Atlantic salmon type I interferon genes revisited.

Type I interferons (IFN-I) play a pivotal role in vertebrate innate immunity against viruses. This study is an analysis of IFN-I genes in an updated version of the Atlantic salmon genome published in 2021 (version Ssal_v3.1), revealing 47 IFN-I genes in the Atlantic salmon genome. The GH1 locus of chromosome (Chr) 3 harbors 9 IFNa genes, 5 IFNb genes, 6 IFNc genes, 11 IFNe genes and 1 IFNf gene. The GH2 locus on Chr6 contains 1 IFNa gene, 12 IFNc genes and 1 IFNf gene while Chr19 carries a single IFNd gene. Intraperitoneal injection of Atlantic salmon presmolts with poly I:C, a mimic of virus double-stranded RNA, significantly up-regulated IFNc genes from both Chr3 and Chr6 in heart, with lower expression in head kidney. IFNe expression increased in the heart, but not in the head kidney while IFNf was strongly up-regulated in both tissues. Antiviral activity of selected IFNs was assessed by transfection of salmon cells with IFN-expressing plasmids followed by infectious pancreatic necrosis virus infection, and by injection of fish with IFN-plasmids followed by measuring expression of the antiviral Mx1 gene. The results demonstrated that IFNc from both Chr3 and Chr6 provided full protection of cells against virus infection, whereas IFNe and IFNf showed lesser protection. IFNc from Chr3 and Chr6 along with IFNe and IFNf, up-regulated the Mx1 gene in the muscle, while only the IFNcs caused induction of Mx1 in liver. Overall, this study reveals that Atlantic salmon possesses an even more potent innate immune defense against viruses than previously understood.

Microbial Danger Signals Control Transcriptional Induction of Distinct MHC Class I L Lineage Genes in Atlantic Salmon.

Antigen processing and presentation by major histocompatibility complex (MHC) molecules is a cornerstone in vertebrate immunity. Like mammals, teleosts possess both classical MHC class I and multiple families of divergent MHC class I genes. However, while certain mammalian MHC class I-like molecules have proven to be integral in immune regulation against a broad array of pathogens, the biological relevance of the different MHC class I lineages in fish remains elusive. This work focuses on MHC class I L lineage genes and reveals unique regulatory patterns of six genes (Sasa-lia, Sasa-lda, Sasa-lca, Sasa-lga, Sasa-lha, and Sasa-lfa) in antimicrobial immunity of Atlantic salmon (Salmo salar L.). Using two separate in vivo challenge models with different kinetics and immune pathologies combined with in vitro stimulation using viral and bacterial TLR ligands, we show that de novo synthesis of different L lineage genes is distinctly regulated in response to various microbial stimuli. Prior to the onset of classical MHC class I gene expression, lia was rapidly and systemically induced in vivo by the single-stranded (ss) RNA virus salmonid alpha virus 3 (SAV3) but not in response to the intracellular bacterium Piscirickettsia salmonis. In contrast, lga expression was upregulated in response to both viral and bacterial stimuli. A role for distinct MHC class I L-lineage genes in anti-microbial immunity in salmon was further substantiated by a marked upregulation of lia and lga gene expression in response to type I IFNa stimulation in vitro. Comparably, lha showed no transcriptional induction in response to IFNa stimulation but was strongly induced in response to a variety of viral and bacterial TLR ligands. In sharp contrast, lda showed no response to viral or bacterial challenge. Similarly, induction of lca, which is predominantly expressed in primary and secondary lymphoid tissues, was marginal with the exception of a strong and transient upregulation in pancreas following SAV3 challenge Together, these findings suggest that certain Atlantic salmon MHC class I L lineage genes play important and divergent roles in early anti-microbial response and that their regulation, in response to different activation signals, represents a system for selectively promoting the expression of distinct non-classical MHC class I genes in response to different types of immune challenges.

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.

