts were detected in the HK of fish infected with LV IPNV compared to HV, suggesting that the HV isolate is able to limit its own replication to a level where the innate immune system is not alerted. Further, ex vivoHK leucocytes derived from fish infected with the two isolates were stimulated with CpG DNA. Significantly, higher IFN levels were found in the LV compared to the HV group in the freshwater phase. This suggests that the viruses attenuate the antiviral host immune response at different levels which may contribute to the observed differences in disease outcome.

Suppressor of cytokine signaling (SOCS) proteins are crucially involved in the control of inflammatory responses through their impact on various signaling pathways including the JAK/STAT pathway. Although all SOCS protein family members are identified in teleost fish, their functional properties in non-mammalian vertebrates have not been extensively studied. To gain further insight into SOCS functions in bony fish, we have identified and characterized the Atlantic salmon (Salmo salar) SOCS1, SOCS2 and CISH genes. These genes exhibited sequence conservation with their mammalian counterparts and they were ubiquitously expressed. SOCS1 in mammalian species has been recognized as a key negative regulator of interferon (IFN) signaling and recent data for the two model fish Tetraodon (Tetraodon nigroviridis) and zebrafish (Danio rerio) suggest that these functions are conserved from teleost to mammals. In agreement with this we here demonstrate a strong negative regulatory activity of salmon SOCS1 on type I and type II IFN signaling, while SOCS2a and b and CISH only moderately affected IFN responses. SOCS1 also inhibited IFNγ-induced nuclear localization of STAT1 and a direct interaction between SOCS1 and STAT1 and between SOCS1 and the Tyk2 kinase was found. Using SOCS1 mutants lacking either the KIR domain or the ESS, SH2 and SOCS box domains showed that all domains affected the ability of SOCS1 to inhibit IFN-mediated signaling. These results are the first to demonstrate that SOCS1 is a potent inhibitor of IFN-mediated JAK-STAT signaling in teleost fish.

Tyk2, a member of the Janus Kinase (JAK) family of protein tyrosine kinases, is required for interferon-α/β binding and signaling in higher vertebrates. Currently, little is known about the role of the different JAKs in signaling responses to interferon (IFN) in lower vertebrates including fish. In this paper we report the identification and characterization of Atlantic salmon (Salmo salar) Tyk2. Four cDNA sequences, two containing an open reading frame encoding full-length Tyk protein and two with an up-stream in frame stop codon, were identified. The deduced amino acid sequences of the salmon full-length Tyk2 proteins showed highest identity with Tyk2 from other species and their transcripts were ubiquitously expressed. Like in mammals the presented data suggests that salmon Tyk2 is auto-phosporylated when ectopically expressed in cells. In our experiments, full-length salmon Tyk2 overexpressed in CHSE-cells phosphorylated itself, while both a kinase-deficient mutant and the truncated Tyk2 (Tyk-short) were inactive. Interestingly, the overexpression of full length Tyk2 was shown to up-regulate the transcript levels of the IFN induced gene Mx, thus indicating the involvement of salmon Tyk2 in the salmon IFN I pathway.

