Publications by authors named "Bertrand Collet"

68 Publications

Antiviral Actions of 25-Hydroxycholesterol in Fish Vary With the Virus-Host Combination.

Front Immunol 2021 24;12:581786. Epub 2021 Feb 24.

Fish Disease Research Unit, Institute for Parasitology, University of Veterinary Medicine Hannover, Hannover, Germany.

Cholesterol is essential for building and maintaining cell membranes and is critical for several steps in the replication cycle of viruses, especially for enveloped viruses. In mammalian cells virus infections lead to the accumulation of the oxysterol 25-hydroxycholesterol (25HC), an antiviral factor, which is produced from cholesterol by the cholesterol 25 hydroxylase (CH25H). Antiviral responses based on CH25H are not well studied in fish. Therefore, in the present study putative genes encoding for CH25H were identified and amplified in common carp and rainbow trout cells and an HPLC-MS method was applied for determination of oxysterol concentrations in these cells under virus infection. Our results give some evidence that the activation of CH25H could be a part of the antiviral response against a broad spectrum of viruses infecting fish, in both common carp and rainbow trout cells . Quantification of oxysterols showed that fibroblastic cells are capable of producing 25HC and its metabolite 7α,25diHC. The oxysterol 25HC showed an antiviral activity by blocking the entry of (CyHV-3) into KFC cells, but not (SVCV) or common carp paramyxovirus (Para) in the same cells, or (VHSV) and (IPNV) into RTG-2 cells. Despite the fact that the CH25H based antiviral response coincides with type I IFN responses, the stimulation of salmonid cells with recombinant type I IFN proteins from rainbow trout could not induce gene expression. This provided further evidence, that the CH25H-response is not type I IFN dependent. Interestingly, the susceptibility of CyHV-3 to 25HC is counteracted by a downregulation of the expression of the gene in carp fibroblasts during CyHV-3 infection. This shows a unique interplay between oxysterol based immune responses and immunomodulatory abilities of certain viruses.
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http://dx.doi.org/10.3389/fimmu.2021.581786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943847PMC
February 2021

Non-Lethal Sequential Individual Monitoring of Viremia in Relation to DNA Vaccination in Fish-Example Using a Salmon Alphavirus DNA Vaccine in Atlantic Salmon .

Vaccines (Basel) 2021 Feb 17;9(2). Epub 2021 Feb 17.

VIM, UVSQ, INRAE, Université Paris-Saclay, Domaine de Vilvert, 78352 Jouy-En-Josas, France.

Traditionally, commercial testing for vaccine efficacy has relied on the mass infection of vaccinated and unvaccinated animals and the comparison of mortality prevalence and incidence. For some infection models where disease does not cause mortality this approach to testing vaccine efficacy is not useful. Additionally, in fish experimental studies on vaccine efficacy and immune response the norm is that several individuals are lethally sampled at sequential timepoints, and results are extrapolated to represent the kinetics of immune and disease parameters of an individual fish over the entire experimental infection period. In the present study we developed a new approach to vaccine testing for viremic viruses in fish by following the same individuals over the course of a DNA vaccination and experimental infection through repeated blood collection and analyses. Injectable DNA vaccines are particularly efficient against viral disease in fish. To date, two DNA vaccines have been authorised for use in fish farming, one in Canada against Infectious Haemorrhagic Necrotic virus and more recently one in Europe against Salmon Pancreatic Disease virus (SPDv) subtype 3. In the current study we engineered and used an experimental DNA vaccine against SPDv subtype 1. We measured viremia using a reporter cell line system and demonstrated that the viremia phase was completely extinguished following DNA vaccination. Differences in viremia infection kinetics between fish in the placebo group could be related to subsequent antibody levels in the individual fish, with higher antibody levels at terminal sampling in fish showing earlier viremia peaks. The results indicate that sequential non-lethal sampling can highlight associations between infection traits and immune responses measured at asynchronous timepoints and, can provide biological explanations for variation in data. Similar to results observed for the SPDv subtype 3 DNA vaccine, the SPDv subtype 1 DNA vaccine also induced an interferon type 1 response after vaccination and provided high protection against SPDv under laboratory conditions when fish were challenged at 7 weeks post-vaccination.
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http://dx.doi.org/10.3390/vaccines9020163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922653PMC
February 2021

Evolution of the IRF Family in Salmonids.

Genes (Basel) 2021 Feb 8;12(2). Epub 2021 Feb 8.

VIM, UVSQ, INRAE, Université Paris-Saclay, 78350 Jouy-En-Josas, France.

Interferon regulatory factors (IRFs) as a family, are major regulators of the innate antiviral response in vertebrates principally involved in regulating the expression of interferons (IFNs) and interferon-stimulated genes (ISGs). To date, nine IRFs have been identified in mammals with a 10th member also found in several avian and fish species. Through genome mining and phylogenetic analysis, we identified and characterised 23 genes in 6 salmonid species. This larger repertoire of IRF in salmonids results from two additional whole-genome duplications which occurred in early teleosts and salmonids, respectively. Synteny analysis was then used to identify and confirm which paralogues belonged to each subgroup and a new nomenclature was assigned to the salmonid IRFs. Furthermore, we present a full set of Real-Time PCR primers for all rainbow trout IRFs, confirmed by sequencing to ensure paralogue specificity. RT PCR was then used to examine the response of all trout genes in vivo, following and poly I:C stimulation, indicating potential functional divergence between paralogues. Overall, this study presents a comprehensive overview of the IRF family in salmonids and highlights some novel roles for the salmonid-specific IRFs in immunity.
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http://dx.doi.org/10.3390/genes12020238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915476PMC
February 2021

The repertoire of vertebrate STAT transcription factors: Origin and variations in fish.

Dev Comp Immunol 2021 Mar 1;116:103929. Epub 2020 Dec 1.

