Salmonid Cell Lines Research Articles

Implementation of mRNA-Lipid Nanoparticle Technology in Atlantic Salmon (Salmo salar).

This study was conducted to investigate whether mRNA vaccine technology could be adapted for the ectothermic vertebrate Atlantic salmon (Salmo salar). Lipid nanoparticle (LNP) technology has been developed and optimized for mRNA vaccines in mammals, stabilizing mRNA and facilitating its delivery into cells. However, its utility at the temperatures and specific biological environments present in ectotherms remains unclear. In addition, it is unknown if modified mRNA containing non-canonical nucleotides can correctly translate in salmonid cells. We used an mRNA transcript coding for enhanced green fluorescence protein, flanked by the untranslated regions of the hemagglutinin-esterase gene of the infectious salmon anemia virus, and a 120-base-long poly(A) tail. The mRNA was generated via in vitro transcription where uridine residues were replaced with N1-methyl-pseudouridines, and then encapsulated in LNPs. When transfected into the salmonid cell line CHH-1, the mRNA-LNP construct induced expression of EGFP. Furthermore, when mRNA-LNPs were injected intramuscularly into salmon, in vivo protein expression was demonstrated via immunohistochemistry. EGFP was observed in cells infiltrating the spaces between muscle cells in a focal inflammatory response. The results indicate that N1-methyl-pseudouridine-modified mRNA encapsulated in LNPs can be used to express antigens of interest in salmonid fish.

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

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. 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. 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. Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

The Viral Hemorrhagic Septicemia Virus (VHSV) Markers of Virulence in Rainbow Trout (Oncorhynchus mykiss).

Viral hemorrhagic septicemia virus (VHSV) is a highly contagious virus leading to high mortality in a large panel of freshwater and marine fish species. VHSV isolates originating from marine fish show low pathogenicity in rainbow trout. The analysis of several nearly complete genome sequences from marine and freshwater isolates displaying varying levels of virulence in rainbow trout suggested that only a limited number of amino acid residues might be involved in regulating the level of virulence. Based on a recent analysis of 55 VHSV strains, which were entirely sequenced and phenotyped in vivo in rainbow trout, several amino acid changes putatively involved in virulence were identified. In the present study, these amino acid changes were introduced, alone or in combination, in a highly-virulent VHSV 23–75 genome backbone by reverse genetics. A total of 35 recombinant VHSV variants were recovered and characterized for virulence in trout by bath immersion. Results confirmed the important role of the NV protein (R116S) and highlighted a major contribution of the nucleoprotein N (K46G and A241E) in regulating virulence. Single amino acid changes in these two proteins drastically affect virus pathogenicity in rainbow trout. This is particularly intriguing for the N variant (K46G) which is unable to establish an active infection in the fins of infected trout, the main portal of entry of VHSV in this species, allowing further spread in its host. In addition, salmonid cell lines were selected to assess the kinetics of replication and cytopathic effect of recombinant VHSV and discriminate virulent and avirulent variants. In conclusion, three major virulence markers were identified in the NV and N proteins. These markers explain almost all phenotypes (92.7%) observed in trout for the 55 VHSV strains analyzed in the present study and herein used for the backward validation of virulence markers. The identification of VHSV specific virulence markers in this species is of importance both to predict the in vivo phenotype of viral isolates with targeted diagnostic tests and to improve prophylactic methods such as the development of safer live-attenuated vaccines.

Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes

Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.

Phylogeny and expression of the tetraspanin CD9 in salmonid cell lines in response to interferon stimulation

Phylogeny and expression of the tetraspanin CD9 in salmonid cell lines in response to interferon stimulation

Polyphenols obtained from Didymosphenia geminata (Lyngbye) Schmith altered the viability and proliferation of salmonids cells lines SHK-1 and CHSE-214

