Publications by authors named "Julia Bejar"

18 Publications

  • Page 1 of 1

Differential immunogene expression profile of European sea bass (Dicentrarchus labrax, L.) in response to highly and low virulent NNV.

Fish Shellfish Immunol 2020 Nov 20;106:56-70. Epub 2020 Jul 20.

Universidad de Málaga, Instituto de Biotecnología y Desarrollo Azul, IBYDA, Departamento de Microbiología, Facultad de Ciencias, 29071, Málaga, Spain. Electronic address:

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (MutDl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of MutDl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection, and therefore, their functional activity could be analysed in future studies. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinants of RGNNV virulence to European sea bass.
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http://dx.doi.org/10.1016/j.fsi.2020.06.052DOI Listing
November 2020

Comparative analysis of marine and freshwater viral haemorrhagic septicaemia virus (VHSV) isolates antagonistic activity.

Comp Immunol Microbiol Infect Dis 2020 Apr 28;69:101426. Epub 2020 Jan 28.

Universidad de Málaga, Instituto de Biotecnología y Desarrollo Azul, IBYDA, Área De Genética, Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, 29071, Málaga, Spain. Electronic address:

Viral Haemorrhagic Septicaemia Virus (VHSV) isolates virulent to marine fish species can replicate in freshwater species, although producing little or no mortality. Conversely, isolates from freshwater fish do not cause disease in marine species. An inverse relationship between VHSV virulence and host mx gene up-regulation has been described for several fish species, suggesting that differences between the antagonistic activity exerted by these isolates might be involved in the outcome of infections. In this study, the antagonistic activity against the type I interferon system of two representative marine and freshwater VHSV isolates has been characterised using RTG-2 cells stably transfected with the luciferase gene under the control of the Senegalese sole mx (ssmx) promoter, RTG pssmx-luc cells. Both isolates exerted a dose-dependent negative effect on the activation of ssmx promoter, showing a notably different minimal viral dose to exert the antagonism. In particular, an inverse relationship between the minimal MOI required and the viral virulence to sole has been recorded, which suggests this parameter as a possible in vivo VHSV virulence marker. Furthermore, the quantification of the endogenous inf I, mx1 and mx3 mRNA has demonstrated differences between both isolates in their antagonistic activity. Besides, a different nv RNA kinetics, which seems to depend on specific cellular factors, has been recorded for both isolates. This knowledge could contribute to the development of efficient tools to fight against viral infections in fish farming. For that purpose, the RTG pssmx-luc cells may be a suitable in vitro tool to identify the molecular mechanisms underlying VHSV-host interactions.
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http://dx.doi.org/10.1016/j.cimid.2020.101426DOI Listing
April 2020

Identification of an interferon-stimulated gene, isg15, involved in host immune defense against viral infections in gilthead seabream (Sparus aurata L.).

Fish Shellfish Immunol 2018 Feb 19;73:220-227. Epub 2017 Dec 19.

Departamento de Biología Celular e Histología, Facultad de Biología, Universidad de Murcia, IMIB-Arrixaca, 30100 Murcia, Spain. Electronic address:

Interferons (IFNs) play a key role in the innate immunity of vertebrates against viral infections by inducing hundreds of IFN-stimulated genes (ISGs), such as isg15. Isg15 is an ubiquitin-like protein, which can conjugate cellular and viral proteins in a process called ISGylation, although it can also act as a cytokine-like protein. Gilthead seabream (Sparus aurata L.) is an important asymptomatic carrier of viral haemorrhagic septicaemia virus (VHSV) and nodavirus, representing a threat to other co-cultivated susceptible species. In order to better understand virus-host interactions in this fish species, this study addresses the identification and molecular characterization of seabream isg15 (sb-isg15). In addition, the modulation of transcript levels of sb-isg15 was analysed in SAF-1 cells and seabream acidophilic granulocytes (AGs) stimulated in vitro with different pathogen-associated molecular patterns (PAMPs) or inoculated with VHSV and striped jack nervous necrosis virus (SJNNV). The full-length cDNA of sb-isg15 gene, encoding a predicted protein of 155 amino acids, was identified and seen to share the same characteristics as other fish and mammalian isg15 genes. Here we report the clear induction of sb-isg15 transcript levels in SAF-1 cells and AGs stimulated with toll-like receptor (TLR) ligands, such as polyinosinic:polycytidylic acid (poly I:C) or genomic DNA from Vibrio anguillarum (VaDNA), respectively. Furthermore, VHSV and SJNNV inoculation induced a significant degree of sb-isg15 transcription in SAF-1 cells and AGs. However, the relative levels of viral RNA transcription showed that SJNNV replication seems to be more efficient than VHSV in both in vitro systems. Interestingly, sb-isg15 transcript induction elicited by VaDNA was reduced in VHSV- and SJNNV-inoculated AGs, suggesting an interference prompted by the viruses against the type I IFN system. Taken together, these findings support the use of seabream AGs as a valuable experimental system to study virus-host interactions, in which sb-isg15 seems to play an important role.
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http://dx.doi.org/10.1016/j.fsi.2017.12.027DOI Listing
February 2018

Molecular characterization and expression analyses of the Solea senegalensis interferon-stimulated gene 15 (isg15) following NNV infections.

Fish Shellfish Immunol 2017 Jul 17;66:423-432. Epub 2017 May 17.

Universidad de Málaga, Departamento de Microbiología, Facultad de Ciencias, Campus de Teatinos s/n, 29071 Málaga, Spain. Electronic address:

Interferons are essential in fish resistance to viral infections. They induce interferon-stimulated genes, such as isg15. In this study, the Senegalese sole isg15 gene (ssisg15) has been characterized. As other isg15, ssisg15 contains a 402-bp intron sited in the 5'-UTR, and the full length cDNA is 1492-bp, including a 480-bp ORF. The expression analyses revealed basal levels of isg15 transcripts, and a clear induction after poly I:C injection, that reached maximum values in brain, head kidney and gills. The ssisg15 induction patterns were similar in RGNNV- and SJNNV-inoculated fish, whereas the reassortant (RG/SJ) isolate, which has higher replication fitness, triggered delayed but higher transcript levels. Furthermore, RG/SJ infection after poly I:C treatment reduced the induction of ssisg15 transcripts, suggesting an antagonistic mechanism against interferon type I system, that might allow an efficient viral replication at the initial steps of the infective process.
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http://dx.doi.org/10.1016/j.fsi.2017.05.040DOI Listing
July 2017

Role of the IFN I system against the VHSV infection in juvenile Senegalese sole (Solea senegalensis).

Vet Res 2016 Jan 8;47. Epub 2016 Jan 8.

Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071, Málaga, Spain.

Senegalese sole is susceptible to marine VHSV isolates but is not affected by freshwater isolates, which may indicate differences regarding virus-host immune system interaction. IFN I induces an antiviral state in fish, stimulating the expression of genes encoding antiviral proteins (ISG). In this study, the stimulation of the Senegalese sole IFN I by VHSV infections has been evaluated by the relative quantification of the transcription of several ISG (Mx, Isg15 and Pkr) after inoculation with marine (pathogenic) and freshwater (non-pathogenic) VHSV isolates. Compared to marine VHSV, lower levels of RNA of the freshwater VHSV induced transcription of ISG to similar levels, with the Isg15 showing the highest fold induction. The protective role of the IFN I system was evaluated in poly I:C-inoculated animals subsequently challenged with VHSV isolates. The cumulative mortality caused by the marine isolate in the control group was 68%, whereas in the poly I:C-stimulated group was 5%. The freshwater VHSV isolate did not cause any mortality. Furthermore, viral RNA fold change and viral titers were lower in animals from the poly I:C + VHSV groups than in the controls. The implication of the IFN I system in the protection observed was confirmed by the transcription of the ISG in animals from the poly I:C + VHSV groups. However, the marine VHSV isolate exerts a negative effect on the ISG transcription at 3 and 6 h post-inoculation (hpi), which is not observed for the freshwater isolate. This difference might be partly responsible for the virulence shown by the marine isolate.
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http://dx.doi.org/10.1186/s13567-015-0299-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705576PMC
January 2016

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

Structural and functional characterization of the Senegalese sole (Solea senegalensis) Mx promoter.