Université Paris-Saclay, INRAE, UVSQ, VIM, 78350, Jouy-en-Josas, France. Electronic address:

The stat gene family diversified during early vertebrate evolution thanks to two rounds of whole genome duplication (WGD) to produce a typical repertoire composed of 6 STAT factors (named 1-6). In contrast, only one or two stat genes have been reported in C. elegans and in D. melanogaster. The main types of STAT found from bony fish to mammals are present in Agnathan genomes, but a typical STAT1-6 repertoire is only observed in jawed vertebrates. Comparative syntenies showed that STAT6 was the closest to the ancestor of the family. An extensive survey of stat genes across fish including polyploid species showed that whole genome duplications did not lead to a uniform expansion of stat genes. While 2 to 5 stat1 are present in salmonids, whose genome duplicated about 35My ago, only one copy of stat2 and stat6 is retained. In contrast, common carp, with a recent whole genome duplication (5-10My), possesses a doubled stat repertoire indicating that the elimination of stat2 and stat6 additional copies is not immediate. Altogether our data shed light on the multiplicity of evolutionary pathways followed by key components of the canonical cytokine receptor signalling pathway, and point to differential selective constraints exerted on these factors.
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http://dx.doi.org/10.1016/j.dci.2020.103929DOI Listing
March 2021

Combining Multiple Approaches and Models to Dissect the Genetic Architecture of Resistance to Infections in Fish.

Front Genet 2020 10;11:677. Epub 2020 Jul 10.

INRAE, UVSQ, VIM, Université Paris-Saclay, Jouy-en-Josas, France.

Infectious diseases represent a major threat for the sustainable development of fish farming. Efficient vaccines are not available against all diseases, and growing antibiotics resistance limits the use of antimicrobial drugs in aquaculture. It is therefore important to understand the basis of fish natural resistance to infections to help genetic selection and to develop new approaches against infectious diseases. However, the identification of the main mechanisms determining the resistance or susceptibility of a host to a pathogenic microbe is challenging, integrating the complexity of the variation of host genetics, the variability of pathogens, and their capacity of fast evolution and adaptation. Multiple approaches have been used for this purpose: (i) genetic approaches, QTL (quantitative trait loci) mapping or GWAS (genome-wide association study) analysis, to dissect the genetic architecture of disease resistance, and (ii) transcriptomics and functional assays to link the genetic constitution of a fish to the molecular mechanisms involved in its interactions with pathogens. To date, many studies in a wide range of fish species have investigated the genetic determinism of resistance to many diseases using QTL mapping or GWAS analyses. A few of these studies pointed mainly toward adaptive mechanisms of resistance/susceptibility to infections; others pointed toward innate or intrinsic mechanisms. However, in the majority of studies, underlying mechanisms remain unknown. By comparing gene expression profiles between resistant and susceptible genetic backgrounds, transcriptomics studies have contributed to build a framework of gene pathways determining fish responsiveness to a number of pathogens. Adding functional assays to expression and genetic approaches has led to a better understanding of resistance mechanisms in some cases. The development of knock-out approaches will complement these analyses and help to validate putative candidate genes critical for resistance to infections. In this review, we highlight fish isogenic lines as a unique biological material to unravel the complexity of host response to different pathogens. In the future, combining multiple approaches will lead to a better understanding of the dynamics of interaction between the pathogen and the host immune response, and contribute to the identification of potential targets of selection for improved resistance.
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http://dx.doi.org/10.3389/fgene.2020.00677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365936PMC
July 2020

Detection of specific Atlantic salmon antibodies against salmonid alphavirus using a bead-based immunoassay.

Fish Shellfish Immunol 2020 Nov 30;106:374-383. Epub 2020 Jul 30.

Faculty of Veterinary Medicine, Norwegian University of Life Sciences (NMBU), Oslo, Norway. Electronic address:

Salmonid alphavirus (SAV) is the etiological cause of pancreas disease (PD) in Atlantic salmon (Salmo salar). Several vaccines against SAV are in use, but PD still cause significant mortality and concern in European aquaculture, raising the need for optimal tools to monitor SAV immunity. To monitor and control the distribution of PD in Norway, all salmonid farms are regularly screened for SAV by RT-qPCR. While the direct detection of SAV is helpful in the early stages of infection, serological methods could bring additional information on acquired SAV immunity in the later stages. Traditionally, SAV antibodies are monitored in neutralization assays, but they are time-consuming and cumbersome, thus alternative assays are warranted. Enzyme-linked immunosorbent assays (ELISAs) have not yet been successfully used for anti-SAV antibody detection in aquaculture. We aimed to develop a bead-based immunoassay for SAV-specific antibodies. By using detergent-treated SAV particles as antigens, we detected SAV-specific antibodies in plasma collected from both a SAV challenge trial and a field outbreak of PD. Increased levels of SAV-specific antibodies were seen after most fish had become negative for viral RNA. The bead-based assay is time saving compared to virus neutralization assays, and suitable for non-lethal testing due to low sample size requirements. We conclude that the bead-based immunoassay for SAV antibody detection is a promising diagnostic tool to complement SAV screening in aquaculture.
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http://dx.doi.org/10.1016/j.fsi.2020.07.055DOI Listing
November 2020

Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system.

BMC Biotechnol 2020 06 23;20(1):35. Epub 2020 Jun 23.

The Roslin Institute, University of Edinburgh, Easter Bush campus, Midlothian, UK.

Background: Genome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques.

Results: In the current study, we developed an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214). As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated.

Conclusions: The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.
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http://dx.doi.org/10.1186/s12896-020-00626-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310381PMC
June 2020

Individual monitoring of immune response in Atlantic salmon Salmo salar following experimental infection with piscine myocarditis virus (PMCV), agent of cardiomyopathy syndrome (CMS).

Dev Comp Immunol 2019 10 31;99:103406. Epub 2019 May 31.