Didymosphenia geminata (Lyngbye) Schmidt, also referred to as Didymo, is an invasive diatom that forms nuisance mats. Since it was first reported in our country in approximately 2010, Didymo has expanded and colonized different rivers in the Zona Austral region of Chile. Its biology and effects on ecosystems are still being studied because Didymo is an invasive algal mat that forms in a range of systems from oligotrophic austral rivers to more subtropical systems. We aimed to evaluate the viability of two salmonid cell lines, CHSE-214 and SHK-1 (somatic and embryonic cell lines, respectively), in dilutions of river water alone and in river water contaminated with Didymo or polyphenols extracted from Didymo under controlled conditions. We developed an artificial river system (2 aquariums/replicate) from five different rivers from the central area (Bio-Bio) and Patagonia area (Futaleufú) of Chile to maintain Didymo in the benthic phase. The Didymo populations were maintained for six months in the water from the rivers, after which samples were obtained. Following the extraction of polyphenols from the Didymo samples maintained in the artificial rivers, toxicity assays (10 assays) were performed to determine cell viability. Our results indicated that the CHSE-214 cells were highly sensitive to increasing concentrations of Didymo extracts. We observed a 50% reduction in cell viability after 24 h of exposure to a 0.01 V/V dilution, and this treatment further reduced the proliferative capacity by 70% after 120 h. The SHK-1 cells were less responsive, showing only a 20% decrease in viability at 24 h and a lower cell proliferation rate (45%) after 120 h, which remained higher than that of the CHSE-214 cells. We conclude that certain cell types are sensitive to Didymo in rivers, suggesting that there are chronic effects on several aquatic species following exposure to these diatom substances. These effects should be further studied using this laboratory model to understand the full impact of Didymo on river ecosystems.

Molecular cloning of MDA5, phylogenetic analysis of RIG‐I‐like receptors (RLRs) and differential gene expression of RLRs, interferons and proinflammatory cytokines after in vitro challenge with IPNV, ISAV and SAV in the salmonid cell line TO

The RIG-I receptors RIG-I, MDA5 and LGP2 are involved in viral recognition, and they have different ligand specificity and recognize different viruses. Activation of RIG-I-like receptors (RLRs) leads to production of cytokines essential for antiviral immunity. In fish, most research has focused on interferons, and less is known about the production of proinflammatory cytokines during viral infections. In this study, we have cloned the full-length MDA5 sequence in Atlantic salmon, and compared it with RIG-I and LGP2. Further, the salmonid cell line TO was infected with three fish pathogenic viruses, infectious pancreatic necrosis virus (IPNV), infectious salmon anaemia virus (ISAV) and salmonid alphavirus (SAV), and differential gene expression (DEG) analyses of RLRs, interferons (IFNa-d) and proinflammatory cytokines (TNF-α1, TNF-α2, IL-1β, IL-6, IL-12 p40s) were performed. The DEG analyses showed that the responses of proinflammatory cytokines in TO cells infected with IPNV and ISAV were profoundly different from SAV-infected cells. In the two aforementioned, TNF-α1 and TNF-α2 were highly upregulated, while in SAV-infected cells these cytokines were downregulated. Knowledge of virus recognition by the host and the immune responses during infection may help elucidate why and how some viruses can escape the immune system. Such knowledge is useful for the development of immune prophylactic measures.

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model.

Piscirickettsia salmonis is a facultative intracellular bacterium that causes the disease called “salmon rickettsial syndrome”. Attempts to control this disease have been unsuccessful, because existing vaccines have not achieved the expected effectiveness and the antibiotics used fail to completely eradicate the pathogen. This is in part the product of lack of scientific information that still lacks on the mechanisms used by this bacterium to overcome infected–cell responses and survive to induce a productive infection in macrophages. For that, this work was focused in determining if P. salmonis is able to modify the expression and the imbalance of IL-12 and IL-10 using an in vitro model. Additionally, we also evaluated the role the antimicrobial peptide hepcidin had in the control of this pathogen in infected cells. Therefore, the expression of IL-10 and IL-12 was evaluated at earlier stages of infection in the RTS11 cell line derived from Oncorhynchus mykiss macrophages. Simultaneously, the hepcidin expression and location was analyzed in the macrophages infected with the pathogen. Our results suggest that IL-10 is clearly induced at early stages of infection with values peaking at 36 hours post infection. Furthermore, infective P. salmonis downregulates the expression of antimicrobial peptide hepcidin and vesicles containing this peptide were unable to merge with the infective bacteria. Our results suggest that P. salmonis is able to manipulate the behavior of host cytokines and likely might constitute a virulence mechanism that promotes intracellular bacterial replication in leukocytes cells lines of trout and salmon. This mechanism involves the generation of an optimum environment for the microorganism and the downregulation of antimicrobial effectors like hepcidin.

