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

BackgroundInterferon (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.MethodsBy 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.ResultsWe 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.ConclusionAside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

Finfish production has seen over three-fold increase in the past 30 years (1990–2020), and Atlantic salmon (A. salmon; salmo salar) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci—cr2 and mmp9 of A. salmon—were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells. [ABSTRACT FROM AUTHOR]

The interleukin-1-receptor-associated kinase 3 (IRAK3) is known in mammals as a negative feedback regulator of NF-κB-mediated innate-immune mechanisms. Our RNA-seq experiments revealed a prototypic 1920-nt sequence encoding irak3 from rainbow trout (Oncorhynchus mykiss), as well as 20 variants that vary in length and nucleotide composition. Based on the DNA-sequence information from two closely related irak3 genes from rainbow trout and an irak3-sequence fragment from Atlantic salmon retrieved from public databases, we elucidated the underlying genetic causes for this striking irak3 diversity. Infecting rainbow trout with a lethal dose of Aeromonas salmonicida enhanced the expression of all variants in the liver, head kidney, and peripheral blood leucocytes. We analyzed the functional impact of the full-length factor and selected structural variants by overexpressing them in mammalian HEK-293 cells. The full-length factor enhanced the basal activity of NF-κB, but did not dampen the TLR2-signaling-induced levels of NF-κB activation. Increasing the basal NF-κB-activity through Irak3 apparently does not involve its C-terminal domain. However, more severely truncated factors had only a minor impact on the activity of NF-κB. The TLR2-mediated stimulation did not alter the spatial distribution of Irak3 inside the cells. In salmonid CHSE-214 cells, we observed that the Irak3-splice variant that prominently expresses the C-terminal domain significantly quenched the stimulation-dependent production of interleukin-1β and interleukin-8, but not the production of other immune regulators. We conclude that the different gene and splice variants of Irak3 from trout play distinct roles in the activation of immune-regulatory mechanisms.

The article discusses a research which described a novel salmon gill poxvirus (SGPV) variant among Atlantic salmon in Eastern Canada. It attempts to isolate and identify the variant. It discusses the results of disease testing, quantitative polymerase chain reaction and transmission electron microscopy.

Double-stranded RNA (dsRNA) is produced by almost all viruses during their replicative cycle and is a potent inducer of the innate antiviral immune response including inducing expression of type I interferons (IFNs) and interferon-stimulated genes (ISGs). During lytic virus infections intracellular dsRNA can escape into the extracellular space, where surface pattern recognition receptors, such as class A scavenger receptors (SR-As) facilitate its binding and entry into neighbouring cells. Studying extracellular dsRNA entry is difficult due to the ubiquitous expression profile and compensatory dsRNA binding characteristics of SR-As; a SR-A deficient cell line has yet to be identified. The present study suggests the Chinook salmon embryonic cell line, CHSE-214, as a model for studying extracellular dsRNA sensing in vitro . CHSE-214 is unable to bind and respond to extracellular dsRNA, can only respond to dsRNA when it is transfected into the cells, and is able to bind dsRNA when overexpressing human SR-AI. The applications for this model could include elucidating: dsRNA binding and entry mechanisms, including sequence and length effects, as well as SR-A and other putative surface dsRNA receptor ligand binding studies. [ABSTRACT FROM AUTHOR]

The mammalian interleukin 1 receptor-like 1 receptor (IL1RL1), commonly known as ST2, is thought to downregulate TLR signalling by sequestering the signalling adapter MYD88 (myeloid differentiation primary response protein 88). ST2 sequences are known in several fish species, but none of them have functionally been examined. We characterised ST2 from rainbow trout ( Oncorhynchus mykiss ) and the structure of its encoding gene. The primary sequence of ST2 is only weakly conserved from fish to human. However, the amino acid sequences forming the interfaces for ST2 and MYD88 interaction are well conserved throughout evolution. High similarity of the gene segmentation unambiguously proves the common ancestry of fish and mammalian ST2. Trout ST2 and trout MYD88 genes were constitutively expressed in embryonic, larval and adult trout. In vivo infection with Aeromonas salmonicida did not modulate the mRNA levels of both factors. Overexpressing trout ST2 in the mammalian HEK-293 reconstitution system of TLR2 signalling quenched the Escherichia coli –induced activation of NF-κB and SAA promoters in a dose-dependent fashion. The expression of GFP-tagged trout ST2 in human HEK-293 or trout CHSE-214 cells surprisingly revealed that (i) ST2 localised abundantly at the nuclear membrane rather than at the cell membrane and (ii) the coexpression of both ST2 and MYD88 allowed the translocation of trout MYD88 from cytoplasm to nucleus, as assessed using confocal microscopy and Western blotting. Hence, we validated that trout ST2 is a dampener of TLR signalling and interacts with MYD88. The spatial distribution of these factors raises questions about how this repressive mechanism functions. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening. [ABSTRACT FROM AUTHOR]

Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 ( SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells ( CHSE-214). Different stages of viral replication were observed under electron microscopy. Virus-like particles were observed inside membrane-bound vesicles as early as 1 h following contact of the virus with the cells. Membrane-dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome-like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive-sense single-stranded RNA (+ss RNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by 'fuzzy'-coated membranes was greater in virus-infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post-infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Antimicrobial peptides, small cysteine-rich molecules, play vital roles in host defense mechanisms against pathogen infection. Recently, tilapia hepcidin (TH)1–5, was characterized, and its antimicrobial functions against several pathogens were reported. Herein, we investigated the antiviral functions of TH1-5 against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells. The presence of TH1-5 enhanced the survival of CHSE-214 cells infected with IPNV. Additionally, the number of plaques formed by the cytopathic effect of IPNV in CHSE-214 cells decreased when IPNV was preincubated with TH1-5. This observation demonstrates the antiviral function of TH1-5. Real-time PCR studies showed the modulation of interleukin, annexin, and other viral-responsive gene expressions by TH1-5. When TH1-5 and IPNV were used to co-treat CHSE-214 cells, then cells were re-challenged with IPNV at 24h, the cells did not survive the IPNV infection. This shows that in the absence of TH1-5, viral re-challenge killed CHSE-214 cells. In conclusion TH1-5 protected CHSE-214 cells against IPNV by direct antimicrobial and immunomodulatory functions. [ABSTRACT FROM AUTHOR]

Abstract: Cathelicidins are antimicrobial peptides indicated as important in the control of the natural microflora as well as in the fight against bacterial invasion in mammals. Little is known about cathelicidins in fish and here the Chinook salmon (Oncorhynchus tshawytscha) embryo cell line (CHSE-214) was used as a model system to study the expression of cathelicidins due to fish pathogenic bacteria. The cDNA of cathelicidin from CHSE-214 cells (csCath) was cloned and shown to be closely related to gene 2 of both rainbow trout and Atlantic salmon. The deducted amino acid sequence showed highest sequence identity to rtCath2 with 95% and 72% for the cathelin and the antibacterial part, respectively. Cathelicidin gene expression was studied and various Gram positive and Gram negative bacteria caused the upregulation of the gene (csCath). Bacterial DNA and protein were shown important for the induction of cathelicidin expression in these cells. LPS (Escherichia coli) also causes the upregulation of cathelicidins, but digestion of the LPS with DNase I before incubation of the cells, totally abolished the upregulation of cathelicidin and suggests DNA contamination in the LPS to be the trigger for this effect. These results could explain the limited responsiveness of fish cells towards pure LPS and confirm previous suggestions that fish cells are less sensitive to LPS than mammalian cells. [Copyright &y& Elsevier]

Abstract: Epithelial (CHSE-214), fibroblast (RTG-2) and macrophage (RTS11) cell lines from Chinook salmon and rainbow trout were tested for their sensitivity to gliotoxin, a fungal metabolite. Gliotoxin treatment for 6 or 24 h caused cell viability to decrease in a dose-dependent manner, with effective concentrations (EC50s) being similar for the three cell lines but varying with exposure time. Under some exposure conditions, hallmarks of apoptosis were detected. Apoptosis was evaluated by the appearance of fragmented nuclei upon H33258 staining and of genomic DNA laddering into 180 bp oligomers. Gliotoxin induced cell detachment in RTG-2 and CHSE-214 cultures, under some conditions. These were the only cultures of these two cell lines in which apoptosis was detected, and apoptotic cells appeared more frequent in the detached population. At the highest concentration, 15 μM, the cells died by an alternative mode, likely necrosis. By contrast, in RTS11 cultures cell detachment was not observed, and apoptosis occurred over a wider concentration range, even 15 μM, reaching levels of over 90%. The preferential death by necrosis for epithelial cells (CHSE-214) and by apoptosis for macrophages (RTS11) could be a beneficial host response to gliotoxin-producing fungi, leading respectively to the development and then resolution of inflammation. [Copyright &y& Elsevier]

