s8ORF2 protein of infectious salmon anaemia virus is a RNA-silencing suppressor and interacts with Salmon salar Mov10 (SsMov10) of the host RNAi machinery.

The infectious salmon anaemia virus (ISAV) is a piscine virus, a member of Orthomyxoviridae family. It encodes at least 10 proteins from eight negative-strand RNA segments. Since ISAV belongs to the same virus family as Influenza A virus, with similarities in protein functions, they may hence be characterised by analogy. Like NS1 protein of Influenza A virus, s8ORF2 of ISAV is implicated in interferon antagonism and RNA-binding functions. In this study, we investigated the role of s8ORF2 in RNAi suppression in a well-established Agrobacterium transient suppression assay in stably silenced transgenic Nicotiana xanthi. In addition, s8ORF2 was identified as a novel interactor with SsMov10, a key molecule responsible for RISC assembly and maturation in the RNAi pathway. This study thus sheds light on a novel route undertaken by viral proteins in promoting viral growth, using the host RNAi machinery.

Protective oral vaccination against infectious salmon anaemia virus in Salmo salar.

Infectious salmon anemia (ISA) is a systemic disease caused by an orthomyxovirus, which has a significant economic impact on the production of Atlantic salmon (Salmo salar). Currently, there are several commercial ISA vaccines available, however, those products are applied through injection, causing stress in the fish and leaving them susceptible to infectious diseases due to the injection process and associated handling. In this study, we evaluated an oral vaccine against ISA containing a recombinant viral hemagglutinin-esterase and a fusion protein as antigens. Our findings indicated that oral vaccination is able to protect Atlantic salmon against challenge with a high-virulence Chilean isolate. The oral vaccination was also correlated with the induction of IgM-specific antibodies. On the other hand, the vaccine was unable to modulate expression of the antiviral related gene Mx, showing the importance of the humoral response to the disease survival. This study provides new insights into fish protection and immune response induced by an oral vaccine against ISA, but also promises future development of preventive solutions or validation of the current existing therapies.

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.

Development of a nanoparticle-based oral vaccine for Atlantic salmon against ISAV using an alphavirus replicon as adjuvant.

Adjuvants used in vaccine aquaculture are frequently harmful for the fish, causing melanosis, granulomas and kidney damage. Along with that, vaccines are mostly administered by injection, causing pain and stress to the fish. We used the DNA coding for the replicase of alphavirus as adjuvant (Ad) of a vaccine against ISAV. The Ad and an inactivated ISAV (V) were loaded in chitosan nanoparticles (NPs) to be administered orally to Atlantic salmon. NP-Ad was able to deliver the DNA ex vivo and in vivo. Oral administration of the NPs stimulated the expression of immune molecules, but did not stimulate the humoral response. Although the vaccination with NP-V results in a modest protection of fish against ISAV, NP-V administered together with NP-Ad caused a protection of 77%. Therefore, the DNA coding for the replicase of alphavirus could be administered orally and can potentiate the immuneprotection of a virine against infection.

Low virulent infectious salmon anaemia virus (ISAV) replicates and initiates the immune response earlier than a highly virulent virus in Atlantic salmon gills.

Observations from the field and experimental evidence suggest that different strains of infectious salmon anaemia virus (ISAV) can induce disease of varying severity in Atlantic salmon. Variation in host mortality and dissemination of ISAV isolates with high and low virulence was investigated using immersion challenge; from which mortality, pathological, immunohistochemical and preliminary molecular results have been previously published. Here, real-time RT-PCR analysis and statistical modelling have been used to further investigate variation in virus load and the response of four select immune genes. Expression of type I and II interferon (IFN), Mx and γIFN induced protein (γIP) to high and low pathogenic virus infection were examined in gill, heart and anterior kidney. In addition, a novel RNA species-specific assay targeting individual RNA types was used to investigate the separate viral processes of transcription and replication. Unexpectedly, the low virulent ISAV (LVI) replicated and transcribed more rapidly in the gills compared to the highly virulent virus (HVI). Subsequently LVI was able to disseminate to the internal organs more quickly and induced a more rapid systemic immune response in the host that may have offered some protection. Contrary to this, HVI initially progressed more slowly in the gills resulting in a slower generalised infection. However HVI ultimately reached a higher viral load and induced a greater mortality.

Salmonid alphavirus-based replicon vaccine against infectious salmon anemia (ISA): impact of immunization route and interactions of the replicon vector.