Antioxidant and anti-inflammatory activities ofPinus radiatabark extract in salmonid cell lines

A fish meal supply shortage is limiting aquaculture development. Currently, plant-based proteins, such as soya bean meal, are being used as an alternative protein source, despite that such a diet can adversely affect fish, such as by inducing an inflammatory response. A possible solution is to include dietary additives in farm diets to counteract negative effects. One such solution originates from pine bark extracts, which present bioactive properties. In this study, the antioxidant and anti-inflammatory properties of Pinus radiata bark extracts were evaluated for the first time in a salmonid cell line. This extract chemically demonstrated antioxidant activity through 2,2-diphenyl-1-picrylhydrazyl (DPPH = 58.4 ± 1.1%) and ferric ion reducing antioxidant power (FRAP = 575 ± 17 mgEqFe(II)·g extract−1) assays. Additionally, the extract showed high flavonoid and phenolic compound contents. Up to 100 mg mL−1, the P. radiata extract showed no cytotoxicity in the CHSE-214 salmonid embryo cell line. Moreover, the antioxidant activity of the extract (50 μg mL−1) was evaluated by a dichlorofluorescein (DCFH) assay in the SHK-1 salmon cell line challenged with an oxidant stimulus (H2O2), showing 58.9% activity. The extract also protected DNA from oxidative damage, as observed through a comet assay. When assessing anti-inflammatory properties in an in vitro inflammation model, the extract significantly reduced the relative expression of the pro-inflammatory cytokines interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8) and of the inducible cyclooxygenase-2 (COX-2) enzyme. These results suggest a potential application of P. radiata bark extract in functional foods in aquaculture.

Differential response of the Senegalese sole (Solea senegalensis) Mx promoter to viral infections in two salmonid cell lines

Mx proteins are main effectors of the antiviral innate immune defence mediated by type I interferon (IFN I). The IFN I response is under a complex regulation; hence, one of the key issues in understanding virus-host interaction is the knowledge of the regulatory mechanisms governing this response. With this purpose, in this study Chinook salmon embryo cells (CHSE-214) and rainbow trout gonad cells (RTG-2) were transiently transfected with a vector containing the luciferase reporter gene under the control of the Senegalese sole Mx promoter. These transfected cells were infected with infectious pancreatic necrosis virus (IPNV), viral haemorrhagic septicaemia virus (VHSV) and epizootic haematopoietic necrosis virus (EHNV) at different doses in order to study the luciferase fold induction in response to viral infections. Transfected CHSE-214 cells infected with EHNV showed significant induction of the luciferase reporter gene, compared to control non-infected cells, at different times post infection (p.i.). The maximum expression was recorded at 24h p.i. in cells inoculated with 5×102TCID50/mL (2.17 folds compared to control cells). In these cells, the infection with IPNV and VHSV did not result in the luciferase expression at any time and doses tested. In transfected RTG-2 cells, VHSV stimulated luciferase expression, obtaining a maximum activity at 48h p.i. in cells infected with 5×102TCID50/mL (2.9 folds compared to control cells), whereas RTG-2 cells infected with IPNV and EHNV did not show significant luciferase activity at any time point. The different induction of the Senegalese sole Mx promoter in CHSE-214 and RTG-2 cells after infection with the same viruses indicates that cell-specific factors are significantly involved in the IFN-signalling response, and, probably, on the success of the strategies of these viruses to escape the IFN mechanisms. The use of these two different cellular systems might be an interesting approach to identify such cellular factors.

