Børre Robertsen

Atlantic Salmon Interferon Genes: Cloning, Sequence Analysis, Expression, and Biological Activity

In this work, we report cDNA cloning of two type I interferons (IFNs) from the head kidney of Atlantic salmon, called SasaIFN-alpha1 (829 bp) and SasaIFN-alpha2 (1290 bp). Both translate into 175 amino acid precursor molecules showing 95% amino acid sequence identity. The precursors have a putative 23 amino acid signal peptide, which suggests that the mature Atlantic salmon IFNs contain 152 amino acids (18.2 kDa). Salmon IFN appears to have five alpha-helices, similar to mammalian and avian type I IFNs, and showed 45% sequence identity with zebrafish IFN, up to 29% identity with mammalian IFN-alpha sequences, and 17%-18% sequence identity with mammalian IFN-beta and chicken type I IFNs. Human embryonic kidney 293 cells transfected with the SasaIFN-alpha1 cDNA gene produced high titers of acid-stable antiviral activity, which protected salmonid cells against infectious pancreatic necrosis virus (IPNV) and also induced Mx protein in the cells. Poly(I)-poly(C) induced two IFN transcripts in head kidney of Atlantic salmon. Genomic IFN sequences contained four introns and five exons, which is different from the intronless type I IFN genes of birds and mammals.

Yeast glucan induces increase in lysozyme and complement-mediated haemolytic activity in Atlantic salmon blood.

The activities of lysozyme, alkaline and acid phosphatase, N-acetyl-glucosaminidase and complement (as spontaneous haemolytic activity) were measured in plasma of Atlantic salmon, Salmo salar L., after intraperitoneal injection of a β -1,3 and β -1,6 linked glucan from Saccharomyces cerevisiae . The glucan has previously been shown to induce resistance of Atlantic salmon to several bacterial pathogens. The glucan treatment resulted in increased activities of lysozyme 1–3 weeks and complement 2–4 weeks after injection. The activities of alkaline phosphatase and N-acetyl-glucosaminidase showed a decrease 1 week after injection and then slowly restored to normal levels, whereas acid phosphatase was only slightly altered during the experimental period. These results indicate that the glucan induces a selective increase in lysozyme rather than a general release of lysosomal enzymes. The time intervals of enhanced activities of lysozyme and complement partially overlapped with the time intervals of expected enhanced protection against pathogens. Thus, induction of increased activities of lysozyme and complement may, at least in part, explain the mechanisms by which glucan induces protection against bacterial pathogens in salmon.

Inhibition of Infectious Pancreatic Necrosis Virus Replication by Atlantic Salmon Mx1 Protein

ABSTRACT Mx proteins form a family of interferon (IFN)-induced GTPases with potent antiviral activity against various single-stranded RNA viruses in mammals and chickens. In fish, alpha/beta IFN has been reported to inhibit the replication of infectious pancreatic necrosis virus (IPNV), but the mode of action has not been elucidated. A correlation between the inhibition of IPNV and Mx protein expression has, however, been observed. To examine whether Atlantic salmon Mx1 protein (ASMx1) possesses antiviral activity against IPNV, CHSE-214 cells constitutively expressing ASMx1 were established. ASMx1 appeared to be localized in the cytoplasm. The ASMx1-expressing clone selected showed a severely reduced IPNV-induced cytopathic effect, which was confirmed by a 500-fold reduction in virus yield. The antiviral activity against IPNV was further confirmed by the inhibition of virus protein synthesis and the reduced accumulation of virus transcripts. The present work further adds to the body of evidence which suggests that antiviral activity is a major functional role of vertebrate Mx proteins. Moreover, the list of viruses inhibited by Mx proteins is extended to include double-stranded RNA viruses.

Yeast β-glucan stimulates respiratory burst activity of Atlantic salmon (Salmo salar L.) macrophages

Previous studies have shown that head kidney macrophages isolated from glucan injected rainbow trout (Oncorhychus mykiss) and Atlantic salmon (Salmo salar L.) have increased ability to kill Aeromonas salmonicida. The present work was aimed at investigating the in vitro effects of glucan on the respiratory burst and bactericidal potential of Atlantic salmon head kidney macrophages. Salmon macrophages were incubated for 1-7 days with various concentrations of yeast beta-glucan (MacroGard) and tested for respiratory burst activity by the reduction of nitroblue-tetrazolium (NBT) after exposure to phorbol myristate acetate (PMA) or opsonized zymosan. The macrophages showed a marked increase in respiratory burst activity 4 to 7 days after addition of glucan. Macrophages treated with 0.1-1 microgram mL-1 gave a maximum respiratory burst response, whereas 10 micrograms mL-1 had no effect and 50 micrograms mL-1 was inhibitory. The glucan also triggered respiratory burst activity directly, but this occurred only at relative high concentrations with a maximal effect at > or = 200 micrograms mL-1. The validity of using the NBT-assay as a measure of respiratory burst activity was confirmed by using inhibitors of O2- production (superoxide dismutase, trifluoperazine and diphenylene iodonium). Despite the stimulatory effect of glucan on the respiratory burst activity of salmon macrophages, these cells did not show increased bactericidal activity against the avirulent and virulent strain of A. salmonicida. Upregulation of burst activity alone is thus apparently not sufficient to enhance bactericidal activity against this pathogen by Atlantic salmon macrophages.

Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties.