A salmonid alphavirus (SAV)-based replicon encoding the infectious salmon anemia virus (ISAV) hemagglutinin-esterase (HE), pSAV/HE, is an efficacious vaccine against infectious salmon anemia (ISA). Delivered intramuscularly (i.m.), the replicon vaccine provides high protection against subsequent ISAV challenge in Atlantic salmon (Salmo salar), and induces a strong innate response locally at the injection site. This may be beneficial and could warrant reduced doses and improved efficacy compared to conventional DNA vaccines. In the present study, we found that intraperitoneal (i.p.) administration of the pSAV/HE replicon vaccine did not induce protection, neither alone or in combination with a sub-potent, inactivated low-dose ISAV vaccine given i.p. No significant differences between the two immunization routes regarding systemic immune responses could be observed. I.m. injection of the replicon vector encoding a non-viral gene or the protective glycoprotein (G protein) from the heterologous viral hemorrhagic septicemia virus (VHSV) induced no protection against ISA. Although the replicons without the ISAV HE did induce IFN-signaling pathways at the muscle injection site similar to the pSAV/HE replicon they did not improve the efficacy of a sub-potent inactivated low-dose ISAV vaccine delivered i.p. Moreover, there was a tendency for reduced efficacy of the pSAV/HE replicon vaccine injected i.m. when co-injected with the replicon encoding the VHSV G protein, which previously, after DNA vaccination, have been reported to induce cross-protection against heterologous virus challenge in fish.

Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor.

Endothelial cells (ECs) line the luminal surfaces of the cardiovascular system and play an important role in cardiovascular functions such as regulation of haemostasis and vasomotor tone. A number of fish and mammalian viruses target these cells in the course of their infection. Infectious salmon anaemia virus (ISAV) attacks ECs and red blood cells (RBCs) of farmed Atlantic salmon (Salmo salar L.), producing the severe disease of infectious salmon anaemia (ISA). The investigation of ISA has up to now been hampered by the lack of a functional marker for ECs in Atlantic salmon in situ. In this study, we report the characterisation and use of a novel monoclonal antibody (MAb) detecting Atlantic salmon ECs (e.g. vessel endothelium, endocardial cells and scavenger ECs) and RBCs. The antibody can be used with immunohistochemistry, IFAT and on Western blots. It appears that the epitope recognised by the antibody is associated with the ISAV cellular receptor. Besides being a tool to identify ECs in situ, it could be useful in further studies of the pathogenicity of ISA. Finally, the detection of an epitope shared by ECs and RBCs agrees with recent findings that these cells share a common origin, thus the MAb can potentially be used to study the ontogeny of these cells in Atlantic salmon.

Immune parameters correlating with reduced susceptibility to pancreas disease in experimentally challenged Atlantic salmon (Salmo salar).

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.

Multiple passage of infectious salmon anaemia virus in rainbow trout, Oncorhynchus mykiss (Walbaum), did not induce increased virus load.

The infectious salmon anaemia virus (ISAV) has not been observed to cause natural disease in farmed rainbow trout, Onchorhynchus mykiss (Walbaum), but may cause high mortality in farmed Atlantic salmon, Salmo salar L. In this study, ISAV was passaged 10 times in succession by intraperitoneal injections of serum from previous passage into naïve rainbow trout. The serum viraemia was monitored by real-time qPCR. The rainbow trout in this study became infected but did not develop ISA. No clinical signs were observed in the rainbow trout in any passage, but replication of ISAV was detected from Day 4 post-infection (p.i.). Neither increased relative virus loads nor histopathological and immunohistochemical findings consistent with ISA were observed. However, the expression of interferon type I and Mx genes were slightly up-regulated in the hearts of some individual fish at day 17 p.i. Sequencing of all open reading frames in the ISAV genome of the 10th passage revealed two nucleotide mutations, one in segment 6 coding for the haemagglutinin-esterase (HE) and one in segment 1 coding for the basic polymerase 2 (PB2). The mutation in HE resulted in an amino acid substitution T/K(312) .

Completion of the full-length genome sequence of the infectious salmon anemia virus, an aquatic orthomyxovirus-like, and characterization of mAbs.