Delayed protein shut down and cytopathic changes lead to high yields of infectious pancreatic necrosis virus cultured in Asian Grouper cells

Inactivated whole virus vaccines represent the majority of commercial preparations used to prevent infectious pancreatic necrosis (IPN) in salmonids today. The production of these vaccines requires high virus concentrations that are resource-demanding. In this study, we describe the cultivation of high yields of IPN virus in Asian Grouper strain K (AGK) cells. The mechanism by which this is achieved was investigated by comparison with commonly used salmonid cell lines (RTG-2 and CHSE-214 cells). The cells were counted before and sequentially after infection. Thereafter, protein shut down, virus yields and apoptosis were assessed. The effects of poly(I:C) pre-treatment and Mx expression on IPNV concentrations were examined and the results show that high virus yields were associated with high cell numbers per unit volume, delayed cell death and apoptosis in AGK cells while the opposite was observed in RTG-2 cells. Poly(I:C) treatment and Mx expression resulted in a dose-dependent inhibition of virus multiplication. The production capacity of AGK and CHSE-214 cells were compared and higher split ratio and shorter split interval of AGK cells documents dramatic differences in virus antigen production capacity. Collectively, the results suggest that high cell numbers and prolonged survival of AGK cells are responsible for the superior virus yields over RTG-2 and higher split ratio/shorter split interval makes AGK superior over CHSE cells.

The Use of Fish‐Derived Cell Lines for Investigation of Environmental Contaminants: An Update Following OECD's Fish Toxicity Testing Framework No. 171

Protocols for evaluating chemical toxicity at the cellular level using fish cell lines are described in this unit. Routine methodologies for growing salmonid cell lines, and using them in aquatic toxicology studies that support the mandate of the Organization for Economic Co-operation and Development (OECD) to reduce the use of whole animals in toxicity testing, are presented. Rapid, simple, cost-effective tests evaluating viability of cells with three indicator dyes per sample provides a broad overview of the sensitivity of cells to chemical contaminants. This fluorometric assay involves: (1) alamar blue for metabolic activity, (2) CFDA-AM for membrane integrity, and (3) neutral red for lysosomal function. These protocols are conveniently performed in semi-unison within the same multiwell plates and read at three different wavelengths. Detailed step-by-step descriptions of the assays, parameters to consider, troubleshooting, and guidelines for data interpretation are provided as essential tools for investigating environmental aquatic contaminants at the cellular level.

Comparison of complete polyprotein sequences of two isolates of salmon alphavirus (SAV) type I and their behaviour in a salmonid cell line

Salmon pancreas disease virus is an alphavirus (family Togaviridae) affecting mainly Atlantic salmon (Salmo salar L.). Both polyprotein sequences of the Scottish isolate (SAV4640) were determined and compared with those of Irish isolate SAVF93-125. High amino acid sequence similarity (99.4%) was found. Six amino acid deletions were found in the E2 gene of SAV4640. SAVF93-125 demonstrated a high viral load in culture despite high Mx expression. Approximately 50% of cells infected with SAVF93-125 exhibited a cytopathic effect by day 8. SAV4640 successfully entered the cells, inducing 10,500-fold higher Mx expression at day 2 compared to SAVF93-25; however, no replication was observed based on results of the nsP1 qRT-PCR.

CpG oligonucleotides bind TLR9 and RRM-Containing proteins in Atlantic Salmon (Salmo salar)

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

MULAN related gene (MRG): A potential novel ubiquitin ligase activator of NF-kB involved in immune response in Atlantic salmon (Salmo salar)

Nuclear factor-kB (NF-kB) is a transcription factor that plays a central role in the regulation of a variety of genes including many involved in bacterial and viral infections. NF-kB is normally sequestered by inhibitory proteins (IkBs) in the cytoplasm of non-stimulated cells. The degradation of IkBs by the ubiquitin proteasome pathway releases NF-kB allowing its translocation to the nucleus where it regulates gene transcription. The Mitochondrial Ubiquitin Ligase Activator of NF-kB, (MULAN), is an E3 ubiquitin ligase involved in controlling activation of NF-kB, and regulating mitochondrial dynamics and apoptosis. We report the characterisation of a novel piscine-specific MULAN related gene (MRG) sequence, its mRNA tissue distribution and expression following in vivo and in vitro challenges. MRG cDNA was identified in Atlantic salmon and its sequence encodes a predicted protein of 274 amino acids. The mRNA of MRG was expressed in multiple tissues, with the highest abundance head kidney. An Aeromonas salmonicida bacterial challenge increased expression of this gene in head kidney, liver and gill tissue at 6h and 24h. In vitro stimulation of a salmonid cell line indicated MRG was increased in expression following stimulation with LPS, PolyI:C and recombinant trout IL-1β for 4h and 24h. These results suggest an active role of MRG in the activation of the NF-kB pathway during early immune responses.