Transcriptome analyses of Atlantic salmon muscle genes induced by a DNA vaccine against salmonid alphavirus, the causative agent of salmon pancreas disease (PD).

Salmonid alphavirus (SAV) is the causative agent of pancreas disease (PD) in farmed Atlantic salmon. A previous study showed that vaccination of pre-smolt salmon with a plasmid encoding the structural polypeptide of SAV gave protection against infection and development of PD accompanied by production of antibodies against the virus. In the present work we analyzed transcript responses in the muscle to vaccination with this plasmid (here named pSAV). The purpose was to shed light on how pSAV might initiate adaptive immune responses in the fish. The work was based on microarray and reverse transcription quantitative PCR analyses of muscle at the injection site 7 days after vaccination. The results showed that pSAV and pcDNA3.3 had similar abilities to up-regulate type I IFN stimulated genes. In contrast, pSAV caused higher up-regulation of IFNγ and several IFNγ inducible genes. Compared to pcDNA3.3, pSAV also gave larger increase in transcripts of marker genes for B-cells, T-cells and antigen presenting cells (APCs), which suggest attraction and role of these cells in the adaptive immune responses elicited by pSAV. Moreover, pSAV caused a stronger up-regulation of the chemokine CXCL10 and the proinflammatory cytokines IL-1ß and TNFα, which may explain attraction of lymphocytes and APCs. The present work shows that the expression profile of genes resulting from vaccination with pSAV is different from the expression profiles obtained previously by vaccination of salmonids with DNA vaccines against infectious salmon anemia virus and infectious hematopoietic necrosis virus.

The role of type I interferons in innate and adaptive immunity against viruses in Atlantic salmon.

Type I IFNs (IFN-I) are cytokines, which play a crucial role in innate and adaptive immunity against viruses of vertebrates. In essence, IFN-I are induced and secreted upon host cell recognition of viral nucleic acids and protect other cells against infection by inducing antiviral proteins. Atlantic salmon possesses an extraordinary repertoire of IFN-I genes encompassing at least six different classes (IFNa, IFNb, IFNc, IFNd, IFNe and IFNf) most of which are encoded by several genes. This review describes recent research on the functions of salmon IFNa, IFNb, IFNc and IFNd. As in mammals, expression of different salmon IFN-I in response to virus infection is dependent on their promoters, properties of the virus and the cell's expression of nucleic acid receptors and interferon regulatory factors (IRFs). While IFNa mainly display local antiviral activity, IFNb and IFNc show systemic antiviral activity. In addition, salmon appears to possess several IFN-I receptors, which show selectivity in binding different IFN-I. This complexity in IFN-I and receptors allows for a large variation in functions of the salmon IFN-I. Studies with intramuscular injection of IFN expression plasmids have recently provided surprising results, which may be of relevance for application of IFN-I in prophylaxis against virus infection. Firstly, injection of IFNc plasmid protected salmon presmolts against virus infection for at least 10 weeks. Secondly, IFN plasmids showed potent adjuvant activity when injected together with a DNA vaccine against infectious salmon anemia virus (ISAV).

Transcriptome analysis of plasmid-induced genes sheds light on the role of type I IFN as adjuvant in DNA vaccine against infectious salmon anemia virus.