We report here the first full-length sequence of the eight ssRNA genome segments of the infectious salmon anemia virus (ISAV, Glesvaer/2/90 isolate), a salmonid orthomyxovirus-like. Comparison of ISAV genome sequence with those of others orthomyxovirus reveals low identity values, and a remarkable feature is the extremely long 5' end UTR of ISAV segments, which all contain an additional conserved motif of unknown function. In addition to the genome nucleotide sequence determination, specific mAbs have been produced through mice immunization with sucrose-purified ISAV. Four mAbs directed against the haemagglutinin-esterase glycoprotein, the nucleoprotein and free or actin-associated forms of the matrix protein have been characterized by (i) indirect fluorescent antibody test; (ii) virus neutralization; (iii) radioimmunoprecipitation and (iv) Western blot assays. These mAbs will potentially be useful for the development of new diagnostic tests, and the nucleotide sequences will help to establish a reverse genetics system for ISAV.

Expression of ssa-miR-155 during ISAV infection in vitro: Putative role as a modulator of the immune response in Salmo salar.

Multiple cellular components are involved in pathogen-host interaction during viral infection; in this context, the role of miRNAs have become highly relevant. We assessed the expression of selected miRNAs during an in vitro infection of a Salmo salar cell line with Infectious Salmon Anemia Virus (ISAV), the causative agent of a severe disease by the same name. Salmon orthologs for miRNAs that regulate antiviral responses were measured using RT-qPCR in an in vitro time-course assay. We observed a modulation of specific miRNAs expression, where ssa-miR-155-5p was differentially over-expressed. Using in silico analysis, we identified the putative mRNA targets for ssa-miR-155-5p, finding a high prevalence of hosts immune response-related genes; moreover, several mRNAs involved in the viral infective process were also identified as targets for this miRNA. Our results suggest a relevant role for miR-155-5p in Salmo salar during an ISAV infection as a regulator of the immune response to the virus.

Genetic markers of the immune response of Atlantic salmon (Salmo salar) to infectious salmon anemia virus (ISAV).

Infectious diseases among fish present an important economic burden for the aquaculture and fisheries industries around the world. For example, the infectious salmon anemia virus (ISAV) infects farmed Atlantic salmon (Salmo salar), and results in millions of dollars of lost revenue to salmon farmers. Although improved management and husbandry practices over the last few years have minimized the losses and the number of outbreaks, the risk of new virulent strains emerging is a looming threat to the viability and sustainability of this industry. A greater understanding of the host-pathogen interactions at the molecular level during the course of an infection thus remains of strategic importance for the development of molecular tools and efficient vaccines capable of minimizing losses in the eventual case of a new outbreak. Using a 32 k cDNA microarray platform (cGRASP), we have identified various signaling pathways and immune regulated genes, which are activated or repressed in Atlantic salmon head-kidney during the course of an ISAV infection. Gene expressions were measured at five different time-points: 6 h, 24 h, 3 d, 7 d and 16 d post-injection. The earliest time points showed only a few differentially expressed genes in ISAV injected fish, relative to sham injected controls, although as time progressed, many additional genes involved in key defense pathways were up-regulated including MHC type I, beta-2 microglobulin, TRIM 25 and CC chemokine 19. During the latest stage of the infection process, many genes related to oxygen transportation were under-expressed, which correlates well with the observed anemia that occurs prior to death in Atlantic salmon infected with virulent strains of ISAV.

In situ localisation of major histocompatibility complex class I and class II and CD8 positive cells in infectious salmon anaemia virus (ISAV)-infected Atlantic salmon.

It is assumed that the mobilisation of a strong cellular immune response is important for the survival of Atlantic salmon infected with infectious salmon anaemia virus (ISAV). In this study, the characterisation of immune cell populations in tissues of non-ISAV infected Atlantic salmon and during the early viraemia of ISAV was undertaken. Immunohistochemical investigations of spleen, head kidney and gills using monoclonal antibodies against recombinant proteins from MHC I, II and CD8 were performed on tissues from Atlantic salmon collected day 17 post-challenge in a cohabitant infection model. The localisations of MHC I and II in control salmon were consistent with previous reports but this study presents novel observations on the distribution of CD8 labelled cell populations in Atlantic salmon including the description of significant mucosal populations in the gills. The distribution of MHC I, MHC II and CD8 positive cell populations differed between control salmon and cohabitant salmon in the early stages of ISAV infection. The changes in MHC I labelled cells differed between organs in ISAV cohabitants but all investigated organs showed a decreased presence of MHC II labelled cells. Together with a clustering of CD8 labelled cells in the head kidney and a reduced presence of CD8 labelled cells in the gills, these observations support the early mobilisation of cellular immunity in the response of Atlantic salmon to ISAV infection. However, differences between the present study and the findings from studies investigating immune gene mRNA expression during ISAV infection suggest that viral strategies to interfere with protein expression and circumvent the host immune response could be operative in the early response to ISAV infection.

