Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

CRISPR-Cas- induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.

Background: Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means. Methods: By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection. Results: We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication. Conclusion: Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

The importance of the Atlantic salmon peritoneal cavity B cell response: Local IgM secreting cells are predominant upon Piscirickettsia salmonis infection.

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.

Infection and microbial molecular motifs modulate transcription of the interferon-inducible gene ifit5 in a teleost fish.

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.

Analysis of the Atlantic salmon genome reveals a cluster of Mx genes that respond more strongly to IFN gamma than to type I IFN.

Mx proteins are antiviral GTPases, which are induced by type I IFN and virus infection. Analysis of the Atlantic salmon genome revealed the presence of 9 Mx genes localized to three chromosomes. A cluster of three Mx genes (SsaMx1 - SsaMx3), which includes previously cloned Mx genes, is present on chromosome (Chr) 12. A cluster of five Mx genes (SsaMx4-SsaMx8) is present on Chr25 while one Mx gene (SsaMx9) is present on Chr9. Phylogenetic and gene synteny analyses showed that SsaMx1-SsaMx3 are most closely related to the main group of teleost Mx proteins. In contrast, SsaMx 4-SsaMx9 formed a separate group together with zebrafish MxD and MxG and eel MxB. The Mx cluster in Chr25 showed gene synteny similar to a Mx gene cluster in the gar genome. Expression of Mx genes in cell lines stimulated with recombinant IFNs showed that Mx genes in Chr12 responded more strongly to type I IFN than to type II IFN (IFN gamma) whilst Mx genes in Chr25 responded more strongly to IFN gamma than to type I IFNs. SsaMx9 showed no response to the IFNs.

Infectious salmon anemia virus segment 7 ORF1 and segment 8 ORF2 proteins inhibit IRF mediated activation of the Atlantic salmon IFNa1 promoter.

Infectious salmon anemia virus (ISAV) is an orthomyxovirus, which may cause multisystemic disease and high mortality of Atlantic salmon (Salmo salar L). This suggests that ISAV encodes proteins that antagonize the type I interferon (IFN-I) system, which is of crucial importance in innate antiviral immunity. To find out how ISAV might inhibit IFN-I synthesis, we have here studied whether the two ISAV proteins s7ORF1 and s8ORF2 might interfere with activation of the IFNa1 promoter mediated by overexpression of interferon regulatory factors (IRFs) or by the IFN promoter activation protein IPS-1. The IRF tested were IRF1, IRF3, IRF7A and IRF7B. Promoter activation was measured using a luciferase reporter assay where Atlantic salmon TO cells were co-transfected with the IFNa1 promoter reporter plasmid together with an IRF plasmid and the s7ORF1 or the s8ORF2 construct or a control plasmid. The results showed that s7ORF1 significantly inhibited IRF3 and IRF7B induced IFN promoter activity, while s8ORF2 significantly inhibited IRF1 and IRF3 induced promoter activity. Neither s7ORF1 nor s8ORF2 inhibited IPS-1 mediated promoter activation. Immunoprecipitation data suggest that both s7ORF1 and s8ORF2 can bind to all four IRFs. Taken together, this study thus shows that the ISAV proteins s7ORF1 and s8ORF2 antagonizes IFN-I transcription activation mediated by the IRFs. As such this work provides further insight into the pathogenic properties of ISAV.

Infectious pancreatic necrosis virus proteins VP2, VP3, VP4 and VP5 antagonize IFNa1 promoter activation while VP1 induces IFNa1.

Infectious pancreatic necrosis virus (IPNV) is one of the major viral pathogens causing disease in farmed Atlantic salmon worldwide. In the present work we show that several of the IPN proteins have powerful antagonistic properties against type I IFN induction in Atlantic salmon. Each of the five IPNV genes cloned into an expression vector were tested for the ability to influence activation of the Atlantic salmon IFNa1 promoter by the interferon promoter inducing protein one (IPS-1) or interferon regulatory factors (IRF). This showed that preVP2, VP3 and VP5 inhibited activation of both promoters, while VP4 only antagonized activation of the IFNa1 promoter. The viral protease VP4 was the most potent inhibitor of IFN induction, apparently targeting the IRF1 and IRF3 branch of the signaling cascade. VP4 antagonism is independent of its protease activity since the catalytically dead mutant VP4K674A inhibited activation of the IFNa1 promoter to a similar extent as wild type VP4. In contrast to the other IPNV proteins, the RNA-dependent RNA polymerase VP1 activated the IFNa1 promoter. The ability to activate the IFN response was disrupted in the mutant VP1S163A, which has lost the ability to produce dsRNA. VP1 also exhibited synergistic effects with IRF1 and IRF3 in inducing an IFNa1-dependent antiviral state in cells. Taken together these results suggest that IPNV has developed multiple IFN antagonistic properties to prevent IFN-induction by VP1 and its dsRNA genome.