A previous study showed that a plasmid expressing IFNa (pIFNa) strongly enhanced protection and antibody production of a DNA vaccine against infectious salmon anemia virus (ISAV) in Atlantic salmon. The vaccine consisted of a plasmid (pHE) expressing the virus hemagglutinin-esterase as an antigen. To increase the understanding of the adjuvant effect of pIFNa, we here compared transcriptome responses in salmon muscle at the injection site at week 1 and 2 after injection of pIFNa, pHE, plasmid control (pcDNA3.3) and PBS, respectively. The results showed that the IFNa plasmid mediates an increase in gene transcripts of at least three major types in the muscle; typical IFN-I induced genes (ISGs), certain chemokines and markers of B- cells, T-cells and antigen-presenting cells. The latter suggests recruitment of cells to the plasmid injection site. Attraction of lymphocytes was likely caused by the induction of chemokines homologous to mammalian CCL5, CCL8, CCL19 and CXCL10. IFN may possibly also co-stimulate activation of lymphocytes as suggested by studies in mammals. A major finding was that both pcDNA3.3 and pHE caused responses similar to pIFNa, but at lower magnitude. Plasmid DNA may thus by itself have adjuvant activity as observed in mammalian models. Notably, pHE had a lower effect on many immune genes including ISGs and chemokines than pcDNA3.3, which suggests an inhibitory effect of HE expression on the immune genes. This hypothesis was supported by an Mx-reporter assay. The present study thus suggests that a main role for pIFNa as adjuvant in the DNA vaccine against ISAV may be to overcome the inhibitory effect of HE- expression on plasmid-induced ISGs and chemokines.

Francisella noatunensis subsp. noatunensis invades, survives and replicates in Atlantic cod cells.

Systemic infection caused by the facultative intracellular bacterium Francisella noatunensis subsp. noatunensis remains a disease threat to Atlantic cod Gadus morhua L. Future prophylactics could benefit from better knowledge on how the bacterium invades, survives and establishes infection in its host cells. Here, facilitated by the use of a gentamicin protection assay, this was studied in primary monocyte/macrophage cultures and an epithelial-like cell line derived from Atlantic cod larvae (ACL cells). The results showed that F. noatunensis subsp. noatunensis is able to invade primary monocyte/macrophages, and that the actin-polymerisation inhibitor cytochalasin D blocked internalisation, demonstrating that the invasion is mediated through phagocytosis. Interferon gamma (IFNγ) treatment of cod macrophages prior to infection enhanced bacterial invasion, potentially by stimulating macrophage activation in an early step in host defence against F. noatunensis subsp. noatunensis infections. We measured a rapid drop of the initial high levels of internalised bacteria in macrophages, indicating the presence and action of a cellular immune defence mechanism before intracellular bacterial replication took place. Low levels of bacterial internalisation and replication were detected in the epithelial-like ACL cells. The capacity of F. noatunensis subsp. noatunensis to enter, survive and even replicate within an epithelial cell line may play an important role in its ability to infect live fish and transverse epithelial barriers to reach the bacterium's main target cells-the macrophage.

Protection of Atlantic salmon against salmonid alphavirus infection by type I interferons IFNa, IFNb and IFNc.

Salmonid alphavirus 3 (SAV3) causes pancreas disease (PD), which is a major problem in Norwegian aquaculture of Atlantic salmon. In this work we studied antiviral activities of salmon type I interferons IFNa, IFNb and IFNc against SAV3 infection in cell culture and in live fish to increase the understanding of the innate immunity of salmon against this virus. Recombinant IFNa, IFNb and IFNc all induced antiviral activity against SAV3 in ASK cells. For in vivo studies, we injected salmon presmolts intramuscularly with plasmids encoding salmon IFNa, IFNb and IFNc or a control plasmid and measured expression of the antiviral protein Mx in pancreas after 2 and 10 weeks and protection against SAV3 infection after 10 weeks. IFNb and IFNc plasmids, but not IFNa plasmid induced Mx expression in pancreas as shown by RT-qPCR and immunohistochemistry. A high level of protection against SAV3 infection by IFNc plasmid was observed by a strong reduction of virus load in serum and by a marked reduction in pathology of pancreas and heart compared to control fish. Lesser but significant protection was observed with IFNb plasmid while no protection was observed after treatment with IFNa plasmid. Taken together, this work suggests that IFNa provides protection of salmon against SAV3 locally in an infected area while IFNb and IFNc provides systemic protection against the virus.

IFN-adjuvanted DNA vaccine against infectious salmon anemia virus: Antibody kinetics and longevity of IFN expression.