Two strains of Atlantic salmon ( Salmon salar ) with different susceptibility to infectious salmon anaemia (ISA) were challenged with salmon pancreas disease virus (SPDV), the etiological agent of salmon pancreas disease (PD), by cohabitation. Serum and tissues were sampled at 0, 1, 3, 6 and 8 weeks post-challenge. Experimental challenge with SAV did not cause mortality, but virus loads and assessment of histopathology indicated that the fish more resistant to ISAV (ISAHi) also was more resistant to PD. Eight weeks post-challenge, the ISAHi strain had higher titres of SAV-neutralising antibodies than the less resistant strain (ISALo). Transcript levels of four adaptive and six innate immune parameters were analysed by real-time RT-PCR in heart, head kidney (HK) and gills of both strains. Secretory IgM (sIgM) and CD8 levels differed most between the two salmon strains. The ISAHi strain had significantly higher levels of sIgM in HK at all samplings, and significantly higher CD8 levels in gills at most samplings. In heart, both sIgM and CD8 levels increased significantly during the challenge, but the increase appeared earlier for the ISALo strain. By hierarchical clustering analysis of mRNA levels, a clear segregation was observed between the two strains prior to the virus challenge. As the viral infection developed, the clustering divide between fish strains disappeared, first for innate and later for adaptive parameters. At eight weeks post-challenge, the divide had however reformed for adaptive parameters. Possible pair-wise correlation between transcript levels of immune parameters was evaluated by a non-parametric statistical test. For innate parameters, the extent of correlation peaked at 3 wpc in all tissues; this came rapidly for ISALo and more gradual for ISAHi. The ISAHi strain tended to show higher correlation for innate parameters in heart and gill than ISALo at early sampling times. For adaptive immune parameters, little correlation was observed in general, except for ISAHi in heart at 6 wpc. Overall, the observed differences in immune parameters may provide important clues to the causes underlying the observed difference in susceptibility to PD.

The CD40L/CD40 signalling pathway is critically involved in the final stage of the maturation of DCs. This paper reports the identification and functional characterization of CD40L and CD40 from Atlantic salmon (Salmo salar). Salmon CD40L is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, while CD40 is a type I membrane-bound receptor with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The salmon CD40L and CD40 were widely expressed, particularly in immune tissues, and while CD40L expression was induced by in vitro stimulation of HKLs with PHA and ConA, CpG increased CD40 expression. A CD40L construct was overexpressed in the CHSE-214 cell line and co-cultivation of the CD40L-CHSE transfectants with HKL induced a rapid and long-lasting upregulation of important costimulatory molecules like CD40, CD83, B7-H1 and the cytokines IL-12p40, IL-10, IL-1β and IFNs, which all are involved in T-helper cell responses. Furthermore, the CD40L transfected cells increased the percentage of HKLs expressing surface MHCIIβ but unlike other APC maturation stimuli, like CpG, they did not reduce the capacity to internalise antigen. Our results provide the first evidence for the existence of a functional CD40L mediated costimulatory pathway in Atlantic salmon.

The SOCS proteins are regulators of JAK/STAT signaling. A number of viral infections has been associated with SOCS upregulation. Here we report that SOCS1 mRNA expression is up-regulated in salmon alphavirus (SAV3) infected TO cells, while infectious pancreatic necrosis virus infection has a negligible effect. SAV3 infected salmon showed increased SOCS1 mRNA levels in heart correlating with increased viral transcripts. Together, the in vitro and in vivo data suggests that SAV3-induced SOCS1 expression may affect the outcome of the virus infection. Using CHSE-214 cells overexpressing SOCS1 we revealed increased SAV3 replication compared to control, suggesting that SOCS1 suppress the antiviral capacity of the cells. In IFNa1-treated cells, the suppression of viral replication was partially rescued by SOCS1 overexpression. The increased viral replication in SOCS1 transgene cells was likely a result of impaired IFN-signaling and the reduced expression of interferon-stimulated genes in the transgene cells underscores this.

MyD88 is an intracellular adaptor protein that transmits signals downstream of immune receptors such as the IL-1 receptor and the majority of the known mammalian toll-like receptors. Homologs of MyD88 have been identified in many vertebrate species; however, the adaptor has been studied mostly in mammals, and little is known about its function in lower vertebrates. The results presented in the current paper demonstrate, for the first time, that the teleost MyD88, through its Toll/Interleukin-1 receptor domain, interacts with SsIRF3 and two SsIRF7 paralogs: transcription factors that are critically involved in the virus-induced IFN responses. The data further highlight the potential of transgenic SsMyD88 to modulate the IRF-induced type I IFN response as the adaptor synergized with SsIRF3 to activate IRF-E/IFN-stimulated response element-containing reporter gene constructs and endogenous myxovirus resistance homolog expression. Microscopy analyses demonstrated that, similar to mammalian MyD88, both endogenous and transgenic SsMyD88 accumulated in intracellular aggregates. However, unlike the endogenous SsMyD88 clusters, which co-localized with endocytosed CpGs and probably represented myddosomes, overexpressed SsMyD88 accumulated in aggresomes. Although these structures accumulated ubiquitinated proteins, they did not associate with the autophagosome markers p62 and light chain 3-like protein, indicating that they are most likely classical aggresomes rather than aggresome-like induced structures, aggregates of ubiquitinated proteins induced by toll-like receptor/MyD88 signaling in antigen-presenting cells. The significance of the accumulation of transgenic MyD88 in aggresomes is currently unknown; nevertheless it is tempting to speculate that it might represent a defense mechanism against the potentially harmful effects of excessive MyD88 signaling.