Fish Shellfish Immunol 2013 Nov 19;35(5):1642-8. Epub 2013 Sep 19.

Department of Genetics, Faculty of Sciences, University of Malaga, 29071 Malaga, Spain; Department of Microbiology, Faculty of Sciences, University of Malaga, 29071 Malaga, Spain.

Mx proteins are one of the most studied interferon-stimulated genes (ISGs). The antiviral activity against different fish viruses has been demonstrated for diverse fish Mx proteins, including the Senegalese sole (Solea senegalensis) Mx protein (SsMx). The aim of the current study is to characterize the structure and functional activity of the SsMx promoter. Several polyclonal cell populations expressing the luciferase reporter gene under the control of the SsMx promoter have been used to determine the ability of this promoter to drive the expression of the luciferase gene after poly I:C stimulation. In addition, the implication of each interferon-stimulated response element (ISRE) in the activation of the promoter has also been analysed. The genomic structure of the Senegalese sole and Japanese flounder Mx promoters (containing three ISREs) differs from the rest of the fish Mx promoters described to date. The ISRE1, the one closest to the start codon, is the main ISRE involved in the SsMx promoter activity, whereas ISRE2 and ISRE3 show a minor additive effect on this activity. Another feature differing SsMx promoter from the rest of the fish Mx promoters is the presence of a 24-bp GC island close to the ATG codon, including one Sp1 binding site, which may constitute the transcriptional start site. Furthermore, the SsMx promoter contains a gamma interferon activation site (GAS) element.
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http://dx.doi.org/10.1016/j.fsi.2013.09.016DOI Listing
November 2013

Antiviral specificity of the Solea senegalensis Mx protein constitutively expressed in CHSE-214 cells.

Mar Biotechnol (NY) 2013 Apr 11;15(2):125-32. Epub 2012 Aug 11.

Department of Microbiology, Faculty of Sciences, University of Malaga, Campus Teatinos, 29071 Malaga, Spain.

Interferons play a key role in fish resistance to viral infections by inducing the expression of antiviral proteins, such as Mx. The aim of the present study was to test the antiviral activity of the Senegalese sole Mx protein (SsMx) against RNA and DNA viruses pathogenic to fish, i.e. the infectious pancreatic necrosis virus (IPNV, dsRNA), the viral haemorrhagic septicaemia virus (VHSV, ssRNA), and the European sheatfish virus (ESV, dsDNA), using a CHSE-214 cell clone expressing this antiviral protein. A strong inhibition of IPNV and VHSV replication was recorded in SsMx-expressing cells, as has been shown by the virus yield reduction and the decrease in the synthesis of the viral RNA encoding the polyprotein (for IPNV) and the nucleoprotein (for VHSV). The titres of these viruses replicating on SsMx-expressing cells were 100 times lower than those recorded on non-transfected cells. In contrast, SsMx did not inhibit ESV replication since no significant differences were observed regarding the virus yield or the major capsid protein gene transcription in transfected and non-transfected cells.
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http://dx.doi.org/10.1007/s10126-012-9478-8DOI Listing
April 2013

Viral nervous necrosis virus persistently replicates in the central nervous system of asymptomatic gilthead seabream and promotes a transient inflammatory response followed by the infiltration of IgM+ B lymphocytes.

Dev Comp Immunol 2012 Jul 5;37(3-4):429-37. Epub 2012 Mar 5.

Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, Murcia, Spain.