Plasmids expressing interferon (IFN) have recently been shown to function as adjuvants in Atlantic salmon when co-injected with a DNA vaccine encoding hemagglutinin-esterase (HE) from infectious salmon anemia virus (ISAV). In this work we have compared the antibody kinetics and the systemic Mx/ISG15 response of fish vaccinated with HE-plasmid using either IFNa plasmid (pIFNa) or pIFNc as adjuvants over a longer time period, i.e. 22 weeks post vaccination (pv). The results showed that the antibody response against ISAV with pIFNa as adjuvant arose earlier (7 weeks pv) than with pIFNc as adjuvant (10 weeks pv), peaked at week 10 and declined at week 22. The antibody response with pIFNc as adjuvant peaked at 16 weeks and kept at this level 22 weeks pv. Fish injected with pIFNc alone expressed high levels of Mx and ISG15 in liver throughout the 22 week period. In contrast, fish injected with pIFNc together with HE-plasmid expressed high levels of Mx and ISG15 in liver for the first 10 weeks, but at week 16 this response was absent in two of three fish at week 16 and was absent in all tested fish at week 22 pv. This suggests that cells expressing HE and IFNc are intact at week 10 pv, but are eliminated by adaptive immune responses after week 10 due to recognition of HE. The longevity of the Mx/ISG15 response in pIFNc treated fish is likely due to the fact that IFNc is a self-antigen of salmon and is not attacked by the adaptive immune system.

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.

Adjuvant activity of fish type I interferon shown in a virus DNA vaccination model.

There is a need for more efficient vaccines to combat viral diseases of Atlantic salmon and other farmed fish. DNA vaccines are highly effective against salmonid rhabdoviruses, but have shown less effect against other viruses. In the present work we have studied if type I IFNs might be used as adjuvants in fish DNA vaccines. For this purpose we chose a DNA vaccine model based on the hemagglutinin-esterase (HE) gene of infectious salmon anemia virus (ISAV) as antigen. Salmon presmolts were injected with a plasmid encoding HE alone or together with a plasmid encoding Atlantic salmon type I IFN (IFNa1, IFNb or IFNc). Sera were harvested after 7-10 weeks for measurements of antibody against ISAV and the fish were challenged with ISAV to measure protective effects of the vaccines. The results showed that all three IFN plasmids delivered together with HE plasmid potently enhanced protection of salmon against ISAV mediated mortality and stimulated an increase in IgM antibodies against the virus. In contrast, HE plasmid alone gave low antibody titers and a minor protection against ISAV. This demonstrates that type I IFNs stimulate adaptive immune responses in fish, which may be a benefit also in other fish DNA vaccines. Quantitative RT-PCR studies showed that the salmon IFNs caused an increased influx of B-cells and cytotoxic T-cells at the muscle injection site, which may in part explain the adjuvant effect of the IFNs.

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.

Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts.

Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

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.

Protection of Atlantic salmon against virus infection by intramuscular injection of IFNc expression plasmid.

In this work we have tested the in vivo antiviral activity of type I interferons (IFNs) in Atlantic salmon by injecting presmolts intramuscularly (i.m.) with plasmids encoding IFNa1, IFNb or IFNc under the control of a CMV promoter, and measured expression of antiviral genes in organs and protection against infection with infectious salmon anemia virus (ISAV) infection. All three IFN plasmids induced expression of antiviral genes (Mx, Viperin, ISG15 and IFIT5) at the muscle injection site while the control plasmid had little effect. Only IFNb and IFNc plasmids induced expression of antiviral genes in head kidney, liver and heart. This suggests that IFNb and IFNc are distributed systemically while IFNa1 is active only at the injection site. Injection of IFNc plasmid was found to induce expression of antiviral genes and receptors for virus RNA (RIG-I, TLR3 and TLR7) in head kidney from 1 to at least 8 weeks. Immunoblotting showed increased expression of ISG15 and Mx protein in liver with time during this time period. Challenge of presmolts with ISAV 8 weeks after injection of IFN plasmids, showed strong protection of the IFNc plasmid injected fish, low protection of the IFNb plasmid injected fish and no protection of the IFNa1 plasmid injected fish. Clues to the difference in protection obtained with IFNb and IFNc plasmids were found by immunohistochemical and immunoblot studies of Mx protein, which indicated that IFNc plasmid stimulated stronger Mx protein expression in heart tissues and liver endothelial cells than IFNb plasmid. Taken together, these data suggest that i.m. injection of the IFNc expression plasmid may be a new method for protecting Atlantic salmon against virus infection.