Viral diseases are one of the main problems and risk factors in aquaculture. At present diseases are diagnosed by detection of pathogens and clinical symptoms. Identification of genes involved in early responses to viruses is important for better knowledge of antiviral defence and development of diagnostic tools. The aim of this study was to search for gene markers common for viral infections in Atlantic salmon based on microarray analyses of a wide range of samples. Gene expression profiles from fish and cell cultures infected with different viruses and treated with the synthetic double-stranded RNA poly(I:C) were compared in order to identify virus responsive genes (VRG). The list of VRG defined in this study contained 117 genes with known or unidentified functions. Several genes, including the most highly ranked one (receptor transporting protein), had not been previously reported to be involved in antiviral defence. VRG were characterized by a rapid induction and low tissue specificity, and their expression levels were related to the viral load. Immunofluorescence analyses of proteins encoded by VRG in cardiac tissue of salmon with the viral disease cardiomyopathy syndrome (CMS) revealed a common expression pattern. In head kidney leukocytes VRG showed comparable or equal responses to CpG and poly(I:C), which mimic respectively bacterial DNA and viral RNA. Most VRG showed highly correlated expression with interferon-a (IFNa). Sequence comparison of salmon VRG with those from other species gave an understanding of the evolution of these genes, which showed a remarkably rapid sequence divergence in comparison with the entire proteome. VRG emerged both before and after separation of teleosts and tetrapods, and among genes found exclusively in fish species there were members of several multigene families: tripartite motif proteins, gig1- and gig2-like proteins. Several VRG, including genes with unknown functions and orthologs to mammalian RNA helicase RIG-I and chemokine C-X-C type 10, were present in cyprinid and salmonid fish but not in the phylogenetically advanced orders, suggesting that they have been lost in the evolution of Teleostei. Apparently, a number of genes involved in antiviral responses in salmon have acquired different functional roles in higher vertebrates.

Many protein kinase functions, including autophosphorylation in trans, require dimerization, possibly by activation segment exchange. Such dimers have been reported for a few autophosphorylating protein kinases, but not for mitogen-activated protein kinases (MAPKs). Activation of MAPKs proceeds not only via the well-characterized action of dual T/Y specificity MAPK kinases, phosphorylating both residues of the MAPK TxY activation loop motif, but also via a noncanonical activation pathway triggered by phosphorylation at Tyr323 and homodimerization. Here, we report the 2. 7-A-resolution structure of p38α MAPK from Salmo salar in a novel domain-swapped homodimeric form. The tyrosines of the conserved sequence YxAPE anchor the swapped activation segments in a configuration suitable for autophosphorylation in trans and provide a model for the noncanonical pathway. In the dimer, a structural unit containing Tyr323 is formed at a dimerization contact region that stabilizes the HRD catalytic loop in a unique inactive geometry. This feature is consistent with the requirement of Tyr323 phosphorylation for the initiation of the noncanonical pathway. Despite the interacting surface of more than 2600 A2, the dimer is not obligate, as gel filtration shows the dimerization to occur only at relatively high concentrations. The transition from monomer to dimer involves a relatively simple hinged displacement of helix EF and adjacent residues. Thus, dimer formation is likely to be transient, compatible with functional requirements for autophosphorylation, allowing further modulation, for example, by scaffolding mechanisms.