Cloning and expression analysis of the Mitochondrial Ubiquitin Ligase Activator of NF-κB (MULAN) in Atlantic salmon ( Salmo salar)

Nuclear factor-κB (NF-κB) is a transcription factor involved in the regulation of a large number of genes including many involved in bacterial and viral infections. NF-κB is normally sequestered by inhibitory proteins (IκBs) in the cytoplasm of non-stimulated cells. The degradation of IκBs by the ubiquitin proteasome pathway leads to the rapid translocation of NF-κB to the nucleous where it regulates gene transcription. The Mitochondrial Ubiquitin Ligase Activator of NF-κB, (MULAN), is an E3 ubiquitin ligase believed to be central in controlling activation of NF-κB, and regulating the mitochondrial dynamics and apoptosis process. We report, for the first time in fish, the characterization of a MULAN cDNA in Atlantic salmon and rainbow trout. The salmonid MULAN sequences encode predicted proteins of 352 amino acids. The mRNA of MULAN was expressed in multiple tissues, with the highest abundance in brain and white muscle. An Aeromonas salmonicida bacterial challenge increased expression of this gene in head kidney, liver and gill both at 6 and at 24 h following the infection. In vitro experiments using the salmonid cell line RTG-2 indicated MULAN was increased in expression following 4 h stimulation with LPS and recombinant trout IL-1β. MULAN expression remained increased 24 h post-stimulation with both LPS and IL-1β, but was down regulated by PolyI:C at this time. These results suggest an active role of the MULAN gene in the activation of the NF-κB pathway during piscine immune responses.

Endogenous Transposases Affect Differently Sleeping Beauty and Frog Prince Transposons in Fish Cells

Fish cells stably expressing exogenous genes have potential applications in the production of fish recombinant proteins, gene-function studies, gene-trapping, and the production of transgenic fish. However, expression of a gene of interest after random integration may be difficult to predict or control. In the past decade, major contributions have been made in vertebrate-gene transfer, by using tools derived from DNA transposons. Among them, the Sleeping Beauty (SB) and Frog Prince (FP) transposons, derived, respectively, from fish and frog genomes, mediate transposition in a large variety of cells, although with different efficiency. This study was aimed at assessing the activities of the SB and the FP transposases in fish cell lines from genetically distant species (CHSE-214, RTG-2, BF-2, EPC, and SAF-1). Their transpositional ability was evaluated by the plasmid-based excision assay, the colony formation assay, and the footprint patterns. The results reveal that while both transposases are active in all cell lines, the transposition rates and the precision of the transposition are overall higher with FP than SB. Our results also indicated a key role of cell-specific host factors in transposition, which was associated with the presence of Tc1-like endogenous transposases; this effect was more accentuated in the two salmonid cell lines transfected with SB. This result agrees with previous studies supporting the use of transposons in heterologous organisms to prevent from genomic instability and from impeding the precise activity of the exogenous transposase.