Aquaculture and Fish Health, Marine Scotland, Aberdeen, Scotland, UK; Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique (INRA), Université Paris-Saclay, Jouy-en-Josas, France. Electronic address:

Piscine myocarditis virus (PCMV) is a double-stranded RNA virus structurally similar to the Totiviridae family. PCMV is the causative agent of cardiomyopathy syndrome (CMS), a severe cardiac disease that affects farmed Atlantic salmon (Salmo salar). A recent study characterized the host immune response in infected salmon through a transcriptome immune profiling, which confirmed a high regulation of immune and anti-viral genes throughout infection with PCMV. Previously we developed a novel model based on repeated non-lethal blood sampling, enabling the individual monitoring of salmonids during an infection. In the present work, we used this model to describe the host immune response in the blood cells of Atlantic salmon after intramuscular infection with PCMV-containing tissue homogenate over a 77-day period. At the final stage heart samples were also collected to verify the PCMV load, the pathological impact of infection and to compare the transcript profiles to blood. The expression level of a range of key immune genes was determined in the blood and heart samples by real-time PCR. Results indicated selected immune genes (mx, cd8α and γip) were up-regulated in the heart tissue of infected animals at the terminal time point, in comparison to the non-infected fish. When analyzing the blood samples over the course of infection, a significant n up-regulation of mx gene was also observed. The time and number of peaks in the kinetics of expression was different between individuals. The PCMV load and CMS pathology was verified by real-time PCR and histopathology, respectively. No pathogen and no pathology could be detected during the course of the experiment except at the terminal stage (viral load by qPCR and pathology by histology). This study emphasizes the value of non-lethal monitoring for evaluating the health status of fish at early stages of infection and in the absence of clinical signs.
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http://dx.doi.org/10.1016/j.dci.2019.103406DOI Listing
October 2019

Viral Resistance and IFN Signaling in STAT2 Knockout Fish Cells.

J Immunol 2019 07 29;203(2):465-475. Epub 2019 May 29.

Marine Scotland, Marine Laboratory, AB11 9DB Aberdeen, United Kingdom; and

IFN belong to a group of cytokines specialized in the immunity to viruses. Upon viral infection, type I IFN is produced and alters the transcriptome of responding cells through induction of a set of IFN stimulated genes (ISGs) with regulatory or antiviral function, resulting in a cellular antiviral state. Fish genomes have both type I IFN and type II IFN (IFN-γ), but no type III (λ) IFN has been identified. Their receptors are not simple counterparts of the mammalian type I/II IFN receptors, because alternative chains are used in type I IFN receptors. The mechanisms of the downstream signaling remain partly undefined. In mammals, members of the signal transducer and activator of family of transcription factors are responsible for the transmission of the signal from cytokine receptors, and STAT2 is required for type I but not type II IFN signaling. In fish, its role in IFN signaling in fish remains unclear. We isolated a Chinook salmon () cell line, GS2, with a stat2 gene knocked out by CRISPR/Cas9 genome editing. In this cell line, the induction of ISGs by stimulation with a recombinant type I IFN is completely obliterated as evidenced by comparative RNA-seq analysis of the transcriptome of GS2 and its parental counterpart, EC. Despite a complete absence of ISGs induction, the GS2 cell line has a remarkable ability to resist to viral infections. Therefore, other STAT2-independent pathways may be induced by the viral infection, illustrating the robustness and redundancy of the innate antiviral defenses in fish.
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http://dx.doi.org/10.4049/jimmunol.1801376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612602PMC
July 2019

IFN Signaling in Inflammation and Viral Infections: New Insights from Fish Models.

Viruses 2019 03 26;11(3). Epub 2019 Mar 26.

INRA, Virologie et Immunologie Moléculaires, Université Paris-Saclay, 78352 Jouy-en-Josas, France.

The overarching structure of the type I interferon (IFN) system is conserved across vertebrates. However, the variable numbers of whole genome duplication events during fish evolution offer opportunities for the expansion, diversification, and new functionalization of the genes that are involved in antiviral immunity. In this review, we examine how fish models provide new insights about the implication of virus-driven inflammation in immunity and hematopoiesis. Mechanisms that have been discovered in fish, such as the strong adjuvant effect of type I IFN that is used with DNA vaccination, constitute good models to understand how virus-induced inflammatory mechanisms can interfere with adaptive responses. We also comment on new discoveries regarding the role of pathogen-induced inflammation in the development and guidance of hematopoietic stem cells in zebrafish. These findings raise issues about the potential interferences of viral infections with the establishment of the immune system. Finally, the recent development of genome editing provides new opportunities to dissect the roles of the key players involved in the antiviral response in fish, hence enhancing the power of comparative approaches.
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http://dx.doi.org/10.3390/v11030302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466407PMC
March 2019

Effects of repeated anaesthesia on gill and general health of Atlantic salmon, Salmo salar.

J Fish Biol 2018 Dec 28;93(6):1069-1081. Epub 2018 Nov 28.

Marine Laboratory, Marine Scotland Science, Aberdeen, UK.