Major histocompatibility complex class II (MHCII) is encoded by polymorphic genes present in vertebrates and expressed predominately in leukocytes. Upon leukocyte differentiation, intracellular MHCII is dynamically redistributed within the cells and it is expressed at maximal levels on mature antigen presenting cells (APCs). In addition, APCs secrete MHCII within endosome-derived vesicles known as exosomes which possess diverse immunomodulatory properties. Genetic and biochemical data have confirmed that piscine leukocytes express the MHCII components as well as costimulatory molecules that are necessary for the function of APCs. However data concerning the biosynthesis and the distribution of the MHCII complex within leukocytes of lower vertebrates is scarce. The presented data demonstrates for the first time that salmon leukocytes secrete vesicles that contain exosomal markers and the abundance of MHCII indicates that these exosomes are released by APCs. The secretion was specifically induced by CpG stimulation in vitro and it was observed only in head kidney leukocytes but not in splenocyte cultures. Flow cytometry revealed that, unlike splenocytes, the majority of the MHCII-positive head kidney leukocytes were Ig-negative and a population of cells expressing high levels of surface MHCII underwent degranulation upon CpG stimulation suggesting that the MHCII-containing exosomes were derived from maturing salmon APCs. Gene expression analyses have further demonstrated that CpG-B, despite its relatively weak proinflammatory activity compared to LPS, induced expression of a larger group of genes involved in regulation of the adaptive immune response.

Infectious pancreatic necrosis virus (IPNV) is a major pathogen in the aquaculture industry worldwide. Factors contributing to IPNV pathogenicity are yet poorly understood. Indications of IPNV being able to evade or counteract innate host defense come from its lack of ability to induce strong type I interferon (IFN) responses in cell culture. We show here that addition of salmon rIFN-alpha1 to cells prior to IPNV infection halts the viral protein synthesis and prevents processing of pVP2 into mature VP2. Furthermore, compared to pre-treatment with IFN-alpha1 the antiviral state in cells infected with IPNV prior to IFN-treatment, was antagonized by IPNV, as detected by higher viral titers, faster viral protein synthesis and also by reduced Mx expression. The longer headstart the virus gets, the more prominent is the weakening of IFN signaling. IPNV VP4 and VP5 inhibit IFN-induced expression from the Mx promoter, indicating that these proteins contribute to the antagonistic effect.

Mitogen activated protein kinase kinase (MKK) 3 and 6 are the main p38 mitogen-activated protein kinase activators in mammals. In the present study, three Atlantic salmon MKK6 orthologs were identified. The deduced amino acid sequences of the salmon MKK6 proteins were highly similar to mammalian MKK6 sequences, and they were ubiquitously expressed. All three were shown to be upstream activators of salmon p38. In cells exposed to sorbitol, sodium arsenite and UV radiation, the different salmon MKK6s were shown to be selectively activated. Thus, our results suggest a specific function of the three salmon MKK6s depending on which stress stimuli the cells are exposed to. Phylogenetic analysis of MKK6 and MKK3 sequences from different species indicate that salmon is unique in having three MKK6 gene copies, whereas other fish species possess one or two MKK6 genes. Interestingly, in contrast to mammals, fish do not have an MKK3 gene. We propose that two major duplication events have occurred for the ancestral MKK3/6 gene: one in tetrapods yielding MKK3 and MKK6, and another one in fish yielding two MKK6 paralogs. The third MKK6 copy found in salmon is probably the result of the salmonid-specific tetraploidization event. In conclusion, we report for the first time in any species the existence of three MKK6 genes displaying distinct expression and activation patterns. Furthermore, MKK3 is dispensable in some vertebrates because it is absent from fish genomes despite being present in chicken and all mammals sequenced so far.