The viral nervous necrosis virus (VNNV) is the causal agent of viral encephalopathy and retinopathy (VER), a worldwide fish disease that is responsible for high mortality in both marine and freshwater species. Infected fish suffer from encephalitis, which leads to abnormal swimming behavior and extensive cellular vacuolation and neuronal degeneration in the central nervous system (CNS) and retina. The marine fish gilthead seabream (Sparus aurata) does not develop VER but it is an asymptomatic carrier of VNNV. In this study, we report that VNNV was able to replicate and persist for up to 3 months in the CNS of the gilthead seabream without causing any neural damage. In addition, we found an early inflammatory response in the CNS that was characterized by the induction of genes encoding pro-inflammatory cytokines, a delayed but persistent induction of anti-inflammatory cytokines, and the infiltration of IgM(+) B lymphocytes, suggesting that local adaptive immunity played a major role in the control of VNNV in the CNS of this species.
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http://dx.doi.org/10.1016/j.dci.2012.02.007DOI Listing
July 2012

Endogenous transposases affect differently Sleeping Beauty and Frog Prince transposons in fish cells.

Mar Biotechnol (NY) 2011 Aug 1;13(4):695-705. Epub 2010 Dec 1.

Department of Genetics, Faculty of Sciences, University of Málaga, Campus de Teatinos, 29071 Málaga, Spain.

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.
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http://dx.doi.org/10.1007/s10126-010-9331-xDOI Listing
August 2011

c-Lysozyme from Senegalese sole (Solea senegalensis): cDNA cloning and expression pattern.

Fish Shellfish Immunol 2008 Nov 22;25(5):697-700. Epub 2008 Aug 22.

Department of Genetics, Faculty of Sciences, University of Málaga, 29071 Málaga, Spain.

Lysozymes are key molecules of innate immunity and proved high bactericidal activity in fish, thus becoming attractive as tools for enhancing fish defences. In this study, a full-length c-type lysozyme cDNA from Senegalese sole (Solea senegalensis) has been cloned and characterized. The cDNA sequence was inferred from two overlapping fragments obtained by RACE-PCR and consisting on 631bp coding for 143 aminoacids. Catalytic and other conserved residues required for lysozyme activity were identified. Pair wise alignments showed the higher identities with c-type lysozyme from other flatfish. Expression patterns under various conditions showed a basal level and a clear upregulation mostly in hematopoietic organs after stimulation with LPS or infection with Photobacterium damselae. This study represents a first step on the genetics and function of the c-lysozyme of Senegalese sole, though disclosing g-DNA structure, allelic variability and antibacterial activity must be requirements prior its immunological properties might have biotechnological applications.
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http://dx.doi.org/10.1016/j.fsi.2008.08.004DOI Listing
November 2008

In vitro inhibition of sole aquabirnavirus by Senegalese sole Mx.

Fish Shellfish Immunol 2008 Feb 5;24(2):187-93. Epub 2007 Nov 5.

Department of Genetics, Faculty of Sciences, University of Málaga, Campus de Teatinos, Málaga, Spain.

Senegalese sole, Solea senegalensis, is a flat fish of growing interest in European aquaculture. In its culture viral infections are constant threats, thus understanding antiviral defences is a key factor for a successful industry. Mx proteins are IFN-induced proteins widespread in eukaryotes; however, their antiviral activity is unclear and the results variable among species. Therefore assessment of the putative Mx antiviral activity in each species is of interest. Our group has recently cloned the Senegalese sole Mx (SsMx) cDNA and in this study its antiviral activity was assessed by infecting CHSE-214 cells expressing recombinant SsMx, with sole aquabirnavirus. The antiviral activity against this pathogen was demonstrated by reduction in induced cytopathic effects, reduction in virus yield and decrease in viral transcripts. These findings contribute to our understanding of fish antiviral mechanisms and open the possibility of using this protein as a tool for fighting viral infections in aquaculture.
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http://dx.doi.org/10.1016/j.fsi.2007.10.010DOI Listing
February 2008

Expression analysis of Mx protein and evaluation of its antiviral activity against sole aquabirnavirus in SAF-1 and TV-1 cell lines.

Vet Immunol Immunopathol 2008 Jan 2;121(1-2):123-9. Epub 2007 Oct 2.

Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Campus Teatinos, 29071 Málaga, Spain.