Fish are the second most widely utilized vertebrate group used for scientific procedures in the United Kingdom, but the development and application of 3Rs (the principles of replacement, reduction, and refinement) in aquaculture disease research lags behind methodologies in place for mammalian studies. With a need for individual monitoring and non-lethal sampling, the effect of repeat anaesthesia on experimental fish needs to be better understood. This study analyses the effect of repeat anaesthesia with MS-222, metomidate and AQUI-S upon the gill and general health of post-smolt Atlantic salmon Salmo salar. A single, lethal dose of anaesthetic was compared with seven anaesthetizing time points over 28 days, terminating in a lethal dose. No anaesthetic showed significant differences in accumulation in the muscle tissue, or changes in plasma glucose after repeated or single dosing. Fish repeatedly anaesthetized with MS-222 or AQUI-S exhibited upregulation of osmoregulatory genes in the gill and AQUI-S-treated individuals showed, histologically, epithelial lifting from the lamellae capillary irrespective of whether they had a single or repeated dose history. No significant changes were seen in inflammatory or stress genes in the head kidney of fish repeatedly anaesthetized with AQUI-S or metomidate, however MS-222 treatment resulted in upregulation of tnfα3. Repeated anaesthesia with MS-222 and metomidate gave a significant decrease and increase in peripheral blood neutrophils, respectively. This study concludes that no increase in cumulative stress or inflammation is induced by the repeated anaesthetization of S. salar with any of the tested anaesthetics, however gill osmotic regulation and blood parameters may be affected.
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http://dx.doi.org/10.1111/jfb.13803DOI Listing
December 2018

DNA vaccination for finfish aquaculture.

Fish Shellfish Immunol 2019 Feb 11;85:106-125. Epub 2018 Jul 11.

Marine Scotland, Aberdeen, United Kingdom; Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique (INRA), Université Paris-Saclay, Jouy-en-Josas, France. Electronic address:

In fish, DNA vaccines have been shown to give very high protection in experimental facilities against a number of viral diseases, particularly diseases caused by rhabdoviruses. However, their efficacy in generating protection against other families of fish viral pathogens is less clear. One DNA vaccine is currently in use commercially in fish farms in Canada and the commercialisation of another was authorised in Europe in 2017. The mechanism of action of DNA vaccines, including the role of the innate immune responses induced shortly after DNA vaccination in the activation of the adaptive immunity providing longer term specific protection, is still not fully understood. In Europe the procedure for the commercialisation of a veterinary DNA vaccine requires the resolution of certain concerns particularly about safety for the host vaccinated fish, the consumer and the environment. Relating to consumer acceptance and particularly environmental safety, a key question is whether a DNA vaccinated fish is considered a Genetically Modified Organism (GMO). In the present opinion paper these key aspects relating to the mechanisms of action, and to the development and the use of DNA vaccines in farmed fish are reviewed and discussed.
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http://dx.doi.org/10.1016/j.fsi.2018.07.012DOI Listing
February 2019

Time-course study of the protection induced by an interferon-inducible DNA vaccine against viral haemorrhagic septicaemia in rainbow trout.

Fish Shellfish Immunol 2019 Feb 30;85:99-105. Epub 2018 Jun 30.

Technical University of Denmark, Denmark. Electronic address:

The highly effective DNA vaccines against diseases caused by fish rhabdoviruses in farmed fish consist of a DNA plasmid vector encoding the viral glycoprotein under the control of a constitutive cytomegalovirus promoter (CMV). Among others, attempts to improve efficacy and safety of these DNA vaccines have focused on regulatory elements of plasmid vectors, which play a major role in controlling expression levels of vaccine antigens. Depending on the context, use of a fish-derived promoter with minimal activity in mammalian cells could be preferable. Another aspect related to the CMV promoter is that constitutive expression of the vaccine antigen may lead to rapid elimination of antigen expressing cells in the fish and thereby potentially reduce the long-term effects of the vaccine. In this study, we compared DNA vaccines with the interferon-inducible Mx promoter from rainbow trout and the CMV promoter, respectively. Plasmid constructs encoding the enhanced green fluorescent protein (EGFP) were used for the in vitro analysis, whereas DNA vaccines encoding the glycoprotein (G) of the viral haemorrhagic septicaemia virus (VHSV) were applied for the in vivo examination. The in vitro analysis showed that while the DNA vaccine with the CMV promoter constitutively drove the expression of EGFP in both fish and human cell lines, the DNA vaccine with the Mx promoter inducibly enhanced the expression of EGFP in the fish cell line. To address the impact on protection, a time-course model was followed as suggested by Kurath et al. (2006), where vaccinated fish were challenged with VHSV at 2, 8 and 78 weeks post-vaccination (wpv). The DNA vaccine with the CMV promoter protected at all times, while vaccination with the DNA vaccine containing the Mx promoter only protected the fish at 8 wpv. However, following induction with Poly (I:C) one week before the challenge, high protection was also evident at 2 wpv. In conclusion, the results revealed a more fish host dependent activity of the trout Mx promoter compared to the traditionally used cross species-active CMV promoter, but improvements will be needed for its application in DNA vaccines to ensure long term protection.
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http://dx.doi.org/10.1016/j.fsi.2018.06.056DOI Listing
February 2019

The potential benefits of repeated measure experiments for fish disease-challenge host-pathogen investigations.

Fish Shellfish Immunol 2019 Feb 2;85:126-131. Epub 2018 Feb 2.

Marine Scotland Science, Aberdeen, AB11 9DB, UK; Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique (INRA), Université Paris-Saclay, Jouy-en-Josas, France. Electronic address:

The utility of molecular response data arising from in-vivo single and repeated measure fish disease-challenge experiments is compared. An in-silico 'experiment' involving the generation of two imaginary immune-molecule quantity response profiles over time for individual animals was carried out. Daily 'observed' molecule quantities were drawn from the 'known' individual response profiles to mimic the results of single and repeated measurement. The results indicate that repeated measure experiments are required to infer individual level response profiles, and that these experiments also provide more accurate summary statistics and data more suited to inferring the dependent ordering of the molecular response. Additionally repeated measure experiments utilise fewer animals than single measure experiments. These results are described alongside a discussion of experimental methodological issues pertinent to the adoption of aquatic animal repeated measure experimental designs. We conclude that investigators need to take particular care when making inferences from single measure experiments and that serious consideration should be given to using repeated measure experiments for in-vivo fish disease-challenge investigations.
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http://dx.doi.org/10.1016/j.fsi.2018.01.033DOI Listing
February 2019

Use of Salmon Cardiac Primary Cultures (SCPCs) of different genotypes for comparative kinetics of mx expression, viral load and ultrastructure pathology, after infection with Salmon Pancreas Disease Virus (SPDV).