The effect of sub-culturing on the basal level of type I interferon (IFN) gene expression in the Salmon Head Kidney (SHK-1) cell line.

Over sub-culturing a cell line generates a selective pressure which can result in key cellular functions being altered such as gene and protein expression. The present study set out to determine whether serial sub-culturing affects the antiviral state of the Salmon Head Kidney (SHK-1) cell line. Cells were cultured under constant conditions and real-time PCR was performed to measure the level of interferon (IFN) and Mx gene expression over different passage numbers. A significant increase in the basal level of IFN and Mx gene expression was recorded at passage number 58 (3 and 14-fold increase versus passage number 53), suggesting a sub-culturing effect on the type I IFN response in SHK-1 cells. Passage dependent variations in morphology and cell sub-populations have been previously observed in SHK-1 cells. Such variations in cell sub-types were suspected to be responsible for the fluctuations in IFN and Mx gene expression recorded in this study.

Peptide-binding motif prediction by using phage display library for SasaUBA*0301, a resistance haplotype of MHC class I molecule from Atlantic Salmon (Salmo salar).

The structure of the peptide-binding specificity of major histocompatibility complex (MHC) class I has been analyzed extensively in human and mouse. For fish, there are no crystallographic models of MHC molecules, neither are there data on the peptide-binding specificity. In this study, we describe for the first time the identification of a fish class I peptide-MHC ligand binding motif. Phage display technology using both 7 mer and 12 mer libraries enabled us to identify peptide ligands with unique specificity that interacts with the recombinant Salmon MHC class I molecule. The recombinant proteins, beta 2m/SasaUBA*0301, were produced in Escherichia coli, in which the carboxyl terminus of beta 2-microglobulin is joined together with a flexible (GGGGS)3 linker to the amino terminus of the heavy chain. One hundred and seven individual phages bound to beta 2m/SasaUBA*0301 were isolated after four rounds of panning from the 7 mer random-peptide library. The peptide encoding sequences were determined and peptide alignment led to the prediction of position-specific anchor residue. A prominent proline at position 2 was observed and we predict that it might be one of the anchors at the N-terminus. Meanwhile, phage display peptide library encoding random 12 mer peptides was also screened against beta 2m/SasaUBA*0301. Eighty-five percentages of the corresponding peptides have an enrichment of leucine, methionine, valine, or isoleucine at the C-terminus. We predict that this particular allele of Salmon class I molecule might have a very similar binding motif at the C-terminus compared with a known mouse class I molecule H2-Kb which has L, or I, V, M at p8. Previous work showed that Atlantic Salmon carrying the allele SasaUBA*0301 are resistant to infectious Salmon aneamia virus and there is a significant association between MHC polymorphism and the disease resistance. Therefore, our study might contribute to designing a peptide vaccine against this viral disease.

Gene expression analyses in Atlantic salmon challenged with infectious salmon anemia virus reveal differences between individuals with early, intermediate and late mortality.

BACKGROUND: Infectious salmon anemia virus (ISAV) causes a multisystemic disease responsible for severe losses in salmon aquaculture. Better understanding of factors that explain variations in resistance between individuals and families is essential for development of strategies for disease control. To approach this, we compared global gene expression using microarrays in fish dying early and late in the time course following infection from a highly pathogenic ISAV. RESULTS: Tissues (gill, heart, liver and spleen) from infected Atlantic salmon (cohabitation, ISAV Glesvaer 2/90 isolate) were collected from three stages over the time course of the experiment; early (EM, 0-10% cumulative mortality (CM), 21-25 days post-infection (DPI)), intermediate (IM, 35-55% CM, 28-31 DPI) and late (LM, 75-85% CM, 37-48 DPI) mortality. Viral loads were equal in EM and IM but dropped markedly in LM fish. Gene expression analyses using a 1.8 K salmonid fish cDNA microarray (SFA2.0) and real-time qPCR revealed a large group of genes highly up-regulated across tissues in EM, which were mainly implicated in innate antiviral responses and cellular stress. Despite equal levels of MHC class I in EM and LM, increase of splenic and cardiac expression of immunoglobulin-like genes was found only in LM while a suite of adaptive immunity markers were activated already in IM. The hepatic responses to ISAV were characterized by difference between EM and LM in expression of chaperones and genes involved in eicosanoid metabolism. To develop classification of high and low resistance phenotypes based on a small number of genes, we processed results from qPCR analyses of liver using a linear discriminant analysis. Four genes (5-lipoxygenase activating protein, cytochrome P450 2K4-1, galectin-9 and annexin A1) were sufficient for correct assignment of individuals to EM, LM and uninfected groups, while IM was inseparable from EM. Three of four prognostic markers are involved in metabolism of inflammatory regulators. CONCLUSION: This study adds to the understanding of molecular determinants for resistance to acute ISAV infection. The most susceptible individuals were characterized by high viral replication and dramatic activation of innate immune responses, which did not provide protection. The ability to endure high levels of infection for sustained periods could be associated with lower inflammatory responses while subsequent protection and viral clearance was most likely conferred by activation of adaptive immunity.

Transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells.

The aquatic orthomyxovirus infectious salmon anemia virus (ISAV) is an important pathogen for salmonid aquaculture, however little is known about protective and pathological host responses to infection. We have investigated intracellular responses during cytopathic ISAV infection in the macrophage-like Atlantic salmon kidney (ASK) cell line by microarray analysis (1.8k SFA2.0 immunochip) and a functional assay for glutathione. Gene transcription changed rapidly and consistently with time and with minor differences between two virus isolates. While several pro-inflammatory and antiviral immune genes were induced, genes involved in cell signaling and integrity were down-regulated, suggesting isolation of infected cells from cell-to-cell interaction and responses to external signals. Differential expression of genes regulating cell cycle and apoptosis implied opposite cues from host cell and virus. This was in pace with massive down-regulation of genes involved in biosynthesis and processing of nucleotides and nucleic acids. Significant down-regulation of several genes involved in metabolism of reactive oxygen species suggested increased oxidative stress, which was confirmed by a functional assay showing reduced levels of glutathione during infection. Testing of expression data against a microarray database containing diverse experiments revealed candidate marker genes for ISAV infection. Our findings provide novel insight into cellular host responses and determinants for acute cytopathic ISAV infection.

Cloning and expression analysis of an Atlantic salmon (Salmo salar L.) tapasin gene.

Loading of the major histocompatibility complex (MHC) class I molecule with peptide is mediated by the multimeric peptide-loading complex in the ER where the glycoprotein tapasin (TAPBP) is required for stabilization of the complex and for control of peptide loading onto MHC class I. To expand our knowledge on antigen presentation genes in Atlantic salmon, we isolated a full-length salmon tapasin cDNA sequence (Sasa-TAPBP). It encoded a 443 bp amino acid sequence with two N-glycosylation sites, two conserved mammalian tapasin signature motifs, two Ig superfamily (IgSf) domains, a transmembrane (TM) domain and an ER-retention KK motif at the C-terminal end, indicative of a similar function as mammalian tapasins. We analysed the regulation of Sasa-TAPBP under immunostimulatory conditions and found an mRNA-upregulation during early infectious salmon anemia virus (ISAV) infection and poly I:C stimulation in vivo and in vitro, in line with our previous findings for other MHC class I pathway genes.

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.

Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon.

Optimization of reference genes for real-time polymerase chain reaction (PCR) studies in fish is strongly needed. We systematically tested beta-actin (ACTB), 18S rRNA (18S), beta(2)-microglobulin (B2M), elongation factor 1-alpha (EF1A), RNA polymerase I and II (RPL1/2), and glycerol 6-phosphate dehydrogenase (G6PDH) for stability in salmon immune relevant tissues and kidney cells (ASK) infected with infectious salmon anemia virus (ISAV), plus in tissues from fish fed thia fatty acids. Transcription of all genes was unchanged in infected and thia fatty acid-treated tissues versus normal tissues. Between tissues, 18S and EF1A were most stable, RPL1 and RPL2 were intermediate, and G6PDH and ACTB and B2M were the least stable. However, only 18S had constant expression in infected cells; the rest significantly down-regulated. Implications of this finding were demonstrated when normalizing major histocompatibility complex (MHC) class I expression in ASK. Software predictions supported a proper normalization is obtained combining 18S, EF1A, and RPL1 in vivo, but for in vitro viral infection assays we recommend using 18S.