The transcription of Mx mRNA after poly I:C induction and sole aquabirnavirus infection has been analysed in SAF-1 and TV-1 cells (derived from gilt-head seabream and turbot, respectively). Both cell lines were stimulated with 10 microg ml(-1) poly I:C and Mx mRNA was analysed by a specific RT-PCR at several times post-induction. The results showed a high level of Mx expression from 12 to 120 h after induction in SAF-1 cells, whereas in TV-1 cells Mx mRNA was only detected at 12 and 24h. The treatment with different concentrations of poly I:C showed that TV-1 cells are less sensitive to this inductor than the SAF-1 cell line. The antiviral activity derived from poly I:C induction has been clearly demonstrated against sole aquabirnavirus on both cell lines. The inoculation of sole aquabirnavirus resulted in the Mx mRNA transcription at 48, 72, and 96 h post-infection (p.i.) in SAF-1 cells. On the contrary, inoculated TV-1 cells only showed a faint Mx mRNA band at 24 and 48 h p.i. This study has established different patterns of Mx expression in both cells under study as a consequence of the poly I:C induction and sole aquabirnavirus infection, and it shows that gilt-head seabream and turbot Mx inhibit sole aquabirnavirus replication.
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http://dx.doi.org/10.1016/j.vetimm.2007.09.008DOI Listing
January 2008

Fish ES cells and applications to biotechnology.

Mar Biotechnol (NY) 2007 Mar-Apr;9(2):117-27. Epub 2006 Nov 6.

Department of Cell Biology and Genetics, Faculty of Sciences, University of Málaga, 29071, Málaga, Spain.

ES cells provide a promising tool for the generation of transgenic animals with site-directed mutations. When ES cells colonize germ cells in chimeras, transgenic animals with modified phenotypes are generated and used either for functional genomics studies or for improving productivity in commercial settings. Although the ES cell approach has been limited to mice, there is strong interest for developing the technology in fish. We describe the step-by-step procedure for developing ES cells in fish. Key aspects include avoiding cell differentiation, specific in vitro traits of pluripotency, and, most importantly, testing for production of chimeric animals as the main evidence of pluripotency. The entire process focuses on two model species, zebrafish and medaka, in which most work has been done. The achievements attained in these species, as well as their applicability to other commercial fish, are discussed. Because of the difficulties relating to germ line competence, mostly of long-term fish ES cells, alternative cell-based approaches such as primordial germ cells and nuclear transfer need to be considered. Although progress to date has been slow, there are promising achievements in homologous recombination and alternative avenues yet to be explored that can bring ES technology in fish to fruition.
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http://dx.doi.org/10.1007/s10126-006-6034-4DOI Listing
October 2007

Cloning and expression analysis of Mx cDNA from Senegalese sole (Solea senegalensis).

Fish Shellfish Immunol 2006 Nov 22;21(5):577-82. Epub 2006 Mar 22.

Department of Genetics, Faculty of Sciences, University of Málaga, Campus de Teatinos, 29071 Málaga, Spain.

Senegalese sole (Solea senegalensis) is a promising fish species of growing interest in European aquaculture. In fish farming, viral infections are a constant threat therefore, understanding fish defence mechanisms is a main priority to avoid economic losses. Mx proteins are involved in the innate antiviral response of fish. They are induced by type I interferons (alpha and beta) and are essential to investigate viral defence mechanisms in fish, due to the difficulty in tracking interferon activity in these species. In this study a full-length Senegalese sole Mx cDNA has been RT-PCR cloned, resulting in 2322bp coding for 623 amino acids. The sequence accounts for the main characteristics of Mx proteins but lacking nuclear localisation signal (NLS), which suggests cytoplasmic localisation. The alignments of Senegalese sole Mx sequence showed the highest identity with the flatfish species, 80.1% identity with flounder and 78.9% with halibut. The spatial and temporal expression pattern has been analysed in control and challenged fish by RT-PCR. In control fish a constitutive level of sole Mx expression has been obtained and a clear induction was observed after treatment with Poly[I:C], which supports a putative role for the Mx in Senegalese sole viral defence. These findings contribute to increasing the knowledge of the role of interferon pathway in fish innate immunity and to develop new tools to fight virus infections in the culture of this species.
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http://dx.doi.org/10.1016/j.fsi.2006.03.003DOI Listing
November 2006

The piscine SAF-1 cell line: genetic stability and labeling.