Fish Shellfish Immunol 2018 Jan 2;72:181-186. Epub 2017 Nov 2.

Royal Dick School of Veterinary Sciences, University of Edinburgh, UK.

In vitro fish based models have been extensively applied in human biomedical research but, paradoxically, less frequently in the research of fish health issues. Farmed Atlantic salmon can suffer from several viral conditions affecting the heart. Therefore, species-specific, cardiac in vitro models may represent a useful tool to help further understanding and management of these diseases. The mechanisms underlying genotype based resistance are complex and usually rely on a combined effect of elements from both the innate and adaptive immune response, which are further complicated by external environmental factors. Here we propose that Salmon Cardiac Primary Cultures (SCPCs) are a useful tool to investigate these mechanisms as the basis for genotypic differences between Atlantic salmon families in susceptibility to cardiotropic viral disease. Using SCPCs produced from two different commercially available Atlantic salmon embryonated ova (Atlantic Ova IPN sensitive" (S) and "Atlantic QTL-innOva IPN/PD" (R)), the influence of host genotype on the viral load and mx expression following Salmon Pancreas Disease Virus infection was assessed over a 15 day period. Both R and S SCPCs groups were successfully infected. A measurable difference between groups of viral nsP1 and host antiviral mx gene expression was observed (i.e. a later, but larger onset of mx expression in the R group). Mx expression peaks were followed by a decrease in viral nsP1 in both groups. Additionally, ultrastructural examination of infected SCPCs allowed the description of degenerative changes at the individual cell level. The SCPC model presents some advantages, over current fish cell culture monolayers and in vivo material, such as the presence of different cell components normally present in the target organ, as well as the removal of a layer of functional complexity (acquired immunity), making it possible to focus on tissue specific, early innate immune mechanisms. These preliminary results highlight the importance of considering genetic origin when selecting the fish source for the production of SCPCs, as well as their usefulness as screening tools for assessment of genotypic differences in disease resistance.
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http://dx.doi.org/10.1016/j.fsi.2017.10.059DOI Listing
January 2018

Atlantic salmon cardiac primary cultures: An in vitro model to study viral host pathogen interactions and pathogenesis.

PLoS One 2017 20;12(7):e0181058. Epub 2017 Jul 20.

Royal Dick School of Veterinary Sciences - University of Edinburgh, Edinburgh, United Kingdom.

Development of Salmon Cardiac Primary Cultures (SCPCs) from Atlantic salmon pre-hatch embryos and their application as in vitro model for cardiotropic viral infection research are described. Producing SCPCs requires plating of trypsin dissociated embryos with subsequent targeted harvest from 24h up to 3 weeks, of relevant tissues after visual identification. SCPCs are then transferred individually to chambered wells for culture in isolation, with incubation at 15-22°. SCPCs production efficiency was not influenced by embryo's origin (0.75/ farmed or wild embryo), but mildly influenced by embryonic developmental stage (0.3 decline between 380 and 445 accumulated thermal units), and strongly influenced by time of harvest post-plating (0.6 decline if harvested after 72 hours). Beating rate was not significantly influenced by temperature (15-22°) or age (2-4 weeks), but was significantly lower on SCPCs originated from farmed embryos with a disease resistant genotype (F = 5.3, p<0.05). Two distinct morphologies suggestive of an ex vivo embryonic heart and a de novo formation were observed sub-grossly, histologically, ultra-structurally and with confocal microscopy. Both types contained cells consistent with cardiomyocytes, endothelium, and fibroblasts. Ageing of SCPCs in culture was observed with increased auto fluorescence in live imaging, and as myelin figures and cellular degeneration ultra-structurally. The SCPCs model was challenged with cardiotropic viruses and both the viral load and the mx gene expression were measurable along time by qPCR. In summary, SCPCs represent a step forward in salmon cardiac disease research as an in vitro model that partially incorporates the functional complexity of the fish heart.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181058PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519056PMC
September 2017

Engineered cell lines for fish health research.

Dev Comp Immunol 2018 03 17;80:34-40. Epub 2017 Jan 17.

Marine Scotland, Marine Laboratory, Aberdeen, UK.

As fish farming continues to increase worldwide, the related research areas of fish disease and immunology are also expanding, aided by the revolution in access to genomic information and molecular technology. The genomes of most fish species of economic importance are now available and annotation based on sequence homology with characterised genomes is underway. However, while useful, functional homology is more difficult to determine, there being a lack of widely distributed and well characterised reagents such as monoclonal antibodies, traditionally used in mammalian studies, to help with confirming functions and cellular interactions of fish molecules. In this context, fish cell lines and the possibility of their genetic engineering offer good prospects for studying functional genomics with respect to fish diseases. In this review, we will give an overview of available permanently genetically engineered fish cell lines, as cell-based reporter systems or platforms for expression of endogenous immune or pathogen genes, to investigate interactions and function. The advantages of such systems and the technical challenge for their development will be discussed.
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http://dx.doi.org/10.1016/j.dci.2017.01.013DOI Listing
March 2018

Production of infectious salmon anaemia virus (ISAV) ribonucleoprotein complexes using a mammalian cell based minigenome system.

J Virol Methods 2017 01 10;239:75-82. Epub 2016 Nov 10.