Several TLR agonists are shown to activate piscine immunity and are interesting adjuvant candidates in vaccine development. To test the outcome of stimulating Atlantic salmon with CpG DNA and poly I:C, ligands for TLR9 and 3, respectively, we have measured the in vivo expression of different immune molecules in spleen and head kidney. The expression profiles for individual treatments with CpGs or poly I:C not only showed similarities but they also displayed unique profiles. When combining them a synergistic up-regulation of the genes interferon (IFN)-alpha1/alpha2, Mx, CXCL10, IL-1beta, IFN-gamma and CD83 was detected. Interestingly, synergies between two different CpG ODNs classes also resulted in pronounced IFN-alpha1/alpha2 and IFN-gamma transcripts levels. To our knowledge this is the first study showing synergy by combining two different TLR9 ligands. In conclusion, detection of dsRNA and CpG DNA in fish may mimic viral recognition, resulting in an enhanced innate immune response that could be used for vaccination.

p38 mitogen-activated protein kinase is activated by environmental stress and cytokines and plays a role in transcriptional regulation and inflammatory responses. In this study, three distinct Atlantic salmon p38 (As-p38) cDNAs were cloned, which all translated into 361 amino acid proteins. The As-p38 protein sequences possessed showed >85% identity to the mammalian homolog, p38alpha. All three contained the conserved phosphorylation motif TGY located in the activation loop of the kinase. Salmon p38 showed ubiquitous tissue distribution, including expression in the immune organs head kidney and spleen. A higher p38 mRNA expression was detected in the ovary compared to other organs suggesting that p38 may perform specific functions within this organ. Western blot analysis with an antibody specific for phosphorylated p38 showed that ectopically expressed As-p38 variants were activated in CHSE-214 cells in response to chemical stress. Furthermore, lipopolysaccharide, CpG oligonucleotides and recombinant trout IL-1beta induced endogenous phosphorylation of p38 in salmon head kidney macrophages in a dose-dependent manner. The importance of p38 for regulation of salmon innate immunity was further demonstrated by the ability of the p38 specific inhibitor SB203580 to completely abolish LPS-stimulated TNF-2 and IL-1beta mRNA expression in the macrophages.

Most commercial vaccines offered to the aquaculture industry include inactivated antigens (Ag) formulated in oil adjuvants. Safety concerns are related to the use of oil adjuvants in multivalent vaccines for fish, since adverse side effects (e.g., adhesions) can appear. Therefore, there is a request for vaccine formulations for which protection will be maintained or improved, while the risk of side effects is reduced. Here, by using an inactivated salmonid alphavirus (SAV) as the test Ag, the combined use of two Toll-like receptor (TLR) ligand adjuvants, CpG oligonucleotides (ODNs) and poly I:C, as well as a genetic adjuvant consisting of a DNA plasmid vector expressing the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) was explored. VHSV-G DNA vaccine was intramuscularly injected in combination with intraperitoneal injection of either SAV Ag alone or combined with the oil adjuvant, Montanide ISA763, or the CpG/polyI:C combo. Adjuvant formulations were evaluated for their ability to boost immune responses and induce protection against SAV in Atlantic salmon, following cohabitation challenge. It was observed that CpG/polyI:C-based formulations generated the highest neutralizing antibody titres (nAbs) before challenge, which endured post challenge. nAb responses for VHSV G-DNA- and oil-adjuvanted formulations were marginal compared to the CpG/poly I:C treatment. Interestingly, heat-inactivated sera showed reduced nAb titres compared to their non-heated counterparts, which suggests a role of complement-mediated neutralization against SAV. Consistently elevated levels of innate antiviral immune genes in the CpG/polyI:C injected groups suggested a role of IFN-mediated responses. Co-delivery of the VHSV-G DNA construct with either CpG/polyI:C or oil-adjuvanted SAV vaccine generated higher CD4 responses in head kidney at 48 h compared to injection of this vector or SAV Ag alone. The results demonstrate that a combination of pattern recognizing receptor (PRR) ligands, such as CpG/polyI:C, increases both adaptive and innate responses and represents a promising adjuvant strategy for enhancing the protection of future viral vaccines.

Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report herein that plasmid DNA and synthetic oliogodeoxynucleotides (ODNs) containing unmethylated CpG induce production of antiviral cytokine activity in Atlantic salmon leucocytes, whereas ODNs with an inverted motif (GpC) or with methylated cytosines have nearly no stimulatory effect. The adherent cell population, representing mainly macrophages, is directly activated by CpG-ODN, while the effect on the non-adherent population is weak. Since the peak antiviral activity in ODN-stimulated leucocytes is seen after 48 h, this might indicate that the unmethylated DNA stimulates the adherent cells to produce co-stimulatory molecules, which in turn stimulates production of antiviral cytokines in the non-adherent cell population. The potent immune activation by CpG ODNs points to possible new applications as adjuvant in fish vaccines.

The Mitogen-activated protein kinases (MAPK) are involved in transmitting intracellular signals downstream of diverse cell surface receptors and mediate the response to ligands such as growth factors, hormones and cytokines. In addition, MAPK are critically involved in the innate immune response to pathogen-derived substances, commonly referred to as pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS) and bacterial DNA rich in CpG dinucleotides. Currently, a great deal of knowledge is available about the involvement of MAPK in the innate immune response to PAMPs in mammals; however, little is known about the role of the different MAPK classes in the immune response to PAMPs in lower vertebrates. In the current study, p38 phosphorylation was induced by CpG oligonucleotides (ODNs) and LPS in primary salmon mononuclear phagocytes. Pre-treatment of the cells with a p38 inhibitor (SB203580) blocked the PAMP-induced p38 activity and suppressed the upregulation of most of the CpG- and LPS-induced transcripts highlighting the role of this kinase in the salmon innate immune response to PAMPs. In contrast to p38, the phosphorylation of extracellular signal-regulated kinase (ERK), a MAPK involved primarily in response to mitogens, was high in resting cells and, surprisingly, incubation with both CpG and control ODNs downregulated the phospho-ERK levels independently of p38 activation. The basal phospho-ERK level and the CpG-inducible p38 phosphorylation were greatly influenced by the length of in vitro incubation. The basal phospho-ERK level increased gradually throughout a 5-day culture period and was PI3K-dependent as demonstrated by its sensitivity to Wortmannin suggesting it is influenced by growth factors. Overall these data indicate that both basal and PAMP-induced activity of MAPKs might be greatly influenced by the differentiation status of salmon mononuclear phagocytes.

Lymph nodes and spleen are major organs where mammalian antigen-presenting cells (APCs) initiate and orchestrate Ag-specific immune responses. Unlike mammals, teleosts lack lymph nodes and an interesting question is whether alternative organs may serve as sites for antigen presentation in teleosts. In the current study, fluorescent ovalbumin (Ova) and CpG oligonucleotides (ODNs) injected intra-abdominally were detected in significant numbers of salmon head kidney (HK) MHCII+ cells over a period of 2 weeks while in spleen the percentage of these was transient and declined from day 1 post injection. In vitro studies further shed light on the properties of the diverse MHCII+ cell types found in HK. The ultrastructure of a subpopulation of MHCII+ cells with a high capacity to endocytose and process Ova indicated that these were able to perform constitutive macropinocytosis. Upon stimulation with CpG ODNs these cells upregulated CD86 and gave very high levels of TNF mRNA indicating that these are professional APCs, related to macrophages and dendritic cells (DCs). A subpopulation of HK granulocytes expressed high levels of surface MHCII and upon CpG stimulation upregulated most of the tested APC marker genes. Although these granulocytes expressed TNF weakly, they had relatively high basal levels of IL-1β mRNA and the CpG stimulation upregulated IL-1β, along with its signaling and decoy receptors, to the highest levels as compared to other HK cell types. Interestingly, the high expression of IL-1β mRNA in the granulocytes correlated with a high autophagy flux as demonstrated by LC3-II conversion. Autophagy has recently been found to be implicated in IL-1β processing and secretion and the presented data suggests that granulocytes of salmon, and perhaps other teleost species, may serve as a valuable model to study the involvement of autophagy in regulation of the vertebrate immune response.