Isolation and characterisation of two cDNAs encoding transglutaminase from Atlantic cod (Gadus morhua).

Two cDNAs encoding transglutaminase (TG) were identified in a subtractive cDNA library prepared from the head kidney of poly I:C stimulated Atlantic cod (Gadus morhua). Full-length TG-1 and TG-2 cDNA were cloned from the head kidney by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The deduced amino acid (aa) sequence for TG-1 was 695 aa with an estimated molecular mass of 78.3 kDa, while TG-2 was a 698 aa protein with an estimated molecular mass of 78.8 kDa. The two proteins were named TG-1 and TG-2 and both possess transglutaminase/protease-like homologous domains (TGc) and full conservation of amino acids cysteine, histidine, and aspartate residues that form the catalytic triad. Sequence analysis showed high similarity (93.1%) with Alaska pollock TG, and the TGs were grouped together with TGs from chum salmon, Japanese flounder, Nile tilapia, and red sea bream in addition to Alaska pollock in phylogenetic analysis. Interestingly, they showed different tissue distribution with highest constitutive expression in reproductive and immunological organs, indicating important roles in these organs. Furthermore, the up-regulation of TG-1 and TG-2 in head kidney after stimulating Atlantic cod with poly I:C suggested a role of TGs in immune response in Atlantic cod.

Infectious salmon anemia virus (ISAV) replication is transiently inhibited by Atlantic salmon type I interferon in cell culture.

Infectious salmon anemia virus (ISAV) is a piscine orthomyxovirus, which causes multisystemic disease in farmed Atlantic salmon that may result in large losses. Previous work has suggested that ISAV is able to resist the antiviral state induced in cells by type I interferon (IFN). These studies were, however, mainly based on cytopathic effect (CPE) reduction assays. Here we have investigated the antiviral activity of Atlantic salmon IFNa1, IFNb and IFNc against ISAV using quantitative PCR (qPCR) of segment 6, Western blot analysis of ISAV proteins and viral yield reduction assays, in addition to CPE reduction assays. Antiviral effects of IFNs were tested against the high virulent strain ISAV4 and the low virulent strain ISAV7 both at the optimum growth temperature 15°C and at 20°C. As expected, IFNa1 showed little protection against CPE development in cells after infection with both strains at 15°C. However, the qPCR and Western blot analysis clearly showed strong inhibition of replication of the virus strains by IFNa1 between 24 and 72h after infection. The inhibitory effect declined four to five days post-infection, which explains the low protection against CPE development 7-10 days later. At 20°C, IFNa1 showed strong protection against CPE development, probably due to slower virus growth. IFNc showed similar antiviral activity as IFNa1 against ISAV4 while IFNb showed lower activity. There were observed differences between ISAV4 and ISAV7 both with respect inhibition by IFNa1 and ability to induce the two IFN-inducible antiviral effector proteins, Mx and ISG15, which may be related to differences in virulence properties and/or adaption to growth in cell culture.

Sequence analysis of the genome of piscine orthoreovirus (PRV) associated with heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar).

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in µ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-ß signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins µ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in µ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.

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.

Antiviral activity of salmonid gamma interferon against infectious pancreatic necrosis virus and salmonid alphavirus and its dependency on type I interferon.

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.