Marine Scotland Science, Marine Laboratory, AB11 9DB, Aberdeen, United Kingdom. Electronic address:

Developments in recombinant virus techniques have been crucial to understand the mechanisms of virulence acquisition and study the replication of many different negatively stranded RNA viruses. However, such technology has been lacking for infectious salmon anaemia virus (ISAV) until recently. This was due in part to the lack of a Polymerase I promoter in Atlantic salmon to drive the production of recombinant vRNA. Therefore, the present study investigated a different alternative to produce ISAV recombinant vRNA, based on Mouse Pol I promoter/terminator sequences and expression in baby hamster kidney (BHK-21) cells. As a first step, a pathogenic ISAV was demonstrated to replicate and produce viable virions in BHK-21 cells. This indicated that the virus could use the mammalian cellular and nuclear machinery to produce vRNA segments and viral proteins, albeit in a limited capacity. Co-transfection of vRNA expressing plasmids with cytomegalovirus (CMV) promoter constructs coding for the three viral polymerase and nucleoprotein led to the generation of functional ribonucleoproteins (RNPs) which expressed either, green fluorescence protein (GFP) or firefly luciferase (FF). Further experiments demonstrated that a 21h incubation at 37°C was optimal for RNPs production. Inhibition by ribavirin confirmed that FF expression was linked to specific RNPs polymerase transcription. The present minigenome system provides a novel and alternative approach to investigate various aspects of ISAV replication and potentially those of other negatively stranded RNA viruses. Expression of RNPs in mammalian cells could also provide a method for the rapid screening of anti-viral compounds targeting ISAV replication.
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http://dx.doi.org/10.1016/j.jviromet.2016.10.017DOI Listing
January 2017

Corrigendum to "In vivo virulence of viral haemorrhagic septicaemia virus (VHSV) in rainbow trout Oncorhynchus mykiss correlates inversely with in vitro Mx gene expression" [Vet. Microbiol. 187 (2016) 31-40].

Vet Microbiol 2016 11 30;195:58-59. Epub 2016 Sep 30.

Aquatic Animal Disease, Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, The Nothe Weymouth, Dorset DT4 8UB, United Kingdom.

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http://dx.doi.org/10.1016/j.vetmic.2016.09.002DOI Listing
November 2016

Individual monitoring of immune responses in rainbow trout after cohabitation and intraperitoneal injection challenge with Yersinia ruckeri.

Fish Shellfish Immunol 2016 Aug 28;55:469-78. Epub 2016 May 28.

Marine Scotland Science, Marine Laboratory, 375 Victoria Road, Aberdeen AB11 9DB, UK. Electronic address:

Yersinia ruckeri, the causative agent of enteric red mouth disease (ERM), is a widely studied pathogen in disease models using rainbow trout. This infection model, mostly based on intraperitoneally injection or bath immersion challenges, has an impact on both components (innate and adaptive) of the fish immune system. Although there has been much attention in studying its host-pathogen interactions, there is still a lack of knowledge regarding the impact of a cohabitation challenge. To tackle this we used a newly established non-lethal sampling method (by withdrawing a small amount of blood) in rainbow trout which allowed the individual immune monitoring before (non-infected) and after infection with Yersinia ruckeri either by intraperitoneal (i.p.) injection or by cohabitation (cohab). A range of key immune genes were monitored during the infection by real-time PCR, and results were compared between the two infection routes. Results indicated that inflammatory (IL-1β1 and IL-8) cytokines and certain antimicrobial peptides (cathelicidins) revealed a different pattern of expression between the two infected groups (i.p. vs cohab), in comparison to adaptive immune cytokines (IL-22, IFN-γ and IL-4/13A) and β-defensins. This suggests a different involvement of distinct immune markers according to the infection model, and the importance of using a cohabitation challenge as a more natural disease model that likely simulates what would occur in the environment.
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http://dx.doi.org/10.1016/j.fsi.2016.05.041DOI Listing
August 2016

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.

Mar Biotechnol (NY) 2016 Aug 28;18(4):449-52. Epub 2016 May 28.

Marine Laboratory, Marine Scotland, 375 Victoria road, Aberdeen, AB11 9DB, UK.

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.
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http://dx.doi.org/10.1007/s10126-016-9708-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007268PMC
August 2016

In vivo virulence of viral haemorrhagic septicaemia virus (VHSV) in rainbow trout Oncorhynchus mykiss correlates inversely with in vitro Mx gene expression.

Vet Microbiol 2016 May 22;187:31-40. Epub 2016 Feb 22.

Aquatic Animal Disease, Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, The Nothe Weymouth, Dorset DT4 8UB, United Kingdom.

The in vitro replication of viral haemorrhagic septicaemia virus (VHSV) isolates from each VHSV genotype and the associated cellular host Mx gene expression were analysed. All the isolates were able to infect RTG-2 cells and induce increased Mx gene expression (generic assay detecting isoforms 1 and 3 [Mx1/3]). A trout pathogenic, genotype Ia isolate (J167), showing high replication in RTG-2 cells (by infective titre and N gene expression) induced lower Mx1/3 gene expression than observed in VHSV isolates known to be non-pathogenic to rainbow trout: 96-43/8, 96-43/10 (Ib); 1p49, 1p53 (II); and MI03 (IVb). Paired co-inoculation assays were analysed using equal number of plaque forming units per ml (PFU) of J167 (Ia genotype) with other less pathogenic VHSV genotypes. In these co-inoculations, the Mx1/3 gene expression was significantly lower than for the non-pathogenic isolate alone. Of the three rainbow trout Mx isoforms, J167 did not induce Mx1 up-regulation in RTG-2 or RTgill-W1 cells. Co-inoculating isolates resulted in greater inhibition of Mx in both rainbow trout cell lines studied. Up-regulation of sea bream Mx in SAF-1 cells induced by 96-43/8 was also lower in co-inoculation assays with J167. The RTG-P1 cell line, expressing luciferase under the control of the interferon-induced Mx rainbow trout gene promoter, showed low luciferase activity when inoculated with pathogenic strains: J167, DK-5131 (Ic), NO-A-163/68 (Id), TR-206239-1, TR-22207111 (Ie), 99-292 (IVa), and CA-NB00-01 (IVc). Co-inoculation assays showed a J167-dose dependent inhibition of the luciferase activity. The data suggest that virulent VHSV isolates may interfere in the interferon pathways, potentially determining higher pathogenicity.
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http://dx.doi.org/10.1016/j.vetmic.2016.02.012DOI Listing
May 2016

Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease.