The present paper describes the effect of lipopolysaccharides (LPS) from Aeromonas salmonicida and other Gram-negative bacteria on the respiratory burst, phagocytosis and bactericidal activity of head kidney macrophages from Atlantic salmon ( Salmo salar L.) in vitro . Macrophages were first cultured in the presence of various concentrations of LPS from A. salmonicida for 1, 2 and 5 days and then tested for respiratory burst activity (reduction of nitroblue tetrazolium) after exposure to phorbol myristate acetate (PMA). The most marked increase in respiratory burst activity of LPS-treated macrophages was observed after 5 days of incubation with 1, 10 and 100 μ g LPS ml −1 . The increase appeared to be dose-dependent with a maximal response at 10 μ g ml −1 . At this LPS-concentration and incubation time the respiratory burst activity was 3·9 times larger in the treated macrophages than in the control macrophages. LPS from three other Gram-negative bacterial salmon pathogens and two non-fish pathogens also enhanced the respiratory burst activity of salmon macrophages. Macrophages incubated with 10 and 50 μ g LPS ml −1 also showed a significant increase in PMA-stimulated H 2 O 2 -production after 5 days of incubation. LPS also stimulated the phagocytic activity of Atlantic salmon macrophages against opsonized and nonopsonized glucan particles, and glutaraldehyde-fixed sheep red blood cells. LPS-treated macrophages showed an increased ability to kill an avirulent A-layer lacking strain of A. salmonicida , but not a virulent A-layer positive strain.

true in mammals. In this study, we were interested finding out whether two important salmon pathogenic RNA viruses, salmon alphavirus (SAV) or infectious pancreas virus, were able to modulate SOCS expression in salmon cells lines. We also tested SOCS expression in SAV challenged Atlantic salmon. Furthermore, we asked the questions whether the negative impact of the salmon SOCS1 on IFN signaling would affect virus replication and the expression of ISGs. As important tools for these studies, CHSE-cells overexpressing SOCS1 or SOCS2 were developed and used for both viral infection studies and to measure ISG expression upon stimulation by IFNs. Results for these experiments will be presented and discussed.

Background Bacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes. Research has found that mammalian TLR9 is a receptor which mediates the immune response to CpG DNA; however, its functional properties in non-mammalian vertebrates are still poorly characterized. Leukocytes isolated from lower vertebrates, including teleosts, respond to CpG DNA and TLR9 has been identified in many fish species; however, the ligand-binding properties of fish TLR9 have, so far, not been studied. The fact that some vertebrates, like chicken, lack TLR9 and use an alternative molecule (TLR21) as a receptor for CpGs has questioned the functional conservation of TLR9 within vertebrates. Results In the current study, TLR9 from Atlantic salmon (SsTLR9) has been found to interact with synthetic oligonucleotides via a CpG-independent but a pH-dependent mechanism. The endogenous receptor, expressed by primary mononuclear phagocytes colocalizes with CpG oligonucleotides (ODNs) in vesicles that appear to be endosomes. When overexpressed in salmonid cell lines, SsTLR9 spontaneously activates ISRE-containing promoters of genes involved in the IFN response; however, the transgenic receptor fails to translocate to CpG-containing endosomes. This indicates that only specific immune cell types have the ability to relocate the receptor to the appropriate cellular compartments where it may become activated by its ligand. In addition, through co-precipitation and mass spectrometry, two salmon proteins - hnRNPA0 and NCOA5, which both contain RNA-binding domains (RRM), were found to bind CpG ODNs, suggesting they may be involved in the CpG response in salmon leukocytes. Conclusion The presented data are the first to demonstrate that the DNA-binding properties of TLR9 are conserved between teleosts and mammals. The current study also identifies additional molecules which may function as mediators of the immunostimulatory properties of foreign DNA.