A cluster of 11 interferon (IFN) genes were identified in the Atlantic salmon genome linked to the growth hormone 1 gene. The genes encode three different IFN subtypes; IFNa (two genes), IFNb (four genes) and IFNc (five genes), which show 22-32% amino acid sequence identity. Expression of the fish IFNs were studied in head kidney, leukocytes or TO cells after stimulation with the dsRNA poly I:C or the imidazoquinoline S-27609. In mammals, poly I:C induces IFN-beta through the RIG-I/MDA5 or the TLR3 pathway, both of which are dependent on NF-kB. In contrast, S-27609 induces mammalian IFN-alpha in plasmacytoid dendritic cells through the TLR7 pathway independent of NF-kappaB. The presence of an NF-kappaB site in their promoters and their strong up-regulation by poly I:C, suggest that salmon IFNa1/IFNa2 are induced through similar pathways as IFN-beta. In contrast, the apparent lack of NF-kappaB motif in the promoter and the strong upregulation by S-27609 in head kidney and leukocytes, suggest that IFNb genes are induced through a pathway similar to mammalian IFN-alpha. IFNc genes showed expression patterns different from both IFNa and IFNb. Taken together, salmon IFNa and IFNb are not orthologs of mammalian IFN-beta and IFN-alpha, respectively, but appear to utilize similar induction pathways.

Elicitors of the production of lignin-like compounds in cucumber hypocotyls

Lignification appears to be a component of the mechanism of disease resistance in cucumber plants to the fungus Cladosporium cucumerinum . A bioassay for elicitors of lignification in cucumber hypocotyls is described which is based upon the detection of lignin-like compounds on the wound surface of treated slices of cucumber hypocotyls using phloroglucinol/HCl. Elicitors of lignification were detected in extracts of cucumber cell walls arid mycelia of C . cucumerinum , and in culture filtrates of the fungus. Of a range of autoclaved polysaccharides and polyanionic compounds only pectin, polygalacturonic acid and chitosan had elicitor activity. Elicitors were released from cucumber cell walls by autoclaving and after partial purification from autoclaved cucumber cell walls were found to be oligomers of galacturonic acid. One of the most active of the cell wall elicitors was enriched in uronic acid (93·5% by weight) and contained low amounts of neutral sugars. It still showed elicitor activity when diluted down to a concentration of 12·5 μg uronic acid units ml −1 . Similar but more active elicitors were obtained by autoclaving polygalacturonic acid. These were found to be oligosaccharides of about 11 galactur-onosyl units which still showed activity when diluted down to a concentration of 0·8 μg uronic acid units ml −1 .

Stimulation of respiratory burst and phagocytic activity in Atlantic salmon (Salmo salar L.) macrophages by lipopolysaccharide

Abstract 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.

Cloning of an Mx cDNA from Atlantic Halibut ( Hippoglossus hippoglossus ) and Characterization of Mx mRNA Expression in Response to Double-Stranded RNA or Infectious Pancreatic Necrosis Virus

Mx proteins are GTPases that are specifically induced by type I interferons (IFN) in vertebrates. Some mammalian Mx proteins have antiviral activity against certain RNA viruses. A 2.3-kb full-length cDNA clone of an Atlantic halibut Mx gene was isolated from a liver cDNA library. The open reading frame (ORF) predicts a 622 amino acid protein of 71.2 kDa possessing a tripartite GTP binding motif, a dynamin signature, and a leucine zipper motif, which are conserved in all known Mx proteins. The C-terminal half contains a putative bipartite nuclear localization signal. The deduced halibut Mx protein showed approximately 76% sequence identity with the Atlantic salmon and rainbow trout Mx proteins, 55% identity with the human MxA, and 48% identity with the chicken Mx protein. Based on sequence comparison of 554-bp Mx cDNA fragments, the Atlantic halibut Mx showed more relationship with the perch and turbot than the salmonid Mx genes. Halibut appears to possess at least two Mx loci, as suggested by Southern blot analysis of genomic DNA. Two halibut Mx transcripts (2.2 kb and 2.6 kb) were strongly induced in vivo by the double-stranded RNA (dsRNA) poly I:C or infectious pancreatic necrosis virus (IPNV) in all organs studied.

Expression of interferon and interferon-induced genes in salmonids in response to virus infection, interferon-inducing compounds and vaccination

Interferons (IFNs) involved in innate immunity against viruses have recently been cloned from Atlantic salmon and rainbow trout. Moreover, several IFN-stimulated genes (ISGs) have been cloned from salmonids although only Mx has been shown to possess antiviral properties. Much less in known about how viruses induce IFNs in salmonids, but synthetic ligands for some of the main mammalian viral sensors also induce IFNs and ISGs in salmonids. Analysis of the promoters of the salmon IFN-alpha1 and IFN-alpha2 genes shows that activation is dependent on both NFkappaB and IRFs similar to human IFN-beta. Furthermore, several IFN-stimulated genes (ISGs) have been cloned from salmonids although only Mx has been shown to possess antiviral properties. The synthetic compounds poly I:C, imidazoquinolines and CpG oligonucleotides induce IFNs and ISGs in salmonids, probably through the same pathways as in mammals. Salmonid viruses show potent ability to stimulate expression of IFN and ISGs in vivo. Differences between viruses in the ability to stimulate host gene expression are often more evident in cell culture, but more work is needed to pinpoint how salmonid viruses antagonize the IFN system of their host. Finally, existing data suggest that IFNs play a role in the early non-specific protection observed after vaccination of salmonids with rhabdoviral DNA vaccines and conventional polyvalent vaccines.

Antiviral Activity of Salmonid Gamma Interferon against Infectious Pancreatic Necrosis Virus and Salmonid Alphavirus and Its Dependency on Type I Interferon

ABSTRACT 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.