Immunostaining of Atlantic salmon (Salmo salar L.) leucocytes

Different salmon leucocyte subpopulations were identified by immunostaining using rabbit antiserum raised against the salmonid cell line TO derived from head kidney leucocytes in combination with other available immunoglobulins. The rabbit anti-TO cell line serum immunostained all isolated leucocytes from head kidney, peripheral blood and spleen, as shown by analyses of these leucocytes by flow cytometry and by fluorescence microscopy. In cytospin preparations, the staining of salmon leucocytes using rabbit anti-TO serum as the primary antibody revealed greater morphological details compared to conventional staining procedures, especially among isolated spleen leucocytes where cells with a morphology usually limited to dendritic cells were seen. Other cells of various shapes and protrusions were also stained although the anti-TO serum did not stain protrusions on all cell types. Among the immunoglobulin positive cells, the thin protrusions were only seen when immunostained using anti-IgM antibody. The same was observed for neutrophils stained using the monoclonal E3D9 antibody. The double staining of cells using rabbit anti-TO serum and monoclonal antibodies specific for IgM positive cells or neutrophils clearly show how the morphology of these cells can be compared with the rest of the leucocyte population. The staining of salmon leucocytes by antiserum to a salmon leucocyte cell line TO provides a tool for staining the total population of salmon immune cells, and can be used in immunofluorescence or confocal microscopy in combinations with labelling of cellular components or pathogens. The detailed morphological characteristics, such as cell protrusions, visualized by the presented staining have not been observed on fish leucocytes by conventional cell staining procedures.

An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV

BackgroundDue to the limited number of species specific antibodies against fish proteins, differential gene expression analyses are vital for the study of host immune responses. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most powerful tools for this purpose. Nevertheless, the accuracy of the method will depend on the careful selection of genes whose expression are stable and can be used as internal controls for a particular experimental setting.FindingsThe expression stability of five commonly used housekeeping genes [beta-actin (ACTB), elongation factor 1-alpha (EF1A), ubiquitin (UBQ), glyceraldehyd-3-phosphate dehydrogenase (GAPDH) and tubulin alpha (TUBA)] were monitored in salmonid cell lines CHSE-214 and RTS11 after infection with two of the most fastidious fish pathogens, the facultative bacterium Piscirickettsia salmonis and the aquabirnavirus IPNV (Infectious Pancreatic Necrosis Virus). After geNorm analysis, UBQ and EF1A appeared as the most stable, although EF1A was slightly upregulated at late stages of P. salmonis infection in RTS11. ACTB instead, showed a good performance in each case, being always considered within the three most stable genes of the panel. In contrast, infection-dependent differential regulation of GAPDH and TUBA was also demonstrated.ConclusionBased on the data presented here with the cell culture models CHSE-214 and RTS11, we suggest the initial choice of UBQ, ACTB and EF1A as reference genes in qRT-PCR assays for studying the effect of P. salmonis and IPNV on the host immune response.

Mitochondrial Antiviral Signaling Protein Plays a Major Role in Induction of the Fish Innate Immune Response against RNA and DNA Viruses

Viral infection triggers host innate immune responses through cellular sensor molecules which activate multiple signaling cascades that induce the production of interferons (IFN) and other cytokines. The recent identification of mammalian cytoplasmic viral RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and their mitochondrial adaptor, the mitochondrial antiviral signaling protein (MAVS), also called IPS-1, VISA, and Cardif, highlights the significance of these molecules in the induction of IFN. Teleost fish also possess a strong IFN system, but nothing is known concerning the RLRs and their downstream adaptor. In this study, we cloned MAVS cDNAs from several fish species (including salmon and zebrafish) and showed that they were orthologs of mammalian MAVS. We demonstrated that overexpression of these mitochondrial proteins in fish cells led to a constitutive induction of IFN and IFN-stimulated genes (ISGs). MAVS-overexpressing cells were almost fully protected against RNA virus infection, with a strong inhibition of both DNA and RNA virus replication (1,000- and 10,000-fold decreases, respectively). Analyses of MAVS deletion mutants showed that both the N-terminal CARD-like and C-terminal transmembrane domains, but not the central proline-rich region, were indispensable for MAVS signaling function. In addition, we cloned the cDNAs encoding a RIG-I-like molecule from salmonid and cyprinid cell lines. Like the case with MAVS, overexpression of RIG-I CARDs in fish cells led to a strong induction of both IFN and ISGs, conferring on fish cells full protection against RNA virus infection. This report provides the first demonstration that teleost fish possess a functional RLR pathway in which MAVS may play a central role in the induction of the innate immune response.