PLoS One 2015 30;10(10):e0142020. Epub 2015 Oct 30.

Aquaculture and Fish Health, Marine Scotland Science, Aberdeen, United Kingdom.

In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142020PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627773PMC
June 2016

The effects of feeding β-glucan to Pangasianodon hypophthalmus on immune gene expression and resistance to Edwardsiella ictaluri.

Fish Shellfish Immunol 2015 Nov 9;47(1):595-605. Epub 2015 Oct 9.

Institute of Aquaculture, University of Stirling, Stirling, Scotland, UK.

Pangasianodon hypophthalmus (striped catfish) is an important aquaculture species and intensification of farming has increased disease problems, particularly Edwardsiella ictaluri. The effects of feeding β-glucans on immune gene expression and resistance to E. ictaluri in P. hypophthalmus were explored. Fish were fed 0.1% fungal-derived β-glucan or 0.1% commercial yeast-derived β-glucan or a basal control diet without glucan. After 14 days of feeding, the mRNA expression of immune genes (transferrin, C-reactive protein, precerebellin-like protein, Complement C3 and factor B, 2a MHC class II and interleukin-1 beta) in liver, kidney and spleen were determined. Following this fish from each of the three diet treatment groups were infected with E. ictaluri and further gene expression measured 24 h post-infection (h.p.i.), while the remaining fish were monitored over 2 weeks for mortalities. Cumulative percentage mortality at 14 days post-infection (d.p.i.) was less in β-glucan fed fish compared to controls. There was no difference in gene expression between dietary groups after feeding for 14 days, but there was a clear difference between infected and uninfected fish at 24 h.p.i., and based on principal component analysis β-glucans stimulated the overall expression of immune genes in the liver, kidney and spleen at 24 h.p.i.
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http://dx.doi.org/10.1016/j.fsi.2015.09.042DOI Listing
November 2015

Individual Monitoring of Immune Response in Atlantic Salmon Salmo salar following Experimental Infection with Infectious Salmon Anaemia Virus (ISAV).

PLoS One 2015 23;10(9):e0137767. Epub 2015 Sep 23.

Aquaculture and Fish Health, Marine Scotland, Aberdeen, Scotland, United Kingdom.

Monitoring the immune response in fish over the progression of a disease is traditionally carried out by experimental infection whereby animals are killed at regular intervals and samples taken. We describe here a novel approach to infectiology for salmonid fish where blood samples are collected repeatedly in a small group of PIT-tagged animals. This approach contributes to the reduction of animals used in research and to improved data quality. Two groups of 12 PIT-tagged Atlantic salmon (Salmo salar) were i.p infected with Infectious Salmon Anaemia Virus (ISAV) or culture medium and placed in 1 m3 tanks. Blood samples were collected at 0, 4, 8, 12, 16, 21 and 25 days post infection. The viral load, immune and stress response were determined in individual fish by real-time quantitative PCR (QPCR) on the blood cells, as well as the haematocrit used as an indicator of haemolysis, a clinical consequence of ISAV infection. "In-tank" anaesthesia was used in order to reduce the stress related to chase and netting prior to sampling. The data were analysed using a statistical approach which is novel with respect to its use in fish immunology. The repeated blood collection procedure did not induce stress response as measured by HSP70 and HSP90 gene expression in the un-infected animals. A strong increase in viraemia as well as a significant induction of Mx and γIP gene expression were observed in the infected group. Interleukin 10 was found induced at the later stage of the infection whereas no induction of CD8 or γ IFN could be detected. These results and the advantages of this approach are discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137767PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580571PMC
June 2016

Identification and expression modulation of a C-type lectin domain family 4 homologue that is highly expressed in monocytes/macrophages in rainbow trout (Oncorhynchus mykiss).

Dev Comp Immunol 2016 Jan 13;54(1):55-65. Epub 2015 Aug 13.

Scottish Fish Immunology Research Centre (SFIRC), School of Biological Sciences, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen, AB24 2TZ, Scotland, UK.

The C-type lectin domain containing (CLEC) receptors including CD209 are expressed in vivo by monocytes, monocyte-derived macrophages and dendritic cells and by in vitro generated monocyte-derived cells. This paper reports the cloning and sequencing of a lectin molecule, CLEC4T1, in rainbow trout that is a homologue of the CLEC4 family. The expression pattern of the CLEC4T1 was investigated in vivo after infection with a bacterial pathogen and in cultured macrophages after modulation with microbial mimics. Trout CLEC4T1 was highly expressed in spleen and head kidney following infection with Yersinia ruckeri. Expression could also be induced in macrophage cultures by LPS but not by Poly I:C, and suggests that the regulation of CLEC4T1 expression in trout varies according to the nature of the stimulant. A polyclonal CLEC4T1 antibody was generated and validated by Western blotting for use in evaluation of CLEC4T1(+) cells by flow cytometry analysis. Freshly isolated adherent trout head kidney cultures, potentially containing macrophages and dendritic cell precursors, showed an increase of CLEC4T1(+) cells (assessed by flow cytometry) upon stimulation with recombinant interleukin-4/13A. The results suggest that CLEC4T1 is a useful marker for further characterisation of monocyte derived antigen presenting cells in fish.
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http://dx.doi.org/10.1016/j.dci.2015.08.005DOI Listing
January 2016

The effects of feeding immunostimulant β-glucan on the immune response of Pangasianodon hypophthalmus.

Fish Shellfish Immunol 2015 Aug 25;45(2):357-66. Epub 2015 Apr 25.

Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, Scotland, UK; Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Near Edinburgh, Scotland, UK.

Immunostimulants are food additives used by the aquaculture industry to enhance the immune response of fish, and although β-glucans are now commonly used for this purpose in aquaculture, little is known about their effects on the immune response of Pangasianodon hypophthalmus. Thus, a variety of immune parameters (e.g. phagocytosis, respiratory burst, lysozyme, complement, peroxidase, total protein, total anti-protease, total IgM, natural antibody titres, and specific IgM titres) was examined in this species after feeding fish with a basal control diet or diets supplemented with 0.05, 0.1, or 0.2 g/kg fungal-derived β-glucan or 0.1% commercial yeast-derived β-glucan, as a positive control diet, for a period of four weeks. The effect of the glucans on disease resistance was then evaluated by experimentally infecting the fish with Edwardsiella ictaluri by immersion and mortalities monitored for 14 days. Samples were collected from fish for analysis at 0, 1, 3, 7, 14, 21 and 28 days post-feeding (dpf), and also at 14 days post infection (dpi). The lowest dose of fungal-derived β-glucan (0.05%) appeared insufficient to effectively stimulate the immune response of the fish, while those fed with the two highest levels of fungal-derived β-glucan had enhanced immune responses compared to the control group. Significantly elevated levels of respiratory burst activity on all days examined (P < 0.05) and lysozyme activity on 7 dpf were found in the group fed 0.2% fungal-derived β-glucan, while plasma anti-protease activity was significantly enhanced (P < 0.05) by 21 dpf, natural antibody titres by 3 dpf and complement activity by 7 dpf and also at 14 dpi in the group fed 0.1% fungal-derived β-glucan. No statistical differences was seen in the level of mortalities between the dietary groups, although the group fed with the control diet had the highest level of mortalities and the groups fed with commercial yeast-derived β-glucan and 0.2% fungal-derived β-glucan the lowest.
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http://dx.doi.org/10.1016/j.fsi.2015.04.025DOI Listing
August 2015

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

Vet Immunol Immunopathol 2014 Oct 25;161(3-4):251-7. Epub 2014 Aug 25.

Universidad de Málaga, Departamento de Genética, Facultad de Ciencias, 29071 Málaga, Spain. Electronic address:

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 × 10(2)TCID50/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 48 h p.i. in cells infected with 5 × 10(2)TCID50/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.
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http://dx.doi.org/10.1016/j.vetimm.2014.08.005DOI Listing
October 2014

Molecular characterisation of four class 2 cytokine receptor family members in rainbow trout, Oncorhynchus mykiss.

Dev Comp Immunol 2015 Jan 3;48(1):43-54. Epub 2014 Sep 3.

Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 2TZ, Scotland, UK.

The interleukin (IL)-10 cytokine family includes IL-10, IL-19, IL-20, IL-22, IL-24, IL-26 and the lambda/type III interferons. They are highly pleiotropic and mediate a variety of activities, including immune suppression and antibacterial immunity. To exert their functions they signal through a heterodimeric receptor composed of a subunit with a long intracellular domain (R1 type receptors; IL-10R1, IL-20R1 or IL-22R1) and a subunit with a short intracellular domain (R2 type receptors; IL-10R2 or IL-20R2). In this study we report the identification of three R1 type receptors (named IL-10R1/CRFB7, IL-20R1a/CRFB8a and IL-20R1b/CRFB8b) and one R2 type receptor (named IL-10R2/CRFB4) in rainbow trout. The nomenclature of the receptors was supported by homology analysis, conserved motifs and phylogenetic tree analysis, confirming they belong to the piscine class 2 cytokine receptor family. For instance, they all displayed the presence of characteristic features, such as conserved fibronectin type-III domains. Expression analysis in tissues collected from healthy fish revealed different patterns of expression for each receptor, suggesting their potential involvement in different types of immune responses. When studying the modulation of the genes in cell lines and primary cultures, a greater effect was observed in the cell lines, where the expression of most receptors was affected by incubation with microbial mimics (LPS and PolyI:C) or the pro-inflammatory cytokine rIFN-γ. In addition, expression of the four receptors was modulated by viral infection, suggesting a potential involvement of such receptors and their ligands in antiviral defence.
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http://dx.doi.org/10.1016/j.dci.2014.08.012DOI Listing
January 2015

Isolation and activity of the promoters for STAT1 and 2 in Atlantic salmon Salmo salar.

Fish Shellfish Immunol 2014 Oct 13;40(2):644-7. Epub 2014 Aug 13.

Marine Scotland, 375 Victoria Road, Aberdeen, AB11 9DB, UK.

Signal Transducer and Activator of Transcription (STAT) 1 and 2 molecules are part of the interferon (IFN) type I and type II (γIFN) signalling pathways, key pathways in the innate immune response. Genomic sequence regions upstream from the 5-prime Salmo salar ORFs were obtained and shown to have functional activity through their incorporation into luciferase reporter constructs and subsequent activation by salmonid alpha virus (SAV). The STAT1 and STAT2 putative promoter regions were also induced by co-transfected plasmids expressing γIFN and IFN type I respectively. Two IFN-induced gene regulatory motifs (GAAANN) associated in a complete Interferon Stimulating Response Element (ISRE) were identified in the STAT1 putative promoter sequence and several GAS elements conforming to Boehm's consensus TTNCNNNAA. Sixteen IFN-induced gene regulatory motifs (GAAANN) could be identified in the STAT2 putative promoter region but no Boehm's GAS element nor ISRE. A palindromic sequence that conforms to Decker's consensus GAS element TTCNNN(N)GAA was identified. The reporter constructs generated here may prove an additional tool for refining knowledge on interferon signalling in fish and the inhibition of such by some fish viral pathogens.
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http://dx.doi.org/10.1016/j.fsi.2014.07.025DOI Listing
October 2014