Atlantic salmon possesses two clusters of type I interferon receptor genes on different chromosomes, which allows for a larger repertoire of interferon receptors than in zebrafish and mammals.

Mammalian type I interferons (IFNs) signal through a receptor composed of the IFNAR1 and IFNAR2 chains. In zebrafish two-cysteine IFNs utilize a receptor composed of CRFB1 and CRFB5, while four-cysteine IFNs signal through a receptor formed by CRFB2 and CRFB5. In the present work two CRFB clusters were identified in different chromosomes of Atlantic salmon. Genes of three CRFB5s, one CRFB1, one CRFB2 and the novel CRFB5x were identified, cloned and studied functionally. All CRFBs were expressed in 10 different organs, but the relative expression of CRFBs varied. Mx-reporter assay was used to study which CRFBs might be involved in receptors for salmon IFNa, IFNb and IFNc. The results of Mx-reporter assays suggest that IFNa signals through a receptor composed of CRFB1a as the long chain and either CRFB5a, CRFB5b or CRFB5c as the short chain; IFNc signals through a receptor with CRFB5a or CRFB5c as the short chain while IFNb may signal through a receptor with CRFB5x as a short chain. Taken together, the present work demonstrates that Atlantic salmon has a more diverse repertoire of type I IFN receptors compared to zebrafish or mammals.

Bovine brain myelin glycerophosphocholine choline phosphodiesterase is an alkaline lysosphingomyelinase of the eNPP-family, regulated by lysosomal sorting.

Glycerophosphocholine choline phosphodiesterase (GPC-Cpde) is a glycosylphosphatidylinositol (GPI)-anchored alkaline hydrolase that is expressed in the brain and kidney. In brain the hydrolase is synthesized by the oligodendrocytes and expressed on the myelin membrane. There are two forms of brain GPC-Cpde, a membrane-linked (mGPC-Cpde) and a soluble (sGPC-Cpde). Here we report the characterisation sGPC-Cpde from bovine brain. The amino acid sequence was identical to ectonucleotide pyrophosphatase/phosphodiesterase 6 (eNPP6) precursor, lacking the N-terminal signal peptide region and a C-terminal stretch, suggesting that the hydrolase was solubilised by C-terminal proteolysis, releasing the GPI-anchor. sGPC-Cpde existed as two isoforms, a homodimer joined by a disulfide bridge linking C414 from each monomer, and a monomer resulting from proteolysis N-terminally to this disulfide bond. The only internal disulfide bridge, linking C142 and C154, stabilises the choline-binding pocket. sGPC-Cpde was specific for lysosphingomyelin, displaying 1 to 2 orders of magnitude higher catalytic activity than towards GPC and lysophosphatidylcholine, suggesting that GPC-Cpde may function in the sphingomyelin signaling, rather than in the homeostasis of acylglycerophosphocholine metabolites. The truncated high mannose and bisected hybrid type glycans linked to N118 and N341 of sGPC-Cpde is a hallmark of glycans in lysosomal glycoproteins, subjected to GlcNAc-1-phosphorylation en route through Golgi. Thus, sGPC-Cpde may originate from the lysosomes, suggesting that lysosomal sorting contributes to the level of mGPC-Cpde on the myelin membrane.

Atlantic salmon type I IFN subtypes show differences in antiviral activity and cell-dependent expression: evidence for high IFNb/IFNc-producing cells in fish lymphoid tissues.

This work reveals distinct roles of the two-cysteine-containing type I IFNs, IFNa and IFNd, and the four-cysteine-containing IFNb and IFNc in antiviral immunity of Atlantic salmon. IFNa and IFNc showed similar antiviral activities and ability to induce antiviral genes, IFNb was less active, and IFNd showed no activity. Expression of IFNs was compared by treatment of cells or fish with the dsRNA polyinosinic-polycytidylic acid [poly(I:C)], which induces IFNs via the viral RNA receptors MDA5 and TLR3/TLR22 and with the imidazoquinoline R848, which induces IFNs via TLR7. Poly(I:C) strongly induced IFNa in cell lines, whereas the other IFNs showed little response, indicating that IFNa is the main IFN subtype induced through the RIG-I/MDA5 pathway. In contrast, IFNb and IFNc are the main IFNs induced through the TLR7 pathway because R848 induced high transcript levels of IFNb and IFNc and low transcript levels of IFNa in the head kidney and spleen. IFNd was constitutively expressed in cells and organs but showed no response to poly(I:C) or R848. Fluorescence in situ hybridization studies showed that poly(I:C) induced IFNa and IFNc in a variety of cells in the head kidney, spleen, gills, liver, and heart, whereas R848 induced coexpression of IFNb and IFNc in distinct cells in head kidney and spleen. These cells are likely to be specialized high IFN producers because they were few in numbers despite high IFNb/IFNc transcript levels in the same organs. High IFN expression in response to TLR7 ligation is a feature shared by mammalian plasmacytoid dendritic cells.