Mar Biotechnol (NY) 2005 Jul-Aug;7(4):389-95. Epub 2005 Jun 4.

Department of Genetics, Faculty of Sciences, University of Málaga, 29071, Málaga, Spain.

Fish cell lines are increasingly important research tools. The SAF-1 cell line, fibroblast-like culture derived from the marine fish gilthead seabream (Sparus aurata), has proved useful in many applications, especially in viral research. For cell lines intended as in vitro models, characterization of their properties and authentication are essential for deeper understanding of their performance and thus more precise experimental design and applicability. In this study we characterized the SAF-1 cell line in terms of genetic stability through time and genetic labeling. Methods for determining stability include telomerase activity, karyotyping, mapping of ribosomal RNA regions, and DNA content. For genetic labeling 12 microsatellite loci were used. The results indicate that telomerase has been activated in the course of SAF-1 development, and the highest levels of telomerase activity correlate with an increase in cell proliferation, thus supporting a permanent cell line. This stability is in agreement with the normal situation presented by the cytogenetic traits and DNA content values, and the genotypic profile allows SAF-1 authentication at the single individual level. This study increases the value of SAF-1 as an in vitro system, which is now one of the few well-characterized cell lines from a marine fish.
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http://dx.doi.org/10.1007/s10126-004-4083-0DOI Listing
April 2006

Mitf expression is sufficient to direct differentiation of medaka blastula derived stem cells to melanocytes.

Development 2003 Dec;130(26):6545-53

Physiological Chemistry I, Biocenter of the University Würzburg, D-97074 Würzburg, Germany.

Embryonic stem (ES) cell lines have provided very useful models to analyse differentiation processes. We present here the development of a differentiation system using ES-like cell lines from medaka. These cells were transfected with the melanocyte specific isoform of the microphtalmia-related transcription factor (Mitf). Mitf is a basic helix-loop-helix-leucine zipper transcription factor whose M isoform is restricted to neural crest derived melanocytes and is essential for the development of these cells in vertebrates from mammals to fish. What is not clear yet is whether Mitf is a downstream factor or a master regulator of melanocyte commitment and differentiation. Expression of Mitf in the ES-like cells from medaka led to the induction of cells that, by morphology, physiology and gene expression pattern, were confirmed to be fully differentiated pigment cells. Mitf expression is therefore sufficient for the proper differentiation of medaka pluripotent stem cells into melanocytes.
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http://dx.doi.org/10.1242/dev.00872DOI Listing
December 2003

An ES-like cell line from the marine fish Sparus aurata: characterization and chimaera production.

Transgenic Res 2002 Jun;11(3):279-89

Departamento de Biología Celular y Genética, Facultad de Ciencias, Universidad de Málaga, Spain.

Embryonic stem (ES) cells provide a unique tool for cell-mediated gene transfer and targeted gene mutations due to the possibility of in vitro selection of desired genotypes. When selected cells contribute to the germ line in chimaeric embryos, transgenic animals may be generated with modified genetic traits. Though the ES cell approach has up to now been limited to mice, there is an increasing interest to develop this technology in both model and commercial fish species, with so far promising results in the medaka and zebrafish. In this study, we present evidence regarding a long-term stable cell line (SaBE-1c), derived from embryonic cells of the aquaculture marine fish Sparus aurata which has been characterized for (i) cell proliferation, (ii) chromosome complement, (iii) molecular markers, and (iv) in vitro tests of pluripotency by alkaline phosphatase (AP) staining, telomerase activity, and induced cell differentiation. These cells have proved their pluripotent capacities by in vitro tests. Furthermore, we have demonstrated their ability to produce chimaeras and to contribute to the formation of tissues from all three embryonic germ layers. These features suggest that SaBE-1c cells have the potential for multiple applications for the ES technology in fish, with the added value of originating from an economically important species.
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http://dx.doi.org/10.1023/a:1015678416921DOI Listing
June 2002