CpG oligonucleotides and polyinosinic:polycytidylic acid (poly I:C) are toll-like receptor (TLR) agonists that mimic the immunostimulatory properties of bacterial DNA and double-stranded viral RNA respectively, and which have exhibited potential to serve as vaccine adjuvants in previous experiments. Here, a combination of CpGs and poly I:C together with water- or oil-formulated Salmonid Alphavirus (SAV) antigen preparations has been used for a vaccine in Atlantic salmon and tested for protection in SAV challenge trial. The results demonstrate that vaccination with a high dose of the SAV antigen induced protection against challenge with SAV which correlated with production of neutralizing antibodies (NAbs). As the high antigen dose alone induced full protection, no beneficial effect from the addition of CpG and poly I:C could be observed. Nevertheless, these TLR ligands significantly enhanced the levels of NAbs in serum of vaccinated fish. Interestingly, gene expression analysis demonstrated that while addition of oil suppressed the CpG/poly I:C-induced expression of IFN-γ, the upregulation of IFNa1 was substantially enhanced. A low dose of the SAV antigen combined with oil did not induce any detectable levels of NAbs either with or without TLR ligands present, however the addition of CpG and poly I:C to the low SAV antigen dose formulation significantly enhanced the protection against SAV suggesting that CpG/poly I:C may have enhanced a cytotoxic response – a process which is dependent on the up-regulation of type I IFN. These results highlight the immunostimulatory properties of the tested TLR ligands and will serve as a ground for further, more detailed studies aimed to investigate their capacity to serve as adjuvants in vaccine formulations for Atlantic salmon.

We investigated the antiviral activity and gene induction properties of interferon gamma (IFN-γ) compared to type I IFN (IFNa1) in Atlantic salmon. IFN-γ protected salmon cells against infectious pancreatic necrosis virus (IPNV)-induced cytopathic effect (CPE), reduced virus titers, and inhibited the synthesis of the viral structural protein VP3. Moreover, IFN-γ showed potent antiviral activity against salmonid alphavirus 3 (SAV3) measured as a reduction in virus nsP1 transcripts. IFN-γ (a type II IFN) had less specific antiviral activity against IPNV than IFNa1, showing a half-maximal effective concentration of 1.6 ng/ml versus 31 pg/ml determined in the CPE reduction assay. Compared to IFNa1, IFN-γ was a more effective inducer of the antiviral protein GBP, several interferon regulatory transcription factors (IRFs), and the chemokine IP-10. The antiviral activity of IFN-γ may also in part be ascribed to upregulation of Mx, ISG15, and viperin. These are typical type I IFN-induced genes in mammals and were also more strongly induced by IFNa1 than by IFN-γ in salmon cells. Fish and mammalian IFN-γ thus show strikingly similar gene induction properties. Interestingly, the antiviral activity of IFN-γ against IPNV and SAV3 and its ability to induce Mx and ISG15 markedly decreased in the presence of neutralizing antiserum against IFNa1. In contrast, antiIFNa1 had no effect on the induction of IRF-1 and IP-10 by IFN-γ. This suggests that the antiviral activity of IFN-γ is partially dependent on IFNa induction. However, because antiIFNa1 could not abolish the IFN-γ-mediated induction of Mx and ISG15 completely, IFN-γ may possibly also induce such genes directly.

Both CpG oligodeoxynucleotides and double-stranded RNA (poly I:C) have documented effects as treatments against several viral diseases in fish. However, as stand-alone treatments their effects have been modest. We have tested here whether CpG and poly I:C, alone or in combination induce protection against Salmonid Alphavirus (SAV), the causative agent of pancreas disease in Atlantic salmon. Our results revealed a significant reduction of viraemia 2 weeks after ip injection of the combined treatment and 1 week after challenge with SAV subtype 3, followed by reduced SAV induced heart pathology 3 weeks later. The SAV titers in blood samples from the combination group were lower as compared to single treatments with either CpG or poly I:C. Surprisingly, reduced SAV levels could also be found in fish as long as 7 weeks after receiving the combination treatment. The expression of IFNγ and to a lesser extent IFNa and Mx was up-regulated in head kidney and spleen 5 days after the fish had been treated with CpG and poly I:C. Furthermore, the complement factor C4 was depleted in serum 8 weeks post treatment, suggesting complement activation leading to C4 consumption. We hypothesize that the CpG/poly I:C-induced protection against SAV3 is mediated by mechanisms involving type I and type II IFN induced antiviral activity and complement mediated protective responses.