Interest in and concern for the quality of the environment has prompted a great deal of research into methods of measuring and assessing changes in it. One problem of major interest is that of increasing amounts of mutagenic/carcinogenic chemicals generated and released into marine and freshwater ecosystems. Numerous techniques involving whole animals and cell culture for these genotoxic changes have been devised to assay specific chemicals. Little has been done to determine the effects of potential genotoxicants on aquatic organisms. The purpose of this study was to investigate if 2,4-Dichloroaniline (2,4-DCA) (CASRN: 554-00-7), induced delayed cell death (DCD) or delayed reproductive cell death a.k.a. as lethal mutations in a teleost cell line, CHSE-214. Delayed expression of cell death in the progeny of cells, which survived a toxic insult, was first shown for ionizing radiation and is one of the signs of induced genomic instability. The survival of cells initially treated with 2,4-DCA and the survival of their progeny were determined. When cells are exposed to a toxic insult, the component cells of a normal appearing survivor colony or clone were commonly thought to have proliferative capacity equivalent to that of the untreated cells. In this study, however, it was found that CHSE-214 cells surviving 2,4-DCA exposure carried heritable lethal defects, which came to light only after numerous apparently successful divisions, in the form of plating efficiencies, which were reduced below those of the untreated, control cells. DCD expression did not appear to be dose-dependent with poor cell survival occurring at the lower end of 2,4-DCA exposure and remained constant until recovering to something like 60% of the controls. A study of the CHSE-214 kinetics post-exposure showed that the apparent reduced growth rate of the cells was due to reduced numbers of reproductively viable cells in the population. Results showed that the expression of DCD occurred persistently post-treatment and was relatively independent of dose once a threshold level of 40 μM (Fig. 1) had been reached. [Copyright &y& Elsevier]

In fish, the synthesis of metallothionein (MT) is increased by a number of heavy metals. The rainbow trout MT-A gene promoter region contains six known metal responsive elements (MREs), that mediate promoter activation by metals. In the present study, two fish cell lines differing in their ability to produce MT, RTG-2 (produce MT protein) and CHSE-214 (produce no detectable MT protein), were used to help elucidate the roles of Zn, Ag and MT in the activation of the MT promoter. The hypothesis tested was that Ag activates the MT-A promoter indirectly by displacing Zn from pre-existing Zn-MT and that this liberated Zn subsequently induces MT synthesis. Both cell lines were transfected with a luciferase reporter gene construct containing the rainbow trout MT-A promoter, exposed to various concentrations of Zn or Ag, and assayed for luciferase activity. CHSE-214 cells showed five times greater production of luciferase than RTG-2 cells when exposed to identical concentrations of Ag. Thus, Ag can likely induce MT transcription without displacing Zn from pre-existing Zn-MT. Furthermore, Ag activated the MT promoter at concentrations 100-fold lower than those required for Zn to initiate transcription, suggesting that zinc displaced from other sites by such low concentrations of Ag would not be sufficient to initiate MT transcription. This interpretation was further supported by radiotracer studies indicating that Ag did not cause a redistribution of 65Zn within either of the two cell types. These combined results indicate that Ag may be a direct inducer of MT. [Copyright &y& Elsevier]

The use of a DNA-binding dye, bisbenzimidazole (H33258), and a microplate fluorometer, CytoFluor 2350, was optimized to measure cell number in Chinook salmon embryo cell cultures (CHSE-214). The uniformity of cell homogenates, which were prepared prior to staining, was evaluated by area scan, which consists of multiple measurements over the total well area. Disruption in 0.01% SDS produced a relatively uniform homogenate and a low background. Homogenates were stained with H33258 at 1 to 10 µg/ml. Linear relationships between cell numbers and fluorescence units were established in 96 well plates, which were read only by standard scan, and in 6, 12, 24 and 48 well plates, which were read by area and standard scan. Area scan provided better relationships. These methods were used to show that in L-15 medium CHSE-214 were able to attach, retain viability, and proliferate with very little exogenous calcium. [ABSTRACT FROM AUTHOR]

The interleukin-1-receptor-associated kinase 3 (IRAK3) is known in mammals as a negative feedback regulator of NF-κB-mediated innate-immune mechanisms. Our RNA-seq experiments revealed a prototypic 1920-nt sequence encoding irak3 from rainbow trout ( Oncorhynchus mykiss ), as well as 20 variants that vary in length and nucleotide composition. Based on the DNA-sequence information from two closely related irak3 genes from rainbow trout and an irak3- sequence fragment from Atlantic salmon retrieved from public databases, we elucidated the underlying genetic causes for this striking irak3 diversity. Infecting rainbow trout with a lethal dose of Aeromonas salmonicida enhanced the expression of all variants in the liver, head kidney, and peripheral blood leucocytes. We analyzed the functional impact of the full-length factor and selected structural variants by overexpressing them in mammalian HEK-293 cells. The full-length factor enhanced the basal activity of NF-κB, but did not dampen the TLR2-signaling-induced levels of NF-κB activation. Increasing the basal NF-κB-activity through Irak3 apparently does not involve its C-terminal domain. However, more severely truncated factors had only a minor impact on the activity of NF-κB. The TLR2-mediated stimulation did not alter the spatial distribution of Irak3 inside the cells. In salmonid CHSE-214 cells, we observed that the Irak3-splice variant that prominently expresses the C-terminal domain significantly quenched the stimulation-dependent production of interleukin-1β and interleukin-8, but not the production of other immune regulators. We conclude that the different gene and splice variants of Irak3 from trout play distinct roles in the activation of immune-regulatory mechanisms., (Copyright © 2019 Rebl, Rebl, Verleih, Haupt, Köbis, Goldammer